CA2236157A1 - Methods for treating photoreceptors using glial cell line-derived neurotrophic factor (gdnf) protein product - Google Patents
Methods for treating photoreceptors using glial cell line-derived neurotrophic factor (gdnf) protein product Download PDFInfo
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Abstract
The present invention relates generally to methods for treating injury or degeneration of retinal neurons, and in particular photoreceptors, by administering glial cell line-derived neurotrophic factor (GDNF). The invention relates specifically to methods for treating retinal conditions or diseases in which vision is lost such as retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, peripheral vitreoretinopathies, photic retinopathies, surgery-induced retinopathies, viral retinopathies, ischemic retinopathies, retinal detachment and traumatic retinopathy.
Description
CA 022361~7 1998-0~-21 METHODS FOR TREATING PHOTORECEPTORS
IJSIN~; GLIAL CELL LINE-DERIYED NEUROTROPHIC FACTOR
(GDNF) PROTEIN PRODUCT
Field of Invention The present invention relales generally to methods for lreating injury or degeneration of retinal neurons by ad~"inist~,i"g glial cell line-derived neufolr~phic factor ~GDNF) protein product. The invention relates specifically 10 to methods for treating pathological conditions, such as inherlted retinal degenerations and age, disease or injury-related retinopathies, in which photoreceptor degeneralion occurs and is responsible for vision loss.
R5~ round of the Invention llecer,lly, several naturally occurring proteinaceous mo'~cules haYe been identified based on their trophic activity on various types of neurons. These molecules are termed "neurotrophic factors". NeL.,ut,ophic factors are endogenous, soluble proteins that play a major role in neuronal survival and growth during develop",ent, as well as in the funclion~l ",ainlenance and 20 plasliclty of mature neurons; see Fallon and Laughlin, Neurotrophlc Factors, Academic Press, San Diego, CA (1993). In view of their ability to promote neuron regeneration and to prevent neuron death and degeneratbn, it has been postulA~ed that neurol,uph c factors might be useful in treating neurodegenerative conditions of the nervous system, such as, for example, 25 Parkinson's disease, Alzheimer's disease, am~olroph c lateral sclerosis and stroke.
Ilerve damage is caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells, including:
(1) physical injury, which causes the degeneration of the axonal processes 30 (which in turn causes nerve cell death) and/or nerve cell bodies near the site of injury, (2) temporary or permanent cess~ti~n of. blood flow (ischemia) to parts of the nervous system, as in stroke, (3) intentional or accidental exposure to neu.uloxins~ such as the cancer and AIDS chemotherapeutic agents cispla~inum and dideoxycytidine, respectively, (4) chronic metabolic dise~cesi such as 35 ~ eles or renal dysfunction, or (5) neurodegenerative ~iseases such as rarl~inson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis.
which result from the degeneration of specific neuronal popul~tions. In order for CA 022361~7 1998-0~-21 WO 97/19694 PCI~/US96/18806 a particular neu~lf~phic factor to be potentially useful in treating nerve dar.,age, the class or classes of da~aged nerve cells must be .esponsNe to the factor; different neufol,ophic factors typically affect clist-"clly ~irlerenl classes of nerve cells. It has been established that all neuron popu'~ions are not responsive to or equally affected by all neurotrophic factors.
The first neurotrophic factor to be identified was nerve growth factor (NGF). NGF is the first ",e",ber of a defined family of trophic factors, called the neurotrophins, that currently includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 ~NT-3), NT-415, and NT-6 (Thoenen, ~rends.
Neurosci., 14:165-170, 1991; Lapchak et al., Rev. NBUrOSCj., 3:1-10, 1993;
~D~h-Yell, Ann. F~ev. Neurosci., 18:223-253, 1g95). These neurotrophins are known to act through the family of trk tyrosine kinase leceplors, i.e., trkA, trkB, trkC, and the low affinity p75 receptor (Lapchak et al., Rev. Neuroscl., 3:1-10, 19g3; Bothwell, Ann. Rev. Neurosci., 18:223-253, 19g5; Chao et al., TINS, 18:321-326, 19g5). In the cenlral nervous system (CNS). the e~,uression of trkA, the receptor for NGF, is almost exclusiYely limited to the cholinergic neurons in the basal forebrain (Venero et al., Neuroreport, 4:959-962, 1993), which also express p75 and trkB. These chol nerg-c neurons are of particular neurologic interest, because cholinergic neuronal degeneration andlordystrophy is a hallmark of Alzheimer's disease (Hefti, ~. NeurobioJ., 25:1418-1435, 1g94; Olson, Neurochem. Jul., 15:1-3, 1994). The basal forebrain cholinergic neurons can be readily identified in morphologic preparations using acetylcholinesterase histochemistry or with immunohistochemistry using ar,libG~y to choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, or to p75 (Batchelor et al., J. Comp. Neurol., 284:187-204, 1989; Kiss et al., Neurosci., 27:731-748, 1988; Woolf et al., Neuroscience, 30 :1 43-1 52, 1 98g) .
Glial cell line-derived neurotrophic factor (GDNF) is a recently discovered protein idenliried and purified using assays based upon its efficacy in pro",oling the survival and stimulating the transmitter phenotype of mesencephalic dopaminergic neurons in vitro (Lin et al., Science, 260:1130-1132, 1993). GDNF is a glycosylated, disulfide-bonded homodimer that is ~i.,l&l~lly related to the transforming growth factor-B (TGF-B) superfamily of neu,otloph c proteins (Krieglstein et al., EMBO J., 14:736-742, 1995;
Poulsen et al., Neuron ,13:1245-1252, 1994). GDNF has been cloned, and the recombinant human GDNF (rhuGDNF) exerts trophic and survival-promoting actions on substantia nigra dopaminergic neurons and spinal cord motor neurons CA 022361~7 1998-0~-21 WO 97/196g4 PC r/uss6tl8806 in vitro, as well as in vivo tBeck et al., Nature, 273:339-341, 1995;
Henderson et al., Science, 266:1130-1132, 1994; Tomac et al., Nature, 273:
335-33~; Yan et al., Nature, 273: 341-343; Zurn et al., Nevroreport,6:113-118, 1994). In vivo, treatment with exogenous GDNF stimulates the dopaminergic phenotype of substantia ni3ra neurons and restores functional deficits induced by axotomy or dopaminergic neurotoxins in animal models of Parl~inson's disease, a neu.udegenerative disease cl,aracle-i~ed by the loss of dopaminergic neurons (Hudson et al., Brain Res. E~ull., 36:425-432, 1995;
Hoffer et al., Neurosci. Lett., t82:107-111, 1994). Although originally thought to be relatively specific for dopa",i"ergic neurons, at least in vitro, subse~llent experiments have found that GDNF has neu,ut~oph ~ efficacy on brain stem and spinal cord cholinergic motor neurons, both in vivo and ~n vitro (Oppenheim et al., Nature, 373:344-346, 1995; Zurn et al., Neuroreport, 6:113-118, 1994; Yan et al., Nature, 373: 341-344, 1995; Henderson et al Scienc~, 266:1062-1064, 1994). GDNF is, therefore, a factor with potential therapeutic benefit in the treatment of degenerative d;sordels of spinal cord motor neurons, such as amyotrophic lateral sclerosis.
Thus, evidence is beginning to emerge indicating that GDNF may have a larger spectrum of neurotrophic targets besides mesencephalic d~pai"i"ergic and sG",alic motor neurons (Yan and Matheson, Nature, 373:341-344, 1995;
Miller et al., Soc. Neurosci. Abstr., 20:1300, 1994). GDNF messenger RNA
(mRNA) has t~een de~ected in muscle and Schwann cells in the peripheral nervous system and in type I astrocytes (Schaas et al., Exp. Neurol., 124:368-371, 1993) in the central nervous system. GDNF mRNA is also ex~lessed in high leYels in the developing rat striatum (Stromberg et al., l~xp. Neurol., 124:401-412, 19g3), and in low levels in regions of the adult rat and human central nervous system, including striatum, hippocampus, cortex and spinal cord (Springer et al., Exp. Neurol., 127:167-170, 1994).
C)f general interest to the present invention is WO93106116 (Lin et al., Syntex-Synergen Neuroscience Joint Venture), published April 1, 1993, which reports lhat GDNF is useful for the treatment of nerve injury, including injury assoc;a~ed with Parkinson's Disease. Also of interest are a report in Schmidt-Kastner et al., Mol. Brain Res., 26:325-330, 1994 that GDNF mRNA became detect~le and was upregulated after pilocarpine-induced seizures; reports in Schaar et al., Exp. Nevrol., 124:368-371, 1993 and Schaar et al.. Exp. Neurol.,130:387-393, 1994 that basal forebrain astrocyles expressed moderate levels of GDNF mRNA under culture conditions, but that GDNF did not alter basal CA 022361~7 1998-05-21 forebrain ChAT activity; and a report in currently pending U.S. Application Seriat Number 08/535.682 fiied September 28, 1995 that GDNF is useful for treating injury or degeneration of basal forebrain cholinergic neurons.
In mammals, a number of ophthalmic neurodegenerative conditions or diseases involve injury or degeneration of photoreceptors. Trophic factors capal~le of promoting the survival or regeneration of these neurons would provide useful therapies for the treatment of such dise~ses.
Photoreceptors are a specialized subset of retinal neurons, that are responsible for vision. Photoreceptors consist of rods and cones which are the photosensili~e cells of the retina. Each rod and cone elabordles a speci~ ed cilium, re~e..ed to as an outer segment, that houses the phototransductiQn machinery. The rods contain a specific light-absor~ing visual pigment, rhodopsin. There are three classes of cones in humans, cha-a~tt:ri~ed by the 15 e~ ression of distinct visual pigments: the blue cone, green cone and red cone pigments. Each type of visual pigment protein is tuned to absorb light maximallyat ~ eren1 wavelengths. The rod rhodopsin ",ed;ales sco ~p;c vision (in dim light), whereas the cone pigments are responsible for photopic vision (in brightlight). The red, blue and green pigments also form the basis of color vision in 20 humans. The visual pigments in rods and cones re~.,ond to light and ~ene,~le an action potential in the output cells, the rod bipolar neurons, which is then relayed by the retinal ganglion neurons to produce a visual stimulus in the visual cortex.
In humans, a number of diseases of the retina involve the progressive 25 degenerdlion and eventual death of photoreceptors, leading inexorably to blindness. Degeneration of photoreceptors, such as by inherited retinal dyslrophies (e.g., retinitis pigmentosa), age-related macular degeneration and other m~u'~pathies, or retinal detac~""ent, are all characterized by the p,ogress:ve atrophy and ioss of function of phot~rec~ptor outer segments. In 30 addilion, death of photoreceptors or loss of photoreceptor function results in partial deafferentation of second order retinal neurons trod bipolar cells and horizontal cells) in patients with retinsl dystrophies, thereby decreasing the overall efficiency of the prop~gation of the electrical signal genetaled by photoreceptors. Trophic factors that are capable of rescuing photoreceptur~
35 from cell death andtor restoring the function of dysfunctional (atrophic or dystrophic) photoreceplor~ may represent useful therapies for the treatment of such conditions.
CA 022361~7 1998-0~-21 WO 97/196g'~ PcrluS96/188o6 There is some evidence that certain protein factors may proil,ote the survival of pholoreceF: rs. For example, photoreceptors can be rescued to some extent Iby basic ~iL"oblasl growth factor (bFGF) in Royal College of Surgeons (RCS) rats and in albino rats that have been da",aged by exposure to cofiala"l light (Faktorovich et al., Nature, 347:83-86, 1g90). RCS rats have an inherited mutation of a gene expressed in the retinal pigment epithelium (RPE), that results in the failure of the RPE to phagocytize the continuously shed portions of the photoreceptor outer seglnents and causes photor~ceptor degeneration and eventually cell death. A single injection of bFGF into the 1 0 vitreous body or into the subretinal space, the extrscellular space surrounding rods and cones, at the onset of the degeneration transiently rescues photoreceptors ~Faktorovich et al., Nature, 347:83-86, 1990 ). In the light-damaged model in albino rats, bFGF in~ected into the su~relinal space or the vitreous body two days prior to the onset of constant illumination sigh'icanlly 1 5 plutecls photoreceptors from light injury and prevents cell death (LaVail et al., Proc. Natl. Acad. Sci. USA, 89:11249-11253, 1992). In this model, photoreceptor survival was also seen with acidic FGF (aFGF), brain-derived neurot,oph'~ factor (BDNF), ciliary neurolfophic factor (CNTF), and interleukin-113 (IL-113). Moderate effects were observed with neurotrophin-3 2û (NT-3~, insulin-like-growth factor ll (IGF-II) and tumor necrosis factor-alpha (TNF-alpha). Nerve growth factor (NGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF) and IGF-I had no effect (LaVail et al., Proc. Natl Acad. Sci. USA, 89:11249-11253, 1992). Also see WO 93/15608, ~aVail et al.
Although bFGF is efficacious in the RCS rat and light-induced darl-age rat models, its therapeutic utility in humans is very limited, due to its hypGIensive, ogen-4 and potent ang;ogen ~ activities. In fact. bFGF injected into the vitreous body causes the invasion of blood-derived ll,acrophages in the inner retina and can produce a massive proliferative vitreoretinopalll~ (Faktorovich et al.. Nature, 347:83-86, 1990). It has also been del~rl"ined, using polyll,erase chain reaction technology, that messenger RNA for GDNF is expressed in the eyes of postnatal day 6 and adult rats, essen~ially associated with the neural retina and the retinal pigment epithelium. The RPE cells produce. store and ~lansporl a variety of factors that are responsible for the survival and functional 35 maint~nance of photoreceptors. The RPE cells are also indispensable to the phototlansduction p~cess: they clear up by phagocytosis the shed tips of the outer seyllle~ of photoreceptors and recycle vitamin A. The transplantation of CA 022361~7 1998-0~-21 WO 97/1s694 PCT/US96/18806 normal RPE cells into retinas of RCS rats prevents photoreceptor cell death (Li and Turner, Exp. Eye f~es., 47:911-917, 1988; Mullen and LaVail, Science, 192:799-801, 1976), suggesting the production by RPE cells of a diffusable trophic factor for photoreceptors.
There continues to exist a need for methods and therapeutic co"",ositions useful for the treatment of photoreceplor cell injury. Such methods and therapeutic cGr"positions would ideally protect the photoreceptors from progressive injury and promote the survival or regeneration of the damaged neuron population, without severe side effects.
SUMMARY OF THE INVENTION
The present invention provides a method for treating vision loss due to photoreceptor degeneration by admin;stering a therapeutically effective amount of glial cell line-derived neurotrophic factor (GDNF) protein product. According toone aspect of the invention, methods are provided for treating vision loss due to photoreceptor degeneration by a.ll"inisteril,g a therapeutically effective amount of GDNF protein product. It is conle",plaled that such GDNF protein products would include a GDNF protein such as that depicted by the amino acid sequence set forth in SEO ID NO:1, as well as variants and derivatives thereof. The invention is based on the novel discovery that adminisl,alion of GDNF protein product pr~",oles the survival and regeneration of damaged photo(eceplor neurons, which are the main population of neurons damaged in retinal degenerations leading to blindness.
GDNF protein product may be administered intraocularly at a dose between about 0.001 mg/day and 10 mglday, preferably at a dose between about 0.01 mgJday and 1 mg/day, and most preferably at a dose bel; een about 0.1 mglday and 0.5 mgJday. It is also conle,,,,ulaled that photoreceptor degeneration or injury may be treated by the administration of a GDNF protein product in conjunction with a second therapeutic agent including, but not limited to, brainderived neurotrophic factor, neurotrophin-3, neurotrophin-415, neurotrophin-6, insulin-like growth factor, ciliary neurotrophic factor, acidic and basic fibroblast growth factors, fibroblast growth factor-5, l~dnsfcr",ing growth factor-B, and ***e-amphetamine regulated transcript. It is also conte",plaled that the delivery means for the ad~";nisl,d~ion of a GDNF protein product in the treatment of ophthalmic condiliGns or dise~ces may advant~geou~ly CA 022361~7 1998-0~-21 involve topical formulations, ocular inserts, ocular injection, ocular implants,cell therapy or gene therapy.
The invention also provides for the use of GDNF protein product in the manufacture of a medicament or pharmaceutical composition for the 5 treatment of injury or degeneration of photoreceptor. Such pharmaceutical compositions include topical, oral or parenteral GDNF protein product formulations. It will also be appreciated by those skilled in the art that the ~";n61,dlion process can be acco".F' shed via cell therapy and gene therapy means, as further desc.ibecl below. In yet another aspect, the t 0 present invention includes a method for providing photoreceptor cells for implantation wherein photorece,ulor cells are cultured in the presence of a GDNF protein product. The invention further includes a composition which contains photoreceptor cells together with a GDNF protein product in amounts to enhance the survival and allow the continued growth and 15 maturalion of the photoreceplor cells. Numerous addi~ional aspects and advantages of the invention will become apparent to those skilled in Ihe art upon ccnsideration of the following detailed description of the invention which describes presently preferred em~odiments thereof.
BRIEF DESCRIPTION OF THE D~AWINGS
Fi3ure 1 depicts the effect of glial cell line-derived neurotrophic factor (GDNF) protein product on photoreceptor survival in cultures of retinal Z5 neurons. Each value is the mean + s.d. of three cultures.
Figure 2 depicts the promotion of photoreceplor neurite outgro.vth by GDNF protein product. The data are expressed as a cumulative frequency distribution plot of the neurite lengths. The percenlage of photoreceptors 30 (ordinate) with neurites longer than a given length in micrometers (Ahscissa) is plotted.
Figure 3 depicts the stimulation of glutamate uptake by GDNF protein product in cultures of photoreceptors. The results are expressed as the 35 ~,~nlages of the glutamate uptake values (in dpmlwell) found in control cultures~ Each data point is the mean + s.d. of 3 wells from a represenl~tive experiment.
CA 022361~7 1998-0~-21 Figure 4 depicts the promotion of photoreceptor survival by GDNF
protein product in cultures of retinal neurons. Changes in pholoreceptor number in response to GDNF protein product treatment in cultures from 18-day-old and 5 39-day-old mice are depicted. Each value is the mean + s.d. of 2-3 cultures.
Figure 5 depicts the promotion of photoreceplor survival by GDNF
protein product in cultures of rd~rd mouse retinas. Photo-eceplor survival was determined by counting the number of arrestin-positive neurons per 6-mm 10 well. Each value is the mean + s.d. of 3-4 cultures.
Figure 6 depicts the effect of GDNF on photoreceptor survival in cultures of chick retina. Photoreceptor survival was determined by counting the number of cones per 6 sq. mm diametrical strips (representing about 21% of the total 15 area of a 6-mm well). Each value is the mean + s.d. of 3 cultures.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the demonstration that GDNF protein product has neurotrophic activity for photoreceptors. Prior to this finding, there was no suggeslion or indicalion that GDNF might have such neu,ut.ùph'~
activity . The present invention provides a method for treating injury or degeneration of retinal neurons, particularly photoreceptors, by administering a26 therapeutically effective amount of glial cell line-derived neulotlophic factor (GDNF) protein product by means of a pharmaceutical composition, the implanlalion of GDNF-ex~ ressing cells. or GDNF gene therapy. the invention may be practiced using any biologically active GDNF protein product, including aGDNF having the amino acid sequence set forth in SEQ ID NO:1, variants, and derivatives thereof.
A unique cell culture technique was developed to provide the retinal neuron Fopul~tions used in assessing the responsiveness of pho~orecep~ors to GDNF protein product admin;~ lion. The culture technique is des.;,i6ed in further detail below. The treatment of these pholoreceptors with a GDNF protein product revealed that in addition to promoting photoreceptor survival, the GDNF
protein product also stimulated the extension of the photoreceptor's axon-like pr~cess, thereby demonstrating an effect on the morphological develop",enl of the CA 022361~7 1998-0~-21 Wo 971196~4 Pcrluss6ll88o6 photoreceptors. Glutamate uptake assays further cle",onsl,dted that GDNF proteinproduc~ treatment enhances the functional differentiation of photoreceptors.
These results indicate that among the potential benelil~ of GDNF protein producltherapy are the pro"~olion of photoreceptor survival, the regeneral;on of the 5 photoreceptors' axons and outer segments and the reslora~ion of visual function.
Thus, the admin;slfalion of a GDNF protein product would benefit cohditions in which vision is lost due to the degeneration of pholoreceplor~, such as inherited retinal degeneralions. age-related macular degeneration, injury-induced retinal degenerations, and retinal dystrophies.
The present invention further demonstrates that GDNF protein product treatment pro",otes photoreceptor surviYal in cultures of retina from mice having an inherited retinal degeneration condition. Studies of pholoreceplors ofrd/rd mice illustrated that GDNF protein product treatment enhanced resislance to the delet~rious effect of the rdfrd mutation on photoreceptors. This indicates 15 that GDNF protein product treatment would be useful in the in the reduction and prevention of photorsceptor degeneration and death and even in the reversal of photoreceptor degeneration in human inherited retinal dise~ces cha~ac1eri~ed by photoreceptor degene,alion, such as, for example, retinitis pigmentosa. As illustrated by the studies described below, GDNF protein product adminisl,alion 20 may benefit a variety of pathological conditions in which photoreceptor degeneration occurs and is responsible for vision loss. These cor~ ,ns include inherited retinal degenerations such as retinitis pigmentosa, Bardet-8iedl sy,,d~u-,,e, Bassen-Kornzweig syndrome (abetalipoproteinemia), Best disease (vitelliform dystrophy), choroidemia, gyrate atrophy, congenital amaurosis, 25 Refsum syndrome, Stargardt disease and Usher syndrome. Other retinopathies that may benefit from GDNF protein product admi-,i~,dtion include age-related macular degeneration (dry and wet forms), diabetic ~etillopall"~, peripheral vitreoretinopathies, photic retinopathies, surgery-induced retinopathies, viral retinopathies (such as HIV retinopathy related to AIDS), ischemic rsti.,û,~alhies, 30 retinal d~lacl,ment and traumatic retinopathy.
According to the currently preferred e",bodi."ents of the present invention, the GDNF protein product is most advantageously administered intraocularly at a dose between about 0.001 mg/day and 10 mglday, and pr~f~rdLly at a dose between about 0.01 mglday and 1 mg/day, and most 35 prsf~ra~ly at a dose between about 0.1 mg/day and 0.5 mg/day. It is further conte,-,,ulaled that the GDNF protein product be adl"i"islered in conjunction orcombination with an effective amount of a second therapeutic agent for treating CA 022361~7 1998-0~-21 WO 9711g694 PCT/US96/18806 retinal degeneration or retinal dysl~ophies. Such second therapeutic agents may include, but are not limited to: mitogens such as insulin, insulin-like growth factors, epidermal growth factor, vasoactive growth factor, pituitary adenylate cyclase activating polypeptide, interferon and somatoslali,~; neu,t,t-ophlc factors such as brain derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5 neurotrophin-6, insulin-like growth factor, ciliary neurotrophic factor, acidic and basic fibroblast growth factors, fibroblast growth factor-5, transforming growth factor-B. and ***e-amphetamine regulated transcript ~CART); and other growth factors such as epidermal growth factor, leukemia inhibitory factor, interleukins, interferons, and colony stimulating factors; as well as molecules and materials which are the functional equivalents to these factors.
The invention also provides for the use of GDNF protein product in pre~t~aration of a medicament for the treatment of injury or degeneration of photoreceptors, including the treatment of the dise~ces and conditions describedabove. Such GDNF protein product pharmaceutical preparations are more fully desc,il,ed below.
As used herein, the term "GDNF protein product" includes purified natural, synthetic or recombinant glial cell line-derived neurotrophic factor, biologically active GDNF variants (including insertion, substitution and deletion variants), and chemically ",o.lified derivatives thereof. Also included are GDNFs that are sul~slantially homologous to the human GDNF having the amino acid sequence set forlh in SEQ ID NO:1. GDNF protein products may exist as homodimers or heterodimers in their biologically active form.
The term "biologically active" as used herein means that the GDNF protein product demon:,~-ates similar neurotrophic properties, but not necessarily all of the same properties, and not necess~rily to the same degree, as the GDNF having the amino acid sequence set forth in SFQ ID NO:1. The selection of the particular neurotluph ~ properties of interest depends upon the use for which the GDNF
protein product is being administered.
The term "substantially homclo~us" as used herein means having a degree of homology to the GDNF having the amino acid sequence set forth in SEQ ID
NO:1 that is prt:~erably in excess of 70%, most prelerably in excess of 8û%, andeven more ~efe~ably in excess of gO% or 95%. For example, the degree of homology betwecn the rat and human protein is about 93%, and it is conte",~.lated that prefer,ed mammalian GDNF will have a similarly high degree of homology.
The pe,~;entage of homology as desc,iLed herein is cfl~cn~ated as the pel~;enlage of amino acid residues found in the smaller of the two sequences which align with CA 022361~7 1998-0~-21 i~entical amino acid residues in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to assist in that alignment, as set forth by Dayhoff, in Atlas of Protein Sequence and Structure v. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), the disc~Qsure of which is hereby incorporated by reference. Also included as substantially hon,~'ogous is any GDNF protein product which may be isolated by virtue of cross-reactivity with antibodies to the GDNF of SEQ ID NO:1 or whose genes may be isolated through hybridization with the gene or with seg",enls of the ~ne encoding the GDNF of SEQ ID NO:1.
1 0 The GDNF protein products according to the present invention may be isolated or generated by any means known to those skilled in the art. Exempiary ll-elllods for producing GDNF protein products useful in the present invention are described in U.S. Patent Application No. 08/182,183 filed May 23, 1994 and its parent applications; PCT Application No. PCT/US92/07888 filed September 17, 19g2, published as WO 93/06116 tLin et al., Syntex-Synergen Neuroscience Joint Venture); European Patent Application No. 92921022.7, published as EP
610 254; and co-owned, co-pending U.S. Applicalion Serial No. 081535,681 filed September 28, 1995 ("Truncated Glial Cell-Line Derived Neurotrophic Factor"), the disclosures of all of which are hereby incorporated by reference.
Naturally-occurring GDNF protein products may be isolated from mammalian neuronal cell preparations, or from a mammalian cell line secreting or e,~,~ressing GDNF. For example, WO93106116 desc,iL)es the isolation of GDNF
from serum-free growth conditioned medium of B49 glioblastoma cells. GDNF
protein products may also be chemically synthesized by any means known to those skilled in the art. GDNF protein products are pr~ferably produced via recombinant techniques because they are capable of achieving comparatively higher amounts of protein at greater purity. Reco",h nant GDNF protein product forms include glycosylated and non-glycosylated forms of the protein, and protein expressed in bacterial, mammalian or insect cell systems.
In general, recombinant techniques involve isolating the genes responsible for coding GDNF, cloning the gene in suitable vectors and cell types, modifying the gene if necessary to encode a desired variant, and eApressing the gene in order to produce the GDNF protein product. Alte-"ati~ely, a nucleotide sequence encoding the desired GDNF protein product may be chemically sy-,ll,esked. It is contemplated that GDNF protein product may be expressed using nucleotide sequences which differ in codon usage due to the degenerac;as of the genetic code or allelic variations. W093106116 describes the isolation and CA 022361~7 1998-0~-21 Wo 97/19694 Pcr/uss6ll88o6 sequencing of a cDNA clone of the rat GDNF gene, and the isolation, sequencing and e~ ssion of a genomic DNA clone of the human GDNF gene. WO93/06116 also desc,iLes vectors, host cells, and culture growth conditions for the e~.,ession of GDNF protein product. Additional vectors suitable for the e~-~.ression of GDNF
protein product in E. colf are disclosed in published European Patent ArF"ca~ionNo. EP 0 423 980 (NStem Cell Factor") published April 24, 1991, the dicc~Qsure of which is hereby incor,u~r~led by reference. The DNA sequence of the gene coding for mature human ~DNF and the arnino acid sequence of the GDNF
is shown in Figure 19 (SEQ ID NO:~) of W093106116. Figure 19 does not show the entire coding sequence for the pre-pro portion of GDNF, but the first 50 amino acids of human pre-pro GDNF are shown in Figure 22 (SEQ ID NO:8) of WO93/061 1 6.
Naturally-occurring GDNF is a disulfide-bonded dimer in its biologically active form. The material isolated after expression in a bacterial system is essentially biologically inactive, and exists as a monomer. Refolding is necessary to produce the b.c'~giG~lly active disulfide-bonded dimer. Processes for the refolding and naturation of the GDNF expressed in bacterial systems are desc-iLed in WO93106116. Standard in vitro assays for the determination of GDNF activity are also described in WO93106116 and in co-owned, co-pending U.S. Application Serial No. 08/535,681 filed September 28, 1g95, and are hereby incorporated by reference.
A ~nNF Vari~nt~
The term HGDNF variants" as used herein includes polypeptides in which amino acids have been deleted from ("deletion variantsn), inserted inlo ("addition variants"), or substituted for ("substitution variants"), residues within the amino acid sequence of naturally-occurring GDNF. Such variants are prepared by introducing approptiale nucleolide changes into the DNA encoding thepolypeptide or by in vi~ro chei"ical synthesis of the desired polypeptide. It will be ap,u,ec;aled by those skilled in the art that many combinations of daletions,insertions, and substitutions can be made provided that the final ",~ u~e possesses GDNF bic!ag.cal activity.
Mutagenesis techniques for the replacement, insertion or deletion of one or more selected amino acid residues are well known to one skilled in the art (e.g., U.S. Patent Number 4,518,584, the disclosure of which is hereby incol~JGrdted by reference.) There are two principal variables in the construction of variants: the location of the mutation site and the nature of the CA 022361~7 1998-0~-21 Wo 97/196~4 PCr/uss6tl88o6 mutation. In designing GDNF variants, the selection of the mutation site and nature of the mutation will depend on the GDNF characteristic(s) to be modified.The sites for mutation can be modified individually or in series, e.g., by (1) s~hstitlJting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target amino acid residue, or (3) inserting amino acid residues adjacent to the locatedsite. ConserYative changes in from 1 to 20 amino acids are p-e1erled. Once the amino acid sequence of the desired GDNF protein product is determined, the nucleic acid sequence to be used in the eA,.ression of the protein is readily determined. N-terminal and C-terminal deletion variants may also be generated by proteolytic enzymes.
For GDNF deletion variants, deletions generally range from about 1 to 30 residues, more usually from about 1 to 10 residues, and typically from about 1 to 5 contiguous residues. N-terminal, C-terminal and internal intrasequence delet-ons are contemplated. Deletions may be introduced into regions of low homology with other TGF-B superfamily members to modify the activity of GDNF.
Deletions in areas of substantial homology with other TGF-B superfamily sequences will be more likely to modify the GDNF biological activity more si~n 'icanll~. The number of consecutive deletions will be sele~,led so as to preserve the tertiary structure of the GDNF protein product in the al1~L:ted domain, e.g., cysteine crosslinking. Non-limiting examples of deletion variants include truncated ~DNF protein products lacking from one to forty N-terminal amino acids of GDNF, or variants lacking the C-terminal residue of GDNF, or combinations thereof, as described in co-owned, co-pending U.S. Application Serial No. 081535,681 filed September 28, 1995, which is hereby incorporated by reference.
For GDNF addition vsriants, amino acid sequence additions typically include N-and/or C-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intemal inl.ase~uence additions of single or multiple amino acid residues. Internal additions may range generally from about 1 to 10 residues, more typically from about 1 to 5 residues, and usually from about 1 to 3 amino acid residues.
Examples of N-terminal addition variants include GDNF with an N-terminal methionyl residue (an artifact of the direct expression of GDNF in bacterial 35 recct-ant cell culture), which is designated IMet~1lGDNF, and fusion of a heter~logoLs N-terminal signal sequence to the N-terminus of G~NF to facili1ale the secrelion of mature GDNF from recombinant host cells. Such signal sequences CA 022361~7 1998-0~-21 generally will be obtained from, and thus be homologous to, the intended host cell species. Additions may also include amino acid sequences derived from the sequence of other neurotrophic factors. A preferred GDNF protein product for use according to the present invention is the recombinant human [Mer1]GDNF.
GDNF substitution variants have at least one amino acid residue of the GDNF amino acid sequence removed and a different residue inserted in its place.
Such substitution variants include allelic variants, which are char~cte.i~ed by na1urally-occurring nucleotide sequence changes in the species popu~tion that may or may not result in an amino acid change. Examples of substitution variants (see, e.g., SEQ ID NO: 50) are disc~osed in co-owned, co-pending U.S.
Application Serial No. 08/535,681, filed September 28, 1g95, and are hereby incorporated by reference.
Specific mutations of the GDNF amino acid sequence may involve ~I~G~ific~ions to a glycosylation site ~e.g., serine, threonine, or asparagine). The absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at any asparagine-linked glycosylation recognition siteor at any site of the molecule that is modified by addition of an O-linl~ed carbohydrate. An asparagine-linked glycosylation recognilion site co",prises a tli~.eplide sequence which is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acW other than Pro. A variety of amino acid substitutions or deletions at one or both of the first or third aminoacid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) result in non-glycosylation at the modified l-ipeplide sequence. Thus, the expression of a,upropriate altered nucleotide sequences produces variants which are not glycosylated at that site. Alte,.,ati~cly, the GDNF amino acid sequence may be ~,odif;ed to add glycosylation sites.
One method for identifying GDNF amino acid residues or regions for mutagenesis is called "alanine scanning mutagenesis" as desc,ibed by Cu"l.i.,~Jl,a", and Wells (Science, 244:1081-1085, 1989). In this method, an amino acid residue or group of target residues are idenli~ied (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatiicly cha,yed amino acid (most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the surrounding aqueous environment in or outside the cell.Those cJo",ail,s demonslrali"g functional sensitivity to the suhstitutions then are refined by introducing acldilional or alternate residues at the sites of substitution. Thus, the target site for introducing an amino acid sequence CA 022361~7 1998-0~-21 WO 97/196~4 PCT/US96/18806 variation is determined, alanine scanning or random mutagenesis is conducted on the c~r-~s~onding target codon or region of the DNA sequence, and the eA~JressedGDNF variants are screened for the optimal combination of desired activity and degree of activity.
The sites of greatest interest for substitutional mutagenesis include sites where the amino acids found in GDNF proteins from various species are sul.stantially different in terms of side-chain bulk, charge, andlor hy.ll~phobicity. Other sites of interest are those in which particular residues of GDNF-like proteins, obtained from various species, are identical. Such positionsare generally important for the biological activity of a protein. Initially, these sites are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of prefer,ed substitutions.If such substitutions result in a change in biological activity, then more suLslantial changes (exemplary substitutions) are introduced, and/or other addilions or deletions may be made, and the resuiting products sc,eened for activity.
Amino Acid Substitutions Preferred F~emplary Ori~in~l Residue Substitutions Substitutions Ala (A) Val Val; Leu; lle Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys ~C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg lle (I) Leu Leu; Val; Met; Ala;
Phe; norleucine Leu (L) lle norleucine; lle; Val;
Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; lle Phe (F) Leu Leu; Val; lle; Ala Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu lle; Leu; Met; Phe;
Ala; norleucine CA 022361~7 1998-0~-21 Conservative l"~clilicalions to the amino acid sequence (and the ~or.esponding ,~,odilic~ions to the encoding nucleic acid sequences) are sYpected to produce GDNF protein products having functional and chelllical ~;lldlaclelistics similar to those of natural GDNF. In contrast, substantial l"oJif-c-ations in the functional andtor chemical cha(acleristics of GDNF protein products may be acco"lplished by selecting substitutions that differ significantly in their effect on Illaintaining (a) the struc2ure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conlurl,,alion, (b) the charge or hylJIoph3bicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:
1) hydrophobic: norleucine, Met, Ala, Val, Leu, lle;
IJSIN~; GLIAL CELL LINE-DERIYED NEUROTROPHIC FACTOR
(GDNF) PROTEIN PRODUCT
Field of Invention The present invention relales generally to methods for lreating injury or degeneration of retinal neurons by ad~"inist~,i"g glial cell line-derived neufolr~phic factor ~GDNF) protein product. The invention relates specifically 10 to methods for treating pathological conditions, such as inherlted retinal degenerations and age, disease or injury-related retinopathies, in which photoreceptor degeneralion occurs and is responsible for vision loss.
R5~ round of the Invention llecer,lly, several naturally occurring proteinaceous mo'~cules haYe been identified based on their trophic activity on various types of neurons. These molecules are termed "neurotrophic factors". NeL.,ut,ophic factors are endogenous, soluble proteins that play a major role in neuronal survival and growth during develop",ent, as well as in the funclion~l ",ainlenance and 20 plasliclty of mature neurons; see Fallon and Laughlin, Neurotrophlc Factors, Academic Press, San Diego, CA (1993). In view of their ability to promote neuron regeneration and to prevent neuron death and degeneratbn, it has been postulA~ed that neurol,uph c factors might be useful in treating neurodegenerative conditions of the nervous system, such as, for example, 25 Parkinson's disease, Alzheimer's disease, am~olroph c lateral sclerosis and stroke.
Ilerve damage is caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells, including:
(1) physical injury, which causes the degeneration of the axonal processes 30 (which in turn causes nerve cell death) and/or nerve cell bodies near the site of injury, (2) temporary or permanent cess~ti~n of. blood flow (ischemia) to parts of the nervous system, as in stroke, (3) intentional or accidental exposure to neu.uloxins~ such as the cancer and AIDS chemotherapeutic agents cispla~inum and dideoxycytidine, respectively, (4) chronic metabolic dise~cesi such as 35 ~ eles or renal dysfunction, or (5) neurodegenerative ~iseases such as rarl~inson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis.
which result from the degeneration of specific neuronal popul~tions. In order for CA 022361~7 1998-0~-21 WO 97/19694 PCI~/US96/18806 a particular neu~lf~phic factor to be potentially useful in treating nerve dar.,age, the class or classes of da~aged nerve cells must be .esponsNe to the factor; different neufol,ophic factors typically affect clist-"clly ~irlerenl classes of nerve cells. It has been established that all neuron popu'~ions are not responsive to or equally affected by all neurotrophic factors.
The first neurotrophic factor to be identified was nerve growth factor (NGF). NGF is the first ",e",ber of a defined family of trophic factors, called the neurotrophins, that currently includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 ~NT-3), NT-415, and NT-6 (Thoenen, ~rends.
Neurosci., 14:165-170, 1991; Lapchak et al., Rev. NBUrOSCj., 3:1-10, 1993;
~D~h-Yell, Ann. F~ev. Neurosci., 18:223-253, 1g95). These neurotrophins are known to act through the family of trk tyrosine kinase leceplors, i.e., trkA, trkB, trkC, and the low affinity p75 receptor (Lapchak et al., Rev. Neuroscl., 3:1-10, 19g3; Bothwell, Ann. Rev. Neurosci., 18:223-253, 19g5; Chao et al., TINS, 18:321-326, 19g5). In the cenlral nervous system (CNS). the e~,uression of trkA, the receptor for NGF, is almost exclusiYely limited to the cholinergic neurons in the basal forebrain (Venero et al., Neuroreport, 4:959-962, 1993), which also express p75 and trkB. These chol nerg-c neurons are of particular neurologic interest, because cholinergic neuronal degeneration andlordystrophy is a hallmark of Alzheimer's disease (Hefti, ~. NeurobioJ., 25:1418-1435, 1g94; Olson, Neurochem. Jul., 15:1-3, 1994). The basal forebrain cholinergic neurons can be readily identified in morphologic preparations using acetylcholinesterase histochemistry or with immunohistochemistry using ar,libG~y to choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, or to p75 (Batchelor et al., J. Comp. Neurol., 284:187-204, 1989; Kiss et al., Neurosci., 27:731-748, 1988; Woolf et al., Neuroscience, 30 :1 43-1 52, 1 98g) .
Glial cell line-derived neurotrophic factor (GDNF) is a recently discovered protein idenliried and purified using assays based upon its efficacy in pro",oling the survival and stimulating the transmitter phenotype of mesencephalic dopaminergic neurons in vitro (Lin et al., Science, 260:1130-1132, 1993). GDNF is a glycosylated, disulfide-bonded homodimer that is ~i.,l&l~lly related to the transforming growth factor-B (TGF-B) superfamily of neu,otloph c proteins (Krieglstein et al., EMBO J., 14:736-742, 1995;
Poulsen et al., Neuron ,13:1245-1252, 1994). GDNF has been cloned, and the recombinant human GDNF (rhuGDNF) exerts trophic and survival-promoting actions on substantia nigra dopaminergic neurons and spinal cord motor neurons CA 022361~7 1998-0~-21 WO 97/196g4 PC r/uss6tl8806 in vitro, as well as in vivo tBeck et al., Nature, 273:339-341, 1995;
Henderson et al., Science, 266:1130-1132, 1994; Tomac et al., Nature, 273:
335-33~; Yan et al., Nature, 273: 341-343; Zurn et al., Nevroreport,6:113-118, 1994). In vivo, treatment with exogenous GDNF stimulates the dopaminergic phenotype of substantia ni3ra neurons and restores functional deficits induced by axotomy or dopaminergic neurotoxins in animal models of Parl~inson's disease, a neu.udegenerative disease cl,aracle-i~ed by the loss of dopaminergic neurons (Hudson et al., Brain Res. E~ull., 36:425-432, 1995;
Hoffer et al., Neurosci. Lett., t82:107-111, 1994). Although originally thought to be relatively specific for dopa",i"ergic neurons, at least in vitro, subse~llent experiments have found that GDNF has neu,ut~oph ~ efficacy on brain stem and spinal cord cholinergic motor neurons, both in vivo and ~n vitro (Oppenheim et al., Nature, 373:344-346, 1995; Zurn et al., Neuroreport, 6:113-118, 1994; Yan et al., Nature, 373: 341-344, 1995; Henderson et al Scienc~, 266:1062-1064, 1994). GDNF is, therefore, a factor with potential therapeutic benefit in the treatment of degenerative d;sordels of spinal cord motor neurons, such as amyotrophic lateral sclerosis.
Thus, evidence is beginning to emerge indicating that GDNF may have a larger spectrum of neurotrophic targets besides mesencephalic d~pai"i"ergic and sG",alic motor neurons (Yan and Matheson, Nature, 373:341-344, 1995;
Miller et al., Soc. Neurosci. Abstr., 20:1300, 1994). GDNF messenger RNA
(mRNA) has t~een de~ected in muscle and Schwann cells in the peripheral nervous system and in type I astrocytes (Schaas et al., Exp. Neurol., 124:368-371, 1993) in the central nervous system. GDNF mRNA is also ex~lessed in high leYels in the developing rat striatum (Stromberg et al., l~xp. Neurol., 124:401-412, 19g3), and in low levels in regions of the adult rat and human central nervous system, including striatum, hippocampus, cortex and spinal cord (Springer et al., Exp. Neurol., 127:167-170, 1994).
C)f general interest to the present invention is WO93106116 (Lin et al., Syntex-Synergen Neuroscience Joint Venture), published April 1, 1993, which reports lhat GDNF is useful for the treatment of nerve injury, including injury assoc;a~ed with Parkinson's Disease. Also of interest are a report in Schmidt-Kastner et al., Mol. Brain Res., 26:325-330, 1994 that GDNF mRNA became detect~le and was upregulated after pilocarpine-induced seizures; reports in Schaar et al., Exp. Nevrol., 124:368-371, 1993 and Schaar et al.. Exp. Neurol.,130:387-393, 1994 that basal forebrain astrocyles expressed moderate levels of GDNF mRNA under culture conditions, but that GDNF did not alter basal CA 022361~7 1998-05-21 forebrain ChAT activity; and a report in currently pending U.S. Application Seriat Number 08/535.682 fiied September 28, 1995 that GDNF is useful for treating injury or degeneration of basal forebrain cholinergic neurons.
In mammals, a number of ophthalmic neurodegenerative conditions or diseases involve injury or degeneration of photoreceptors. Trophic factors capal~le of promoting the survival or regeneration of these neurons would provide useful therapies for the treatment of such dise~ses.
Photoreceptors are a specialized subset of retinal neurons, that are responsible for vision. Photoreceptors consist of rods and cones which are the photosensili~e cells of the retina. Each rod and cone elabordles a speci~ ed cilium, re~e..ed to as an outer segment, that houses the phototransductiQn machinery. The rods contain a specific light-absor~ing visual pigment, rhodopsin. There are three classes of cones in humans, cha-a~tt:ri~ed by the 15 e~ ression of distinct visual pigments: the blue cone, green cone and red cone pigments. Each type of visual pigment protein is tuned to absorb light maximallyat ~ eren1 wavelengths. The rod rhodopsin ",ed;ales sco ~p;c vision (in dim light), whereas the cone pigments are responsible for photopic vision (in brightlight). The red, blue and green pigments also form the basis of color vision in 20 humans. The visual pigments in rods and cones re~.,ond to light and ~ene,~le an action potential in the output cells, the rod bipolar neurons, which is then relayed by the retinal ganglion neurons to produce a visual stimulus in the visual cortex.
In humans, a number of diseases of the retina involve the progressive 25 degenerdlion and eventual death of photoreceptors, leading inexorably to blindness. Degeneration of photoreceptors, such as by inherited retinal dyslrophies (e.g., retinitis pigmentosa), age-related macular degeneration and other m~u'~pathies, or retinal detac~""ent, are all characterized by the p,ogress:ve atrophy and ioss of function of phot~rec~ptor outer segments. In 30 addilion, death of photoreceptors or loss of photoreceptor function results in partial deafferentation of second order retinal neurons trod bipolar cells and horizontal cells) in patients with retinsl dystrophies, thereby decreasing the overall efficiency of the prop~gation of the electrical signal genetaled by photoreceptors. Trophic factors that are capable of rescuing photoreceptur~
35 from cell death andtor restoring the function of dysfunctional (atrophic or dystrophic) photoreceplor~ may represent useful therapies for the treatment of such conditions.
CA 022361~7 1998-0~-21 WO 97/196g'~ PcrluS96/188o6 There is some evidence that certain protein factors may proil,ote the survival of pholoreceF: rs. For example, photoreceptors can be rescued to some extent Iby basic ~iL"oblasl growth factor (bFGF) in Royal College of Surgeons (RCS) rats and in albino rats that have been da",aged by exposure to cofiala"l light (Faktorovich et al., Nature, 347:83-86, 1g90). RCS rats have an inherited mutation of a gene expressed in the retinal pigment epithelium (RPE), that results in the failure of the RPE to phagocytize the continuously shed portions of the photoreceptor outer seglnents and causes photor~ceptor degeneration and eventually cell death. A single injection of bFGF into the 1 0 vitreous body or into the subretinal space, the extrscellular space surrounding rods and cones, at the onset of the degeneration transiently rescues photoreceptors ~Faktorovich et al., Nature, 347:83-86, 1990 ). In the light-damaged model in albino rats, bFGF in~ected into the su~relinal space or the vitreous body two days prior to the onset of constant illumination sigh'icanlly 1 5 plutecls photoreceptors from light injury and prevents cell death (LaVail et al., Proc. Natl. Acad. Sci. USA, 89:11249-11253, 1992). In this model, photoreceptor survival was also seen with acidic FGF (aFGF), brain-derived neurot,oph'~ factor (BDNF), ciliary neurolfophic factor (CNTF), and interleukin-113 (IL-113). Moderate effects were observed with neurotrophin-3 2û (NT-3~, insulin-like-growth factor ll (IGF-II) and tumor necrosis factor-alpha (TNF-alpha). Nerve growth factor (NGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF) and IGF-I had no effect (LaVail et al., Proc. Natl Acad. Sci. USA, 89:11249-11253, 1992). Also see WO 93/15608, ~aVail et al.
Although bFGF is efficacious in the RCS rat and light-induced darl-age rat models, its therapeutic utility in humans is very limited, due to its hypGIensive, ogen-4 and potent ang;ogen ~ activities. In fact. bFGF injected into the vitreous body causes the invasion of blood-derived ll,acrophages in the inner retina and can produce a massive proliferative vitreoretinopalll~ (Faktorovich et al.. Nature, 347:83-86, 1990). It has also been del~rl"ined, using polyll,erase chain reaction technology, that messenger RNA for GDNF is expressed in the eyes of postnatal day 6 and adult rats, essen~ially associated with the neural retina and the retinal pigment epithelium. The RPE cells produce. store and ~lansporl a variety of factors that are responsible for the survival and functional 35 maint~nance of photoreceptors. The RPE cells are also indispensable to the phototlansduction p~cess: they clear up by phagocytosis the shed tips of the outer seyllle~ of photoreceptors and recycle vitamin A. The transplantation of CA 022361~7 1998-0~-21 WO 97/1s694 PCT/US96/18806 normal RPE cells into retinas of RCS rats prevents photoreceptor cell death (Li and Turner, Exp. Eye f~es., 47:911-917, 1988; Mullen and LaVail, Science, 192:799-801, 1976), suggesting the production by RPE cells of a diffusable trophic factor for photoreceptors.
There continues to exist a need for methods and therapeutic co"",ositions useful for the treatment of photoreceplor cell injury. Such methods and therapeutic cGr"positions would ideally protect the photoreceptors from progressive injury and promote the survival or regeneration of the damaged neuron population, without severe side effects.
SUMMARY OF THE INVENTION
The present invention provides a method for treating vision loss due to photoreceptor degeneration by admin;stering a therapeutically effective amount of glial cell line-derived neurotrophic factor (GDNF) protein product. According toone aspect of the invention, methods are provided for treating vision loss due to photoreceptor degeneration by a.ll"inisteril,g a therapeutically effective amount of GDNF protein product. It is conle",plaled that such GDNF protein products would include a GDNF protein such as that depicted by the amino acid sequence set forth in SEO ID NO:1, as well as variants and derivatives thereof. The invention is based on the novel discovery that adminisl,alion of GDNF protein product pr~",oles the survival and regeneration of damaged photo(eceplor neurons, which are the main population of neurons damaged in retinal degenerations leading to blindness.
GDNF protein product may be administered intraocularly at a dose between about 0.001 mg/day and 10 mglday, preferably at a dose between about 0.01 mgJday and 1 mg/day, and most preferably at a dose bel; een about 0.1 mglday and 0.5 mgJday. It is also conle,,,,ulaled that photoreceptor degeneration or injury may be treated by the administration of a GDNF protein product in conjunction with a second therapeutic agent including, but not limited to, brainderived neurotrophic factor, neurotrophin-3, neurotrophin-415, neurotrophin-6, insulin-like growth factor, ciliary neurotrophic factor, acidic and basic fibroblast growth factors, fibroblast growth factor-5, l~dnsfcr",ing growth factor-B, and ***e-amphetamine regulated transcript. It is also conte",plaled that the delivery means for the ad~";nisl,d~ion of a GDNF protein product in the treatment of ophthalmic condiliGns or dise~ces may advant~geou~ly CA 022361~7 1998-0~-21 involve topical formulations, ocular inserts, ocular injection, ocular implants,cell therapy or gene therapy.
The invention also provides for the use of GDNF protein product in the manufacture of a medicament or pharmaceutical composition for the 5 treatment of injury or degeneration of photoreceptor. Such pharmaceutical compositions include topical, oral or parenteral GDNF protein product formulations. It will also be appreciated by those skilled in the art that the ~";n61,dlion process can be acco".F' shed via cell therapy and gene therapy means, as further desc.ibecl below. In yet another aspect, the t 0 present invention includes a method for providing photoreceptor cells for implantation wherein photorece,ulor cells are cultured in the presence of a GDNF protein product. The invention further includes a composition which contains photoreceptor cells together with a GDNF protein product in amounts to enhance the survival and allow the continued growth and 15 maturalion of the photoreceplor cells. Numerous addi~ional aspects and advantages of the invention will become apparent to those skilled in Ihe art upon ccnsideration of the following detailed description of the invention which describes presently preferred em~odiments thereof.
BRIEF DESCRIPTION OF THE D~AWINGS
Fi3ure 1 depicts the effect of glial cell line-derived neurotrophic factor (GDNF) protein product on photoreceptor survival in cultures of retinal Z5 neurons. Each value is the mean + s.d. of three cultures.
Figure 2 depicts the promotion of photoreceplor neurite outgro.vth by GDNF protein product. The data are expressed as a cumulative frequency distribution plot of the neurite lengths. The percenlage of photoreceptors 30 (ordinate) with neurites longer than a given length in micrometers (Ahscissa) is plotted.
Figure 3 depicts the stimulation of glutamate uptake by GDNF protein product in cultures of photoreceptors. The results are expressed as the 35 ~,~nlages of the glutamate uptake values (in dpmlwell) found in control cultures~ Each data point is the mean + s.d. of 3 wells from a represenl~tive experiment.
CA 022361~7 1998-0~-21 Figure 4 depicts the promotion of photoreceptor survival by GDNF
protein product in cultures of retinal neurons. Changes in pholoreceptor number in response to GDNF protein product treatment in cultures from 18-day-old and 5 39-day-old mice are depicted. Each value is the mean + s.d. of 2-3 cultures.
Figure 5 depicts the promotion of photoreceplor survival by GDNF
protein product in cultures of rd~rd mouse retinas. Photo-eceplor survival was determined by counting the number of arrestin-positive neurons per 6-mm 10 well. Each value is the mean + s.d. of 3-4 cultures.
Figure 6 depicts the effect of GDNF on photoreceptor survival in cultures of chick retina. Photoreceptor survival was determined by counting the number of cones per 6 sq. mm diametrical strips (representing about 21% of the total 15 area of a 6-mm well). Each value is the mean + s.d. of 3 cultures.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based on the demonstration that GDNF protein product has neurotrophic activity for photoreceptors. Prior to this finding, there was no suggeslion or indicalion that GDNF might have such neu,ut.ùph'~
activity . The present invention provides a method for treating injury or degeneration of retinal neurons, particularly photoreceptors, by administering a26 therapeutically effective amount of glial cell line-derived neulotlophic factor (GDNF) protein product by means of a pharmaceutical composition, the implanlalion of GDNF-ex~ ressing cells. or GDNF gene therapy. the invention may be practiced using any biologically active GDNF protein product, including aGDNF having the amino acid sequence set forth in SEQ ID NO:1, variants, and derivatives thereof.
A unique cell culture technique was developed to provide the retinal neuron Fopul~tions used in assessing the responsiveness of pho~orecep~ors to GDNF protein product admin;~ lion. The culture technique is des.;,i6ed in further detail below. The treatment of these pholoreceptors with a GDNF protein product revealed that in addition to promoting photoreceptor survival, the GDNF
protein product also stimulated the extension of the photoreceptor's axon-like pr~cess, thereby demonstrating an effect on the morphological develop",enl of the CA 022361~7 1998-0~-21 Wo 971196~4 Pcrluss6ll88o6 photoreceptors. Glutamate uptake assays further cle",onsl,dted that GDNF proteinproduc~ treatment enhances the functional differentiation of photoreceptors.
These results indicate that among the potential benelil~ of GDNF protein producltherapy are the pro"~olion of photoreceptor survival, the regeneral;on of the 5 photoreceptors' axons and outer segments and the reslora~ion of visual function.
Thus, the admin;slfalion of a GDNF protein product would benefit cohditions in which vision is lost due to the degeneration of pholoreceplor~, such as inherited retinal degeneralions. age-related macular degeneration, injury-induced retinal degenerations, and retinal dystrophies.
The present invention further demonstrates that GDNF protein product treatment pro",otes photoreceptor surviYal in cultures of retina from mice having an inherited retinal degeneration condition. Studies of pholoreceplors ofrd/rd mice illustrated that GDNF protein product treatment enhanced resislance to the delet~rious effect of the rdfrd mutation on photoreceptors. This indicates 15 that GDNF protein product treatment would be useful in the in the reduction and prevention of photorsceptor degeneration and death and even in the reversal of photoreceptor degeneration in human inherited retinal dise~ces cha~ac1eri~ed by photoreceptor degene,alion, such as, for example, retinitis pigmentosa. As illustrated by the studies described below, GDNF protein product adminisl,alion 20 may benefit a variety of pathological conditions in which photoreceptor degeneration occurs and is responsible for vision loss. These cor~ ,ns include inherited retinal degenerations such as retinitis pigmentosa, Bardet-8iedl sy,,d~u-,,e, Bassen-Kornzweig syndrome (abetalipoproteinemia), Best disease (vitelliform dystrophy), choroidemia, gyrate atrophy, congenital amaurosis, 25 Refsum syndrome, Stargardt disease and Usher syndrome. Other retinopathies that may benefit from GDNF protein product admi-,i~,dtion include age-related macular degeneration (dry and wet forms), diabetic ~etillopall"~, peripheral vitreoretinopathies, photic retinopathies, surgery-induced retinopathies, viral retinopathies (such as HIV retinopathy related to AIDS), ischemic rsti.,û,~alhies, 30 retinal d~lacl,ment and traumatic retinopathy.
According to the currently preferred e",bodi."ents of the present invention, the GDNF protein product is most advantageously administered intraocularly at a dose between about 0.001 mg/day and 10 mglday, and pr~f~rdLly at a dose between about 0.01 mglday and 1 mg/day, and most 35 prsf~ra~ly at a dose between about 0.1 mg/day and 0.5 mg/day. It is further conte,-,,ulaled that the GDNF protein product be adl"i"islered in conjunction orcombination with an effective amount of a second therapeutic agent for treating CA 022361~7 1998-0~-21 WO 9711g694 PCT/US96/18806 retinal degeneration or retinal dysl~ophies. Such second therapeutic agents may include, but are not limited to: mitogens such as insulin, insulin-like growth factors, epidermal growth factor, vasoactive growth factor, pituitary adenylate cyclase activating polypeptide, interferon and somatoslali,~; neu,t,t-ophlc factors such as brain derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5 neurotrophin-6, insulin-like growth factor, ciliary neurotrophic factor, acidic and basic fibroblast growth factors, fibroblast growth factor-5, transforming growth factor-B. and ***e-amphetamine regulated transcript ~CART); and other growth factors such as epidermal growth factor, leukemia inhibitory factor, interleukins, interferons, and colony stimulating factors; as well as molecules and materials which are the functional equivalents to these factors.
The invention also provides for the use of GDNF protein product in pre~t~aration of a medicament for the treatment of injury or degeneration of photoreceptors, including the treatment of the dise~ces and conditions describedabove. Such GDNF protein product pharmaceutical preparations are more fully desc,il,ed below.
As used herein, the term "GDNF protein product" includes purified natural, synthetic or recombinant glial cell line-derived neurotrophic factor, biologically active GDNF variants (including insertion, substitution and deletion variants), and chemically ",o.lified derivatives thereof. Also included are GDNFs that are sul~slantially homologous to the human GDNF having the amino acid sequence set forlh in SEQ ID NO:1. GDNF protein products may exist as homodimers or heterodimers in their biologically active form.
The term "biologically active" as used herein means that the GDNF protein product demon:,~-ates similar neurotrophic properties, but not necessarily all of the same properties, and not necess~rily to the same degree, as the GDNF having the amino acid sequence set forth in SFQ ID NO:1. The selection of the particular neurotluph ~ properties of interest depends upon the use for which the GDNF
protein product is being administered.
The term "substantially homclo~us" as used herein means having a degree of homology to the GDNF having the amino acid sequence set forth in SEQ ID
NO:1 that is prt:~erably in excess of 70%, most prelerably in excess of 8û%, andeven more ~efe~ably in excess of gO% or 95%. For example, the degree of homology betwecn the rat and human protein is about 93%, and it is conte",~.lated that prefer,ed mammalian GDNF will have a similarly high degree of homology.
The pe,~;entage of homology as desc,iLed herein is cfl~cn~ated as the pel~;enlage of amino acid residues found in the smaller of the two sequences which align with CA 022361~7 1998-0~-21 i~entical amino acid residues in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to assist in that alignment, as set forth by Dayhoff, in Atlas of Protein Sequence and Structure v. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), the disc~Qsure of which is hereby incorporated by reference. Also included as substantially hon,~'ogous is any GDNF protein product which may be isolated by virtue of cross-reactivity with antibodies to the GDNF of SEQ ID NO:1 or whose genes may be isolated through hybridization with the gene or with seg",enls of the ~ne encoding the GDNF of SEQ ID NO:1.
1 0 The GDNF protein products according to the present invention may be isolated or generated by any means known to those skilled in the art. Exempiary ll-elllods for producing GDNF protein products useful in the present invention are described in U.S. Patent Application No. 08/182,183 filed May 23, 1994 and its parent applications; PCT Application No. PCT/US92/07888 filed September 17, 19g2, published as WO 93/06116 tLin et al., Syntex-Synergen Neuroscience Joint Venture); European Patent Application No. 92921022.7, published as EP
610 254; and co-owned, co-pending U.S. Applicalion Serial No. 081535,681 filed September 28, 1995 ("Truncated Glial Cell-Line Derived Neurotrophic Factor"), the disclosures of all of which are hereby incorporated by reference.
Naturally-occurring GDNF protein products may be isolated from mammalian neuronal cell preparations, or from a mammalian cell line secreting or e,~,~ressing GDNF. For example, WO93106116 desc,iL)es the isolation of GDNF
from serum-free growth conditioned medium of B49 glioblastoma cells. GDNF
protein products may also be chemically synthesized by any means known to those skilled in the art. GDNF protein products are pr~ferably produced via recombinant techniques because they are capable of achieving comparatively higher amounts of protein at greater purity. Reco",h nant GDNF protein product forms include glycosylated and non-glycosylated forms of the protein, and protein expressed in bacterial, mammalian or insect cell systems.
In general, recombinant techniques involve isolating the genes responsible for coding GDNF, cloning the gene in suitable vectors and cell types, modifying the gene if necessary to encode a desired variant, and eApressing the gene in order to produce the GDNF protein product. Alte-"ati~ely, a nucleotide sequence encoding the desired GDNF protein product may be chemically sy-,ll,esked. It is contemplated that GDNF protein product may be expressed using nucleotide sequences which differ in codon usage due to the degenerac;as of the genetic code or allelic variations. W093106116 describes the isolation and CA 022361~7 1998-0~-21 Wo 97/19694 Pcr/uss6ll88o6 sequencing of a cDNA clone of the rat GDNF gene, and the isolation, sequencing and e~ ssion of a genomic DNA clone of the human GDNF gene. WO93/06116 also desc,iLes vectors, host cells, and culture growth conditions for the e~.,ession of GDNF protein product. Additional vectors suitable for the e~-~.ression of GDNF
protein product in E. colf are disclosed in published European Patent ArF"ca~ionNo. EP 0 423 980 (NStem Cell Factor") published April 24, 1991, the dicc~Qsure of which is hereby incor,u~r~led by reference. The DNA sequence of the gene coding for mature human ~DNF and the arnino acid sequence of the GDNF
is shown in Figure 19 (SEQ ID NO:~) of W093106116. Figure 19 does not show the entire coding sequence for the pre-pro portion of GDNF, but the first 50 amino acids of human pre-pro GDNF are shown in Figure 22 (SEQ ID NO:8) of WO93/061 1 6.
Naturally-occurring GDNF is a disulfide-bonded dimer in its biologically active form. The material isolated after expression in a bacterial system is essentially biologically inactive, and exists as a monomer. Refolding is necessary to produce the b.c'~giG~lly active disulfide-bonded dimer. Processes for the refolding and naturation of the GDNF expressed in bacterial systems are desc-iLed in WO93106116. Standard in vitro assays for the determination of GDNF activity are also described in WO93106116 and in co-owned, co-pending U.S. Application Serial No. 08/535,681 filed September 28, 1g95, and are hereby incorporated by reference.
A ~nNF Vari~nt~
The term HGDNF variants" as used herein includes polypeptides in which amino acids have been deleted from ("deletion variantsn), inserted inlo ("addition variants"), or substituted for ("substitution variants"), residues within the amino acid sequence of naturally-occurring GDNF. Such variants are prepared by introducing approptiale nucleolide changes into the DNA encoding thepolypeptide or by in vi~ro chei"ical synthesis of the desired polypeptide. It will be ap,u,ec;aled by those skilled in the art that many combinations of daletions,insertions, and substitutions can be made provided that the final ",~ u~e possesses GDNF bic!ag.cal activity.
Mutagenesis techniques for the replacement, insertion or deletion of one or more selected amino acid residues are well known to one skilled in the art (e.g., U.S. Patent Number 4,518,584, the disclosure of which is hereby incol~JGrdted by reference.) There are two principal variables in the construction of variants: the location of the mutation site and the nature of the CA 022361~7 1998-0~-21 Wo 97/196~4 PCr/uss6tl88o6 mutation. In designing GDNF variants, the selection of the mutation site and nature of the mutation will depend on the GDNF characteristic(s) to be modified.The sites for mutation can be modified individually or in series, e.g., by (1) s~hstitlJting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target amino acid residue, or (3) inserting amino acid residues adjacent to the locatedsite. ConserYative changes in from 1 to 20 amino acids are p-e1erled. Once the amino acid sequence of the desired GDNF protein product is determined, the nucleic acid sequence to be used in the eA,.ression of the protein is readily determined. N-terminal and C-terminal deletion variants may also be generated by proteolytic enzymes.
For GDNF deletion variants, deletions generally range from about 1 to 30 residues, more usually from about 1 to 10 residues, and typically from about 1 to 5 contiguous residues. N-terminal, C-terminal and internal intrasequence delet-ons are contemplated. Deletions may be introduced into regions of low homology with other TGF-B superfamily members to modify the activity of GDNF.
Deletions in areas of substantial homology with other TGF-B superfamily sequences will be more likely to modify the GDNF biological activity more si~n 'icanll~. The number of consecutive deletions will be sele~,led so as to preserve the tertiary structure of the GDNF protein product in the al1~L:ted domain, e.g., cysteine crosslinking. Non-limiting examples of deletion variants include truncated ~DNF protein products lacking from one to forty N-terminal amino acids of GDNF, or variants lacking the C-terminal residue of GDNF, or combinations thereof, as described in co-owned, co-pending U.S. Application Serial No. 081535,681 filed September 28, 1995, which is hereby incorporated by reference.
For GDNF addition vsriants, amino acid sequence additions typically include N-and/or C-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intemal inl.ase~uence additions of single or multiple amino acid residues. Internal additions may range generally from about 1 to 10 residues, more typically from about 1 to 5 residues, and usually from about 1 to 3 amino acid residues.
Examples of N-terminal addition variants include GDNF with an N-terminal methionyl residue (an artifact of the direct expression of GDNF in bacterial 35 recct-ant cell culture), which is designated IMet~1lGDNF, and fusion of a heter~logoLs N-terminal signal sequence to the N-terminus of G~NF to facili1ale the secrelion of mature GDNF from recombinant host cells. Such signal sequences CA 022361~7 1998-0~-21 generally will be obtained from, and thus be homologous to, the intended host cell species. Additions may also include amino acid sequences derived from the sequence of other neurotrophic factors. A preferred GDNF protein product for use according to the present invention is the recombinant human [Mer1]GDNF.
GDNF substitution variants have at least one amino acid residue of the GDNF amino acid sequence removed and a different residue inserted in its place.
Such substitution variants include allelic variants, which are char~cte.i~ed by na1urally-occurring nucleotide sequence changes in the species popu~tion that may or may not result in an amino acid change. Examples of substitution variants (see, e.g., SEQ ID NO: 50) are disc~osed in co-owned, co-pending U.S.
Application Serial No. 08/535,681, filed September 28, 1g95, and are hereby incorporated by reference.
Specific mutations of the GDNF amino acid sequence may involve ~I~G~ific~ions to a glycosylation site ~e.g., serine, threonine, or asparagine). The absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at any asparagine-linked glycosylation recognition siteor at any site of the molecule that is modified by addition of an O-linl~ed carbohydrate. An asparagine-linked glycosylation recognilion site co",prises a tli~.eplide sequence which is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acW other than Pro. A variety of amino acid substitutions or deletions at one or both of the first or third aminoacid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) result in non-glycosylation at the modified l-ipeplide sequence. Thus, the expression of a,upropriate altered nucleotide sequences produces variants which are not glycosylated at that site. Alte,.,ati~cly, the GDNF amino acid sequence may be ~,odif;ed to add glycosylation sites.
One method for identifying GDNF amino acid residues or regions for mutagenesis is called "alanine scanning mutagenesis" as desc,ibed by Cu"l.i.,~Jl,a", and Wells (Science, 244:1081-1085, 1989). In this method, an amino acid residue or group of target residues are idenli~ied (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatiicly cha,yed amino acid (most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the surrounding aqueous environment in or outside the cell.Those cJo",ail,s demonslrali"g functional sensitivity to the suhstitutions then are refined by introducing acldilional or alternate residues at the sites of substitution. Thus, the target site for introducing an amino acid sequence CA 022361~7 1998-0~-21 WO 97/196~4 PCT/US96/18806 variation is determined, alanine scanning or random mutagenesis is conducted on the c~r-~s~onding target codon or region of the DNA sequence, and the eA~JressedGDNF variants are screened for the optimal combination of desired activity and degree of activity.
The sites of greatest interest for substitutional mutagenesis include sites where the amino acids found in GDNF proteins from various species are sul.stantially different in terms of side-chain bulk, charge, andlor hy.ll~phobicity. Other sites of interest are those in which particular residues of GDNF-like proteins, obtained from various species, are identical. Such positionsare generally important for the biological activity of a protein. Initially, these sites are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of prefer,ed substitutions.If such substitutions result in a change in biological activity, then more suLslantial changes (exemplary substitutions) are introduced, and/or other addilions or deletions may be made, and the resuiting products sc,eened for activity.
Amino Acid Substitutions Preferred F~emplary Ori~in~l Residue Substitutions Substitutions Ala (A) Val Val; Leu; lle Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys ~C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg lle (I) Leu Leu; Val; Met; Ala;
Phe; norleucine Leu (L) lle norleucine; lle; Val;
Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; lle Phe (F) Leu Leu; Val; lle; Ala Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu lle; Leu; Met; Phe;
Ala; norleucine CA 022361~7 1998-0~-21 Conservative l"~clilicalions to the amino acid sequence (and the ~or.esponding ,~,odilic~ions to the encoding nucleic acid sequences) are sYpected to produce GDNF protein products having functional and chelllical ~;lldlaclelistics similar to those of natural GDNF. In contrast, substantial l"oJif-c-ations in the functional andtor chemical cha(acleristics of GDNF protein products may be acco"lplished by selecting substitutions that differ significantly in their effect on Illaintaining (a) the struc2ure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conlurl,,alion, (b) the charge or hylJIoph3bicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:
1) hydrophobic: norleucine, Met, Ala, Val, Leu, lle;
2) neutral hydrophilic: Cys, Ser, Thr;
3) acidic: Asp, ~;lu;
4) basic: Asn, Gln, His, Lys, Arg;
5) residues that influence chain orientation: Gly, Pro; and 6) aro",alic; Trp, Tyr, Phe.
Non-conservative substitutions may involve the excl~ange of a member of one of these classes for another. Such substituted residues may be introduced into regions of the GDNF protein that are homologous with other TGF-B
superfamily ~.rutcins, or into the non-homologous regions of the molecule.
B. GDNF Deriv~tives Chemically modified derivatives of GDNF or GDNF variants may be p-eparecl by one of skill in the art given the disclosures herein. The chemical " ~3eties most suitable for derivatization include water soluble polymers. A
water soluble polymer is desirable b~c~se the protein to which it is attached does not prec-,l,i1alA in an aqueous en~ or""enl, such as a physiological environment. Preferably, the polymer will be pharmaceutically accepl~le for the preparalion of a therapeutic product or composition. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymerlprotein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considera1;ons. The effectiveness of 1he derivatization may be ascertained by a~ ,;nislering the derivative, in the desired form (i.e., by osmotic pump, or, CA 022361~7 1998-0~-21 WO 97/lsCs4 PcrtuS96/18806 more preferably, by injection or infusion, or, further formulated for oral, pulmonary or other de!ivery routes), and determining its effectiveness.
Suitable water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycollpropylene glycol, 5 carboxymethylcellulose, dextran, polyvinyl alcohol. polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, proptopylene glycol hol"opolymers, polypropylene oxide/ethylene oxide co-polymers, 10 polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advanlages in manufacturing due to its stability in water.
The polymer may be of any molecular weight, and may be branched or u.~branched. For polyethylene glycol, the preferred molecular weight ranges 15 from about 2 kDa to about 100 kDa for ease in har,Jli"g and manufacturing (the term "about" indioali"g that in prepa~lions of polyethylene glycol, some ",~lecules will weigh more, some less, than the stated molecular weight). Other skes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in ~0 handling, the degree or lack of antigenicity and other known effects of poiyethylene glycol on a therapeutic protein or variant).
The number of polymer molec~ s so attached may vary, and one skilled in the art will be able to asce,lain the effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of deriv~ti~tion~
25 with the same or different chemical moieties (e.g., polymers, such as di~rerenl weights of polyethylene glycols). The proportion of polymer ",~'e ules to protein (or peptide) molecules will vary, as will their concer,lralions in the feaotion mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be 30 determined by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is brar,ched or unbranched, and the reaction conditions.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic35 domains of the protein. There are a number of alla~:l,r"ent ",etl,Gds available to those skilled in the art. See for example, EP 0 401 384, the disclQsure of whichis hereby i,,cor,uordlad by reference ~coupling PEG to G-CSF), see also Malik et CA 022361~7 1998-0~-21 WO 97/19694 PCI'/US96rl8806 al., Exp. ~lematot. 20:1028-1035 (1992) (reporting pegylation of GM-CSF
using tresyl chloride). For example, polyethylene glyco~ may be covalently bound through amino acid residues via a reactive group, such as, a free amino orcarboxyl group. Reactive groups are those to which an activated polyethylene 5 glycol ",o'ecule may be bound. The amino acid residues having a free amino group may inciude Iysine residues and the N-terminal amino acid residue. Those having a free carboxyl group may include aspa~lic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for allachil)g the polyethylene glycol molecule(s~. For 10 therapeutic purposes, allacr""ent at an amino group, such as ~llach",ent at the N-terminus or Iysine group is preferred. Attachment at residues important for ,t:ceptor binding should be avoided if receptor binding is desired.
One may specifically desire an N-terminal chemically ",odiried protein.
Using polyethylene glycol as an illusl~alion of the present compositions, one may 15 select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (orpeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of-obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated prepa(alion (i.e., 20 separali"g this moiety from other monopegylated moieties if necessary) may beby purification of the N-terminally pegylated material from a popu'~tion of pegylated protein molecules. Selective N-terminal chemical ",odificalion may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (iysine versus the N-terminal) 25 available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. For exar"F!e, one may selectively N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pKa diflerences bet~een 30 the ~-amino group of the Iysine residues and that of the c~-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no siy";licanl ",odification of other reactive groups, such as the Iysine side chain 35 amino groups, occurs. Using reductive alkylation, the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for CA 022361~7 1998-0~-21 Wo 97/19694 PCT/US96tl8806 coupling to the protein. Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.
The present invention contemplates use of derivatives which are prokaryote-expressed GDNF, or variants thereof, linked to at least one 5 polyethylene glycol molecule, as well as use of GDNF, or variants thereof, attached to one or more polyethylene glycol molecules via an acyl or alkyl linkage.
Pegylation may be carried out by any of the pegylation reactions known in the art. See, for example: Focus on Growth Factors, 3 (2): 4-10 (1992); EP 0 154 316, the disclosure of which is hereby incorporated by reference; EP 0 401 384; and the other publications cited herein that relate to pegylation. The pegylation may be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
Pegylation by acytation generally involves reacting an active ester derivative of polyethylene glycol with the GDNF protein or variant. Any known or subsequently discovered reactive PEG molecule may be used to carry out the pegylation of GDNF protein or variant. A preferred activated PEG ester is PEG
eslt:rified to N-hydroxysuccinimide. As used herein, "acylation" is contem~lated20 to include without li",itation the lollo~;"g types of linkages between the tl,srhpeutic protein and a water soluble polymer such as PEG: amide, c&,l,a",dle, urethane, and the like. See Bioconjugate Chem., 5:133-140 (1994). Reaction condi~ions may be _cle_led from any of those known in the pegylation art or those subsequently developed, but should avoid conditions of temperature, solvent, and25 pH that would inactivate the GDNF or variant to be nlGd;fie~.
Pegylation by acylation will generally result in a poly-pegylated GDNF
protein or variant. Preferably, the connecting linkage will be an amide. Also preferabiy, the resulting product will be substantially only (e.g., > 95%) mono-, di- or tri-pegylated. I lo Arevcr, some species with higher degrees of pegylation 30 may be formed in amounts depending on the specific reaction conditions used. If desired, more purified pegylated species may be separated from the mixture, particularly unreacted species, by standard purification techniques, including, among others, dialysis, salting-out, ultrafiltration, ion-exchange chro",alography, gel filtration chromatography and electrophoresis.
3'i Pegylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with the GDNF protein or variant in the presence of a reducingagent. Pegylation by alkylation can also result in poly-pegylated GDNF protein CA 02236l~7 l998-0~-2l Wo g7/19694 Pcr/uss6/18806 or variant. In addition one can manipulate the reaction conditions to favor pegylation substantially only at the a-amino group of the N-terminus of the GDNF protein or variant (i.e. a mono-pegylated protein). In either case of "-onopeyylation or polypegylation the PEG groups are preferably allached to the protein via a -CH2-NH- group. With particular reference to the -CH2- group this type of linkage is referred to herein as an alkyl linkage.
Derivatization via reductive alkylation to produce a monopegylated product exploits differential reactivity of different types of primary amino groups (Iysine versus the N-terminal) available for derivali~alion. The reaction is pe,f~,l",ed at a pH which allows one to take advantage of the pKa differences between the E-amino groups of the Iysine residues and that of the ~-amino group of the N-terminal residue of the protein. By such selective derivatization allachl~lent of a water soluble polymer that conlains a reactive group such as an aldehyde to a protein is cor,l-~'ed: the conjugation with the polymer takes plsce predominantly at the N-terminus of the protein and no significant modification of other reactive groups such as the Iysine side chain amino groups occurs. In one important aspect, the present invention contemplates use of a substantially ho."ogeneous preparation of ",onopolymer/GDNF protein (or variant) conjugate molecules (meaning GDNF
protein or variant to which a polymer molecule has been allached substantially only (i.e., > 95%) in a single location). More specifically if polyethylene glycol is used, the present invention also encompasses use of pegylated GDNF protein orvariant lacking possibly antigenic linking groups and having the polyethylene glycol molecule directly coupled to the GDNF protein or variant.
Thus it is conle",plaled that GDNF protein products to be used in acco,dance with the present invention may include pegylated GDNF protein or variants wllerei n the PEG group(s) is (are) attached via acyl or alkyl groups.
As disc~ssed above, such products may be mono-pegylated or poly-pegylated (e.g., containing 2-6 and preferably 2-5 PEG groups). The PEG groups are generally attached to the protein at the oL- or E-amino groups of amino acids but it is also conler"~,laled that the PEG groups could be attached to any amino group attached 10 the protein which is suflicien11y reactive to become allacl,ed to a PEG
group under suitable reaction conditions.
The polymer molecules used in both the acylation and alkylation ~,,oal;l,es may be selected from among water soluble polymers as described above. The polymer selected should be modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation preft:rdbly so CA 022361~7 1998-0~-21 Wo 97/19694 Pcrluss6/l8806 that the degree of polymerization may be controlled as provided for in the present .,.ell,ods. An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S. Patent 5,252,714). The polymer may be 5 ~ranched or unbranched. For the acylation reactions, the polymer(s) selEc1ed should have a single reactive ester group. For the present reductive alkylation,the polymer(s) selected should have a single reactive aldehyde group. Generally.the water soluble polymer will not be selected from naturally-occurring glycosyl residues since these are usually made more conveniently by mammalian 10 recombinant expression systems. The polymer may be of any molecular weight, and may be branched or ul)brdncl)ed.
A particularly preferred water-soluble polymer for use herein is polyethylene glycol. As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other pr.l~ ,s, such as 5 mono-(C1 -C10) alkoxy- or aryloxy-polyethylene glycol.
In general, chemical derivatization may be performed under any suitable condition used to reacl a biologically active substance with an activated polymer molecule. M_lhods for p(epa-i"g pegylated GDNF protein or variant will generally comprise the steps of (a) reacting a GDNF protein or variant with 20 polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the protein becomes allached to one or more PEG
groups, and (b) obtaining the reaction product(s). In general, the optimal reaction conclilions for the acylation reactions will be determined case-by-casebased on known parameters and the desired result. For example, the larger the 25 ratio of PEG:protein, the greater the percenlage of poly-pegylated product.
Reductive alkylation to produce a substantially homogeneous popu~~~ion of mono-polymer/GDNF protein (or variant) conjugate molecule will generally co,.,~ri~e the steps of: (a) reacting a GDNF protein or variant with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to permit 30 sel~ctiJc ",oJi~icaticn of the a-amino group at the amino terminus of said GDNF
protein or variant; and (b) obtaining the reaction product(s).
For a substantially homogeneous population of mono-polymer/GDNF
protein ~or variant) conjugate molecules, the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble 35 polymer moiety to the N-terminus of GDNF protein or variant. Such reaction conditions generally provide for pKa differences between the Iysine amino groupsand the ~-amino group at the N-terminus (the pKa being the pH at which 50% of CA 022361~7 1998-0~-21 WO 97/lg6g4 PCT/US96/18806 the amino groups are protonated and 50% are not). The pH also affects the ratio of polymer to protein to be used. In general. if the pH is lower, a larger excess of polymer to protein will be desired (i.e., the less reactive the N-terminal ~-amino group, the more polymer needed to achieve optimal conditions). If the 5 pH is higher, the polymer:protein ratio need not be as large (i.e., more reactive groups are available, so fewer polymer molecules are needed). For purposes of the present invention, the pH will generally fall within the range of 3-9, preferably 3-6.
Another important consideration is the molecular weight of the polymer.
10 In general, the higher the molecular weight of the polymer, the fewer polymermolecules may be attached to the protein. Similarly, b,~nching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. In general, for the pegylation reactions contemplated 15 herein, the preler,ed average molecular weight is about 2 kDa to about 100 kDa.
The prefer,t:d average molecular weight is about 5 kDa to about 50 kDa, particularly preferably about 12 kDa to about 25 kDa. The ratio of water-soluble polymer to GDNF protein or variant will generally range from 1:1 to 100:1, preferably (for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1 to 5:1.
Using the conditions indicated above, reductive alkylation will provide for selective attachment of the polymer to any GDNF protein or variant having an a-amino group at the amino terminus, and provide for a substantially homogenous preparation of monopolymer/GDNF protein (or variant) conjugate.
25 The term "".onopolymer/GDNF protein (or variant) conjugate" is used here to mean a composition co""~ri~ed of a single polymer molecule allached to a " ~lecu'e of GDNF protein or GDNF variant protein. The monopolymer/GDNF
protein ~or variant) conjugate preferably will have a polymer molecule located at the N-terminus, but not on Iysine amino side groups. The preparation will 30 preferably be greater than 90% monopolymer/GDNF protein (or variant) conjugate, and more preferably greater than 95% monopolymer/GDNF protein (or variant) con,ugate. with the remainder of observable molecules being unreacted (i.e., protein lacking the polymer moiety).
For the present reductive alkylation, the reducing agent should be stable 35 in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. F~refer,ad reducing agents may be 5el~Ctel:l from sodium borohydride. sodium cyanoborohydride, dimethylamine CA 022361~7 1998-0~-21 WO 97tl96g4 PCT/US96/18806 borane, trimethylamine borane and pyridine borane. A particularly preferred reducing agent is sodium cyanoborohydride. Other reaction parameters, such as solYent, reaction times. temperatures, etc., and means of purification of products, can be determined case-by-case based on the published information relating to derivatization of proteins with water soluble polymers (see the publications cited herein).
G t~DNF Protein Product Ph~rma~euti~l Cornpositions GDNF protein product pharmaceutical co",posi~;ons typically include a therapeutically effective amount of a GDNF protein product in admixture with oneor more pharmaceutically and physiologically acceptable formulation materials.
Suitable formulation materials include, but are not limited to, antioxidants, preserva1iYes, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents. excipients and/or pharmaceutical adjuvants. For example, a suitable vehicle may be water for injection, physiological saline solution, or artificialCSF, possi~,ly supplemented with other materials common in cG",posilions for parenteral adm;r,;st,alion. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
The primary solvent in a vehicle may be either aqueous or non-aqueous in nature. In addition, the vehicle may contain other pharmaceutically-acce~Jtable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
Similarly, the vehicle may contain still other pharmaceutically-accept~
excipients for ",o lifying or maintaining the rate of release of GDNF protein product, or for promoting the absorption or penetration of GDNF protein product across the me,.ll"dnes of the eye. Such excipients are those sulJalances usuallyand cuslo",a,ily employed to formulate dosages for parenteral adl"inis~ralion ineither unit dose or multi-dose form.
Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or Iyophilized powder. Such formulations may be stored either in a ready to use form or in a form, e.g., Iyophilized, requiring reconstitution prior to administration .
~5 The optimal pharmaceutical formulations will be determined by one skilled in the art depending upon considerations such as the route of aJI";.~ial~alion and desired dosage. See for example, Remlngton's Pharmaceutical CA 022361~7 1998-0~-21 Wo 97/19694 PCr/uss6/l88o6 Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vjl~o clearance of the present GDNF proteins, variants and derivatives.
Other effective administration forms, such as parenteral slow-release formulations, inhalant mists, or orally active formulations are also envisioned.For example, in a sustained release formulation, the GDNF protein product may be bound to or incorporated into particulate preparalions of polymeric compounds (such as polylactic acid, polyglycolic acid, etc.) or li~.oso",es.
Hylauronic acid may also be used, and this may have the effect of p,u,l,oting suslained duration in the circulation. The GDNF protein product pharmaceutical composition also may be formulated for parenteral administration, e.g., by intraocular infusion or injection, and may also include slow-release or sustained circulation formulations. Such parenterally administered therapeutic compositions are typically in the form of a pyrogen-free, parenterally a~ept~le aqueous solution co."prising the GDNF protein product in a pha""aceutically ~ceptAble vehicle. One preferred vehicle is sterile distilled water.
It is also contemplated that certain formulations containing GDNF protein product are to be administered orally. GDNF protein product which is administered in this fashion may be encapsulated and may be formulated with or without those carriers customarily used in the compounding of solid dosage forms. The capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is ".~xl",i~ed and pre-systemic degladalion is mi"i",ked. Additional excipients may be included to facilitate absorption of GDNF protein product. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, susper,ding agents, tablet disintegrating agents, and binders may also be employed.
The formulation of topical ophthalmic preparations, including ophthalmic solutions, suspensions and ointments is well known to those skilled in the art (see Re",ington~s Pharmaceutical Sciences, 18th Edition, Chapter 86, pages 1581-1592 Mack Publishing Company, 1990). Other modes of a~"inislrd~-on are available, including intracameral injections (which may be made directly inlo the anterior chamber or directly into the vitreous chamber), subconjunctival injections and retrobulbar injections, and methods and means CA 022361~7 1998-0~-21 - 2~ -for producing ophthalmic preparations suitable for such modes of ad~ ,tration are also well known.
As used in this application, Uextraocular'' refers to the ocular surface and the (extemal) space between the eyeball and the eyelid. Examples of extraocular regions include the eyelid fornix or cul-de-sac, the conjunctival surface and the comeal surface. This location is extemal to all ocular tissue and an invasive procedure is not required to access this region. Examples of extraocular systemsinclude inserts and "topically" applied drops, gels or ointments which may be used to deliver therapeutic material to these regions. Extraocular devices are 1 0 generally easily removable, even by the patient.
The fo"~ ;ng patents disclose extraocular systems which are used to nisler drugs to the extraocular regions. Higuchi et al. disclQses in U.S.
Patent Numbers 3,981,303, 3,986,510 and 3,995,635, a biodegradable ocular insert which contains a drug. The insert can be made in different shapes for 1 5 retention in the cul-de-sac of the eyeball, the extraocular space between 1he eyeball and the eyelid. Several common biocompatible polymers are d;sclosed as suitable for use in ~db,icat;ng this device. These polymers include zinc alginate, poly (lactic acid), poly (vinyl alcohol), poly (anhydrides) and poly (glycolic acid). The patents also describe membrane coated devices with reduced permeation to the drug and hollow chambers holding the drug formulation.
Theeuwes, U.S. Patent Number 4,217,898, discloses microporous reservoirs which are used for controlled drug delivery. These devices are placedextraocularly in the ocular cul-de-sac. Among the polymer systems of interest include poly (vinylchloride)-co-poly (vinyl acetate) copolymers. Kautman 2~ discloses in U.S. Patent Numbers 4,865,846 and 4,882,150 an ophthalmic drug delivery system which con~a;"s at least one bio-erodibte material or ointment carrier for the conjunctival sac. The patent discloses polymer systems, such as,poly (lactide), poly (glycolide), poly (vinyl alcohol) and cross linked collagen, as suitable delivery systems.
In the presently described use of GDNF protein product for the treatment of retinal disease or injury it is also advantageous that a topically applied ophthalmic formulation include an agent to promote the penetration or transport of the therapeutic agent Into the eye. Such agents are known in the art. For example. Ke et al., U.S. 5,221,696 ~i,close the use of materials to enhance the penetration of ophthalmic preparations through the cornea.
Intraocular systems are those systems which are sui~able for use in any tissue compartment within, between or around the tissue layers of the eye itself.
CA 022361~7 1998-0~-21 These locations include subconjunctival (under the ocular rnucous membrane adjacent to the eyeball), orbital (behind the eyeball), and intracameral ~withinthe chambers of the eyeball itself). In contrast to extraocular systems, an invasive procedure consisting of injection or implantation is required to access5 these regions.
The fol'~ g patents disclose intraocular devices. Wong, U.S. Patent Number 4,853,224, discloses microencapsulated drugs for introduction into the chamber of the eye. Polymers which are used in this system include polyesters and polyethers. Lee, U.S. Patent Number 4,863,457, discloses a biodeg,ddable 10 device which is surgically implanted intraocularly for the sustained release of therapeutic agents. The device is designed for surgical i",,i~lantalion under the conjunctiva (mucous membrane of the eyeball). Krezancaki, U.S. Patent Number 4,188,373, discloses a pharmaceutical vehicie which gels at human body temperature. This vehicle is an aqueous suspension of the drug and gums or 15 cellulose derived synthetic derivatives. Haslam et al. discloses in U.S. Patent Numbers 4,474,751 and 4,474,752 a polymer-drug system which is liquid at room temperature and gels at body temperature. Suit~ble polymers used in this system include polyoxyethylene and polyoxy propylene. Davis et al. disclose in U.S. 5,384,333 a biodegradable injectable drug delivery polymer which 20 provides long term drug release. The drug co"lposition is made up of a pharmaceutically active agent in a biodegradable polymer matrix, where the polymer matrix is a solid at temperatures in the range 20~ to 37~C and is flowable at temperatures in the range 38~ to 52~C. The drug delivery polymer is not limited to the delivery of soluble or liquid drug formulations. For example,25 the polymer can be used as a matrix for stabilizing and retaining at the site of injection drug-containing microspheres, liposomes or other particulate-bound drugs.
A particularly suitable vehicle for intraocular injection is sterile distilled water in which the GDNF protein product is formulated as a sterile, 30 isotonic solution, properly preserved. Yet another ophthalmic preparation mayinvolve the formulation of the GDNF protein product with an agent, such as injectable microspheres or liposomes, that provides for the slow or sustained release of the protein which may then be delivered as a depot injection. Other suitable means for the intraocular introduction of GDNF protein product 35 includes, i",planlable drug delivery devices or which contain the GDNF protein product.
CA 022361~7 1998-0~-21 Wo 97/19694 Pcr/uss6/l8806 The ophthalmic preparations of the present invention, particularly topical prepa~alions, may include other components, for example ophthalmically acce~t~ble preservatives, tonicity agents, cosolvents, wetting agenls, complexing agents, buffering agents, antimicrobials, antioxidants and surfactants, as are 5 well known in the art. For example, suitable ~onicity enhancing agents includealkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol and the like. Sufficient tonicity enhancing agent is advantageously added so that the formulation to be instilled into the eye is hypotonic or subalar,lially isotonic. Suitable preservatives include, but are not limited to, benzalkonium 10 chloride, thimerosal, phenethyl alcohol, methylparaben, propyl,u~ldben, chlo,l,exi~i.le, sorbic acid and the like. Hydrogen peroxide may also be used aspreservative. Suitable cosolvents include, but are not limited to, glycerin, propylene glycol and polyethylene glycol. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta -cyclodextrin or hydroxypropyl- beta -15 cyclcdextrin. Suitable surfactants or wetting agents include, but are not limitedto, sorbitan esters, polysorbates such as polysorbate 80, troilletl,&l"ine, lecithin, cholesterol, tyloxapol and the like. The buffers can be conventional buffers such as borate, citrate, phosphate, bicarbonate, or Tris-HCI.
The formulation components are prese!nt in concenllalions that are 20 ~cep~Allle to the extraocular or intraocular site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8.
Additional formulation components may incllJde materials which provide for the prolonged ocular residence of the extraocularly administered therapeutic25 agent so as to maximize the topical contact and promote absorbtion. Sui'?~blQmaterials include polymers or gel forming materials which provide for increased v;3cosily of the ophthalmic preparation. Chitosan is a particularly suitable material as an ocular release-rate controlling agent in sustained release liquidophthalmic drug formulations (see U.S. 5,422,116, Yen, et. al.) The suitability 30 of the formulations of the instant invention for controlled release (e.g., sustained and prolonged delivery) of an ophthalmic treating agenl in the eye can be clete,l"ined by various procedures known in the art, e.g., as described in Journal of Controlled Release, 6:367-373, 19~7, as well as variations thereof.
Yet another ophthalmic preparation may involve an effective quantity of 35 GDNF protein product in a mixture with non-toxic ophthalmically acce~ ;P~!e excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other appropriate vehicle, ophthalmic solutions can CA 022361~7 1998-0~-21 WO 97/196g4 Pcr/us96/18806 be prepared in unil dose form. Suitable excipienls include, but are nol limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lul,licaling agents such as magnesium stearale, stearic acid. or talc D. ~.lnlinisl~ation/Delivery of (~DNF Protein Product The GDNF protein product may be administered parenterally via a subcutaneous, inlramuscular, intravenous, transpulmonary, transdermal, intrathecal or illl,acerebral route. For the treatment of ophthalmic conditions,10 the GDNF protein product may also be advantageously administered extraocularly or intraocularly, as described above, by topical a~l.calion, inserts, injection,i"lpla"ls, cell therapy or gene Iherapy. For example, slow-releasing i",plan~
containing the neurotrophic factor embedded in a biodegradable polymer matrix can deliver GDNF protein product. GDNF protein product may be admi";slered 15 exlfacerebrally in a form that has been modified chemically or packaged so that it passes the blood-brain barrier, or il may be administered in connection with one or more agents capable of promoting penetration of GDNF protein product across the barrier. Similarly, the GDNF protein product may be administered intraocularly, or it may be administered extraocularly in connection with one or20 more agents capable of promoting penetration or transport of GDNF protein product across the me,llbranes of the eye. The frequency of dosing will depend on the pha""acokinetic parameters of the GDNF protein product as formulated, and the route of administration.
The specific dose may be calculated according to considerdlions of body 25 weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in theart and is within the ambit of tasks routinely pe,lc,r",ed, especially in light of the dosage inforrnation and assays r~isclosed herein. Approp, iale dos~es may be30 asce,lailled through use of the estatjl;shed assays for determining dos~ges utilized in conjunction with appropriate dose-response data. According to the currently pfe~er,ed en-bodi."ents of the present invention, the GDNF protein product is most advantageously a tm;.~;slered intraocutarly at a dose between about 0.001 mg/day and 10 mg/day, and preferably at a dose between about 35 0.01 mg/day and 1 mg/day, and most preferably at a dose between about 0.1 mg/day and 0.5 mg/day. It will be appreciated by those skilled in the art that the dosage used in intraocularly administered formulations will be CA 022361~7 1998-0~-21 rninuscule as compared lo that used in a systemic injection or oral administration .
The final dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician 5 conside,il,g various factors which modify the action of drugs e.g. the age, condition, body weight sex and diet of the patient the severity of any infectiontime of administration and other clinical factors. As studies are conducted further infor",dlion will emerge rega,ding the appropriate dosage levels for thetreatment of various diseases and conditions.
It is envisioned that the continuous administration or sustained delivery of GDNF may be advantageous for a given treatment. While continuous a~l"in;~l,dlion may be accomplished via a mechanical means such as with an infusion pump, it is contemplated that other modes of continuous or near continuous adl"i.);sllalion may be p(acliced. For example chemical derivatization or encapsulation may result in sustained release forms of the protein which have the effect of continuous presence in predictable amounts based on a determined dosage regimen. Thus GDNF protein products include proteins derivatized or otherwise formulated to effectuate such conlinuous administration .
GDNF protein product cell therapy e.g. intraocular i",planlai:cn of cells producing GDNF protein product is also conte",~lated. This embodiment wouW
involve i""~lanting into patients cells aF~!e of synthesizing and secrelil-g a biologically active form of GDNF protein product. Such GDNF protein product-producing cells may be cells that are natural producers of GDNF protein product (analogous to B49 glioblastoma cells) or may be reco",b nanl cells whose ability to produce GDNF protein product has been augmented by l.ansf~".-ation with a gene encoding the desired GDNF protein product in a vector suitable for promoting its e,~,uression and secretion. In order to minimize a po1ential immuncl~cal reaction in patients being admi~islered GDNF protein product of a foreign species it is preler,~d that the natural cells producing GDNF
protein product be of human origin and produce human GDNF protein product.
Likewise it is prefer-ed that the recombinant cells producing GDNF protein product be l,anslor",ed with an expression vector containing a gene encoding a human GDNF protein product. Ill,planled cells may be encarsu~ted to avoid infil~,dlion of surrounding tissue. Human or non-human animal cells may be implanted in patients in biocompatible semipermeable polymeric enclosures or ",~",br;~l,es that allow release of GDNF protein product but lhat prevent CA 022361~7 1998-0~-21 wo 97/19694 Pcrluss6/l88o6 destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue. Such an implant, for example. may be attached to the sclera to produce and release GDNF protein product directly intothe vitreous humor.
It is also contemplated that the patient's own cells may be l.dn~forl"ed ex t~ivo to produce GDNF protein product and would be directly implanted without enoal~su~ Qn. The cells would be transformed with an ap~rupriale vector and ~,ar,~ lanled back into the patient's retina where they would produce and release the desired GDNF protein or GDNF protein variant.
Photoreceptor cell l,dnsplantation studies designed to replace defective or lost cells due to retinal disease or damage have been performed successfully in animal models of retinal degeneration (Silverman and Hughes, Invest.
Ophthalmoi. Vis. Sci.. 30:1684-1690, 1989; Gouras et al., Neuro-Oph~haimol., 10:165-t76, 1990). It is contemplated that photoreceptor cells may be 15 obtained from donor eyes and maintained in culture as desc~ ed herein. The cells would then be used as a source of purified photoreceplor~ to be t,ansplanted via the subretinal space into the retina of patients suffering from retinal disease or da",age. These patients will be treated with immunosuppressive therapies to eliminate immunological responses and rejection of the grafted cells. The ex vivo 20 donor retinas will be cultured in the presence of GDNF, in order to enhance their growth and survival. The patients that will receive photorecep:or cell ~ansplants will be treated with intravitreal injections of GDNF, that will be needed to promote the survival and the maturation of the grafted photo.eceptor~.GDNF protein product in vivo gene therapy is also envisioned. by 25 introducing the gene coding for GDNF protein product into targeted cells via local injection of a nucleic acid construct or other d~.propriate delivery vectors.
(Hefti, J. Neurobiol., 25:1418-1435, 1994). For example, a nucieic acid sequence encoding a GDNF protein product may be contained in an adeno-~csoc:~led virus vector or adenovirus vector for delivery to the retinal 3û cells. Alternative viral vectors inc1ude, but are not limited to, retrovirus,herpes simplex virus and papilloma virus vectors. Physical transfer, either in vivo or ex vivo as appropriate, may also be achieved by liposome-mediated transfer, direct injection (naked DNA), receptor-mediated transfer (ligand-DNA
complex), electroporation, calcium phosphate precipitation or microparticle 35 bG,.IL,afd",ent ~gene gun).
The methodology for the membrane encarsulation of living cells is familiar to those of ordinary skill in the art, and the preparation of the CA 022361~7 1998-0~-21 encP~rsu~ated cells and their implanlation in patients may be accomplished without undue experimentation. See, e.g., U.S. Patent Numbers 4,892,538, 5,011,472, and 5,106,627. each of which is specifically incorporated herein by reference. A system for enc~rsul~ting living cells is described in PCT
Applioalion WO 91110425 of Aebischer et al., specifically incorporated herein by reference. See also, PCT A~pllc2 -~n WO 91~10470 of Aebischer et al., Winn et al.. Expsr. NeuroJ., 113:322-329, 1991, Aebischer et al., Exper. Neurol., 111:26~-275, 1991; Tresco et al., ASAIO, 38:17-23, 1992, each of which is sp~ifically incorporated herein by reference. Additional implantable devices are described in WO 93121902 (International Application No.
PCT/US93103850) which is incorporated herein by reference. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible particles or beads and depot injections, are also known to those skilled in the art.
It should be noted that the GDNF protein product formulations desc,iLed herein may be used for veterinary as well as human applicalions and that the term "patient" should not be construed in a limiting manner. In the case of veterinary a~ F'i~tians, the dosage ranges should be the same as speci~ied above.
Other aspects and advantages of the present invention will be under~lood upon considerat,on of the ~oll~ ,;ng illustrative examples. The examples addressthe effect of GDNF protein product on both normal and mutated retinal neurons.
In addition. the examples set forth unique techniques for culturing retinal cells.
EXAMPLES
MATFP'AI .S AND MErHODS
The materials used in the following Examples were obtained as follows.
Cell Culture Mer~i~
High glucose Dulbecco's Modified Eagle's Medium (DMEM; #11965-092), Ham's F12 medium (F12; #11765-021), Leibovitz~s L15 medium without sodium bicarbonate (#41300-039); B27 medium supplement (#17504-010), penicillinlstreptomycin (#15070-014), L-glutamine (#25030-016), Dulbecco's phosphate-buffered saline (D-PBS; #14190-052), Hank's balanced salt solution with calcium and magnesium salts (HBSS;
CA 022361~7 1998-0~-21 #24020-026), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; #15630-015~, mouse laminin (#23017-015), bovine serum albumin and fractionV (#110-18-017) were all from GIBCO/BRL, Grand Island, NY. Heat-inactivated horse serum was from HyClone, Logan, Utah. Poly-L-ornithine hydrobromide (P-3655), bovine insulin (1-5500), hùman transferrin (T-2252), putrescine (P-6024), progesterone (P-6149) and sodium selenite (S-9133) were all from Sigma Chemical Company, Saint-Louis, MO. Papain, deoxyribonuclease I (DNAase) and ovalbumin (Papain ~issociation system) were from Worthington Biochemicals, Freehold, NJ. Falcon sterile g6-well microplates (#3072), tissue culture plastic ware and polypropylene centrifuge tubes were from Beckton-Dickinson, Oxnard, CA. Nunc Lab-Tek tissue culture chamber coverglasses (#136439) were from Baxter, Irvine, CA.
Nitex 20 llm nylon mesh (#460) was from Tetko, Elmsford, NY. The 4"
dissecting forceps and 4" dissecting scissors were from Roboz Surgical, Washington, DC.
Antibodies. Radioicotopes and Rel~ted Re~n~
Polyclonal rabbit antibody and mouse monoclonal rho4D2 anti-bovine rhodopsin antibodies were from the University of British Columbia, Vancouver, Canada. The polyclonal rabbit antibody was directed to the following synthetic peptide sequence of the rod-specific protein arreslin: Val-Phe-Glu-Glu-Phe-Ala-Arg-Gln-Asn-Leu-Lys-Cys (SEQ ID NO:2). Biotinylated horse anti-mouse IgG, biotinylated goat anti-rabbit IgG, peroxidase-conjugated avidin/biotin con~plex and Texas Red-conjugated streptavidin (ABC Elite; kit PK-6100) were from Vector Laboratories, Burlingame, CA. Fluorescein isothiocyanate con,ug~ted rabbit anti-mouse immunoglobulins were from Dako Corporation (Carpinteria, CA). 3',3'-diaminobenzidine was from Cappel Laboratories, West Chester, PA. Superblock blocking buffer in PBS (#37515) was from Pierce, Rockford, IL. Triton X-100 (X100), Nonidet P-40 (N6507) and hydrogen peroxide (30%, v/v; H1009) were from Sigma. L-13,4-3H]-Glutamic acid (NET-490; 40-80 CiJmmol) was from New England Nuclear, Boston, MA.
Optiphase Supermix scintillation cocktail was from Wallac, Turku, Finland.
White ViewPlate-96 microplates (#600~182) were from Packard Instruments Corporation, Meriden, CT. All other reagents were obtained from Sigma Chemical Company (Saint-Louis, MO), unless otherwise specified.
CA 022361~7 1998-0~-21 WO g7/196g4 PCr/USg6/18806 - 3~ -Prep~r~tion of Media A basal medium was prepared as a 1:1 mixture of DMEM and F12 medium, and was supplemented with B27 medium supplement added as a 50-fold concentrated stock solution. The B27 medium supplement consials of biotin, L-5 carnitine, corticosterone, ethanolamine, D(+)-galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, selenium, T3 (triodo-l-thyronine, DL-alpha-tocopherol (vitamin E), DL-alpha-tocopherol acetate, bovine serum albumin, cat~l~ce~ insulin, superoxide dismutase and transferrin. L-glutamine was added at a final concentldl:on of about 2 mM, penicillin at about 100 IUII, and streptomycin at about 100 mg/l.
Heat-inactivated horse serum was added to a final concer,lralion of about 2.5 percent, D-glucose was added to a final concentration of about 5 9/l, HEPES
buffering agent was added to a final concentration of about 20 mM, bovine insulin was added to a final concentration of about 2.5 mglml, and human ~,ansler-in 15 was added to a final concehl-alion of about 0.1 mg/ml. After mixing, the pH was adjusted to about 7.3 and the medium was kept at 4~C. The media were p(epare~l fresh just before use in order to minimize inter-experimental variations.
Plastic pipettes and containers were used throughout to minimize protein adsorption.
,DNF Protein Product Solutions Purified human recombinant GDNF protein products were prepared as 1 mg/ml solutions in D-PBS (phosphate buffered saline prepared with distilled water) conlail~ing five percent bovine serum albumin. The solutions were stored 25 at -85~C in aliquots. Serial dilutions were prepared in 96-well microplates.
Ten microliters of ten-fold concentrated GDNF protein product solutions were added to cell cultures containing culture medium (90 1l1). Control cultures received D-PBS with 5 percent albumin (10 ~11). The GDNF protein product treatments were initiated one hour after cells were seeded and, in some 30 i"~lances, repeated every other day.
Cultllre Substratum To encourage optimal attachment of photoreceptors on substratum, outer segment outgrowth and neurite outgroJ/Ih, the microtiter plate surfaces (the 35 culture substratum) were modified by sequentiat coating with poly-L-ornithinefbl'~ ed by laminin in accordance with the following procedure. The plate surfaces were completely covered with a 0.1 mglml sterile solution of CA 022361~7 1998-0~-21 polyornithine in 0.1 M boric acid (pH 8.4) for at least one hovr at room temperature, followed by a sterile wash with Super-O water. The water wash was then aspirated and a 1 ,ugtml solution of mouse laminin in PBS was added andinc~ ed at 37~C for two hours. These procedures were conducted just before using the plates in order to ensure reproducibility of the results.
Prer~r~tion of Chick and Mouse Photorece~tor Cultllres Seventeen-day-old White Leghorn chick embryos and 5-day-old C57BI16 mouse pups (obtained from Jackson Laboratories, Bar Harbor, Maine) were killed by decapitation and the eyes were dissected sterilely into L15 medium (without sodium bicarbonate). A maximum of 24 eyes were processed per ex, eri",ent. The eyes were hemisected, and the lens and vitreous were removed.
The neural retinas were carefully removed and dissected free of the pigment epithelium, cut into small (about 1 s~uare mm or less) fragments and placed intoice-cold D-PBS. The cells were collected, and then transferred into 10 ml dissoc;~lion medium (120 units papain and 2000 units DNAase in HBSS). The cells were incubated for 45 minutes at about 37~C on a rotary pldlkJr", shaker at about 200 rpm. The cells were then dispersed by tritùration through fire-polished Pasteur pipettes, sieved through a 20 llm Nitex nylon mesh to discard undissoci~ted tissue, and centrifuged for five minutes at 200 x 9 using an IE
clinical centrifuge. The resulting cell pellet was resuspended into HBSS
containing ovalbumin and about 500 units DNAase, layered on top of a 4 percent ovalbumin solution (in HBSS) and centrifuged for about 10 minutes at 500 x 9.
The final pellet was resuspended in complete culture medium (see above), adjusted to about 15,000 cells/ml, and seeded in 90 1l1 aliquots into the 6 mm-wells of 96-well microplates previously coated with polyornithine and laminin.
Attachment of cells occurred rapidly, and the plating efficiency was about 75 percen{.
Cultures of photoreceptors from adult mouse retina Cultures of adult photoreceptors were obtained by seeding dissociated retinal cells from post-natal day 18 to 39 mice on top of pre-established monolayers of post-natal day 5 mouse retinal glial cells or rat retina pigment epithelium cells. Dissociation procedures and culture media were the same as described above. Cultures of retinal glial cells and pigmenl epithelium cells were established in tissue culture flasks (225 crn2 Costar flasks) and grown until confluence was reached. The cells were then detached by a short (about two CA 022361~7 1998-0~-21 Wo 97/19694 Pcr/uss6/18806 minute) incubation with 0.1% trypsin and plated in 96-well microplates or 16-well glass coverslip chambers. Dissociated adult retinal cells were added after about 3 to 5 days.
Cultures of photoreceptors from rd/rd mouse retina Rd/rd C57BI16 mice (obtained from Jackson Laboratories, Bar Harbor, Maine) have an inherited photoreceptor degeneration resulting from the ex~J,ession of a mutation in the beta subunit of phos,uhodiesterase tan enzyme locz~;~e~ in the outer segments and involved in the photol,dnsduction processes~.
These mice provide a useful model to study the role of trophic factors on lesioned pholoreceFt,rs. Photoreceptor death in rd/rd mice peaks at around 10 days after birth. Cultures of rd/rd photoreceptors were established from 5-day-old mice and maintained in cultures for eight days, covering ~he period of maximal photoreceptor death. The dissociated retinal cells were seeded on top of a pre-established monolayer of retinal glial cells (see above) at a density of about 10,000 cells per 6-mm well and were maintained in the culture medium desc-ibed above.
Immunohistochemistry of Photoreceptors To characterize mouse photoreceptors, an indirect immunoperoxidase method described by Louis et al. (J. Pharmacol. Exp. Therap., 262:1274-1283, 1992; Science, 259:689-692, 1993) was used, with slight modifications as follows. Cul~ures of photoreceptors were fixed for about 30 minutes at room te",perall~re with 4 percent paraformaldehyde in D-PBS, pH 7.4, followed by three washes in D-PBS (200 111 per 6-mm well). The fixed cultures were then incub~ted in Superblock blocking buffer in PBS, containing one percent Nonidet P-40 to increase the penetration of the antibodies. The anti--l,odopsi"
antibodies (rabbit and mouse) were then applied at a dilution of between 1:1000-1:4000 in the same buffer, and the cultures were incubated for one hour at 37~C on a rotary shaker. After three washes with D-PBS, the photorecPF~,r-bound antibodies were detected using goat-anti-rabbit or horse-anti-mouse biotinylated IgG (Vectastain kit from Vector Laboratories, Burlingame, CA) at about a 1:500 dilution: these secondary an~iL~d!~s were inc~b~ted with the cells for about one hour at 37~C, the cells were then washeclthree times with D-PBS. The secondary antibodies were then labeled with an avidin-biotin-peroxidase complex diluted at 1:500, and the cells were incubated for about 45 minutes at 37~C. After three more washes with D-PBS, the labeled CA 022361~7 1998-0~-21 cell cultures were reacted for 5-20 minutes in a solution of 0.1 M Tris-HCI, pH
Non-conservative substitutions may involve the excl~ange of a member of one of these classes for another. Such substituted residues may be introduced into regions of the GDNF protein that are homologous with other TGF-B
superfamily ~.rutcins, or into the non-homologous regions of the molecule.
B. GDNF Deriv~tives Chemically modified derivatives of GDNF or GDNF variants may be p-eparecl by one of skill in the art given the disclosures herein. The chemical " ~3eties most suitable for derivatization include water soluble polymers. A
water soluble polymer is desirable b~c~se the protein to which it is attached does not prec-,l,i1alA in an aqueous en~ or""enl, such as a physiological environment. Preferably, the polymer will be pharmaceutically accepl~le for the preparalion of a therapeutic product or composition. One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymerlprotein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considera1;ons. The effectiveness of 1he derivatization may be ascertained by a~ ,;nislering the derivative, in the desired form (i.e., by osmotic pump, or, CA 022361~7 1998-0~-21 WO 97/lsCs4 PcrtuS96/18806 more preferably, by injection or infusion, or, further formulated for oral, pulmonary or other de!ivery routes), and determining its effectiveness.
Suitable water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycollpropylene glycol, 5 carboxymethylcellulose, dextran, polyvinyl alcohol. polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, proptopylene glycol hol"opolymers, polypropylene oxide/ethylene oxide co-polymers, 10 polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advanlages in manufacturing due to its stability in water.
The polymer may be of any molecular weight, and may be branched or u.~branched. For polyethylene glycol, the preferred molecular weight ranges 15 from about 2 kDa to about 100 kDa for ease in har,Jli"g and manufacturing (the term "about" indioali"g that in prepa~lions of polyethylene glycol, some ",~lecules will weigh more, some less, than the stated molecular weight). Other skes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in ~0 handling, the degree or lack of antigenicity and other known effects of poiyethylene glycol on a therapeutic protein or variant).
The number of polymer molec~ s so attached may vary, and one skilled in the art will be able to asce,lain the effect on function. One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of deriv~ti~tion~
25 with the same or different chemical moieties (e.g., polymers, such as di~rerenl weights of polyethylene glycols). The proportion of polymer ",~'e ules to protein (or peptide) molecules will vary, as will their concer,lralions in the feaotion mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be 30 determined by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is brar,ched or unbranched, and the reaction conditions.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic35 domains of the protein. There are a number of alla~:l,r"ent ",etl,Gds available to those skilled in the art. See for example, EP 0 401 384, the disclQsure of whichis hereby i,,cor,uordlad by reference ~coupling PEG to G-CSF), see also Malik et CA 022361~7 1998-0~-21 WO 97/19694 PCI'/US96rl8806 al., Exp. ~lematot. 20:1028-1035 (1992) (reporting pegylation of GM-CSF
using tresyl chloride). For example, polyethylene glyco~ may be covalently bound through amino acid residues via a reactive group, such as, a free amino orcarboxyl group. Reactive groups are those to which an activated polyethylene 5 glycol ",o'ecule may be bound. The amino acid residues having a free amino group may inciude Iysine residues and the N-terminal amino acid residue. Those having a free carboxyl group may include aspa~lic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for allachil)g the polyethylene glycol molecule(s~. For 10 therapeutic purposes, allacr""ent at an amino group, such as ~llach",ent at the N-terminus or Iysine group is preferred. Attachment at residues important for ,t:ceptor binding should be avoided if receptor binding is desired.
One may specifically desire an N-terminal chemically ",odiried protein.
Using polyethylene glycol as an illusl~alion of the present compositions, one may 15 select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (orpeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of-obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated prepa(alion (i.e., 20 separali"g this moiety from other monopegylated moieties if necessary) may beby purification of the N-terminally pegylated material from a popu'~tion of pegylated protein molecules. Selective N-terminal chemical ",odificalion may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (iysine versus the N-terminal) 25 available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. For exar"F!e, one may selectively N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pKa diflerences bet~een 30 the ~-amino group of the Iysine residues and that of the c~-amino group of the N-terminal residue of the protein. By such selective derivatization, attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no siy";licanl ",odification of other reactive groups, such as the Iysine side chain 35 amino groups, occurs. Using reductive alkylation, the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for CA 022361~7 1998-0~-21 Wo 97/19694 PCT/US96tl8806 coupling to the protein. Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.
The present invention contemplates use of derivatives which are prokaryote-expressed GDNF, or variants thereof, linked to at least one 5 polyethylene glycol molecule, as well as use of GDNF, or variants thereof, attached to one or more polyethylene glycol molecules via an acyl or alkyl linkage.
Pegylation may be carried out by any of the pegylation reactions known in the art. See, for example: Focus on Growth Factors, 3 (2): 4-10 (1992); EP 0 154 316, the disclosure of which is hereby incorporated by reference; EP 0 401 384; and the other publications cited herein that relate to pegylation. The pegylation may be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
Pegylation by acytation generally involves reacting an active ester derivative of polyethylene glycol with the GDNF protein or variant. Any known or subsequently discovered reactive PEG molecule may be used to carry out the pegylation of GDNF protein or variant. A preferred activated PEG ester is PEG
eslt:rified to N-hydroxysuccinimide. As used herein, "acylation" is contem~lated20 to include without li",itation the lollo~;"g types of linkages between the tl,srhpeutic protein and a water soluble polymer such as PEG: amide, c&,l,a",dle, urethane, and the like. See Bioconjugate Chem., 5:133-140 (1994). Reaction condi~ions may be _cle_led from any of those known in the pegylation art or those subsequently developed, but should avoid conditions of temperature, solvent, and25 pH that would inactivate the GDNF or variant to be nlGd;fie~.
Pegylation by acylation will generally result in a poly-pegylated GDNF
protein or variant. Preferably, the connecting linkage will be an amide. Also preferabiy, the resulting product will be substantially only (e.g., > 95%) mono-, di- or tri-pegylated. I lo Arevcr, some species with higher degrees of pegylation 30 may be formed in amounts depending on the specific reaction conditions used. If desired, more purified pegylated species may be separated from the mixture, particularly unreacted species, by standard purification techniques, including, among others, dialysis, salting-out, ultrafiltration, ion-exchange chro",alography, gel filtration chromatography and electrophoresis.
3'i Pegylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with the GDNF protein or variant in the presence of a reducingagent. Pegylation by alkylation can also result in poly-pegylated GDNF protein CA 02236l~7 l998-0~-2l Wo g7/19694 Pcr/uss6/18806 or variant. In addition one can manipulate the reaction conditions to favor pegylation substantially only at the a-amino group of the N-terminus of the GDNF protein or variant (i.e. a mono-pegylated protein). In either case of "-onopeyylation or polypegylation the PEG groups are preferably allached to the protein via a -CH2-NH- group. With particular reference to the -CH2- group this type of linkage is referred to herein as an alkyl linkage.
Derivatization via reductive alkylation to produce a monopegylated product exploits differential reactivity of different types of primary amino groups (Iysine versus the N-terminal) available for derivali~alion. The reaction is pe,f~,l",ed at a pH which allows one to take advantage of the pKa differences between the E-amino groups of the Iysine residues and that of the ~-amino group of the N-terminal residue of the protein. By such selective derivatization allachl~lent of a water soluble polymer that conlains a reactive group such as an aldehyde to a protein is cor,l-~'ed: the conjugation with the polymer takes plsce predominantly at the N-terminus of the protein and no significant modification of other reactive groups such as the Iysine side chain amino groups occurs. In one important aspect, the present invention contemplates use of a substantially ho."ogeneous preparation of ",onopolymer/GDNF protein (or variant) conjugate molecules (meaning GDNF
protein or variant to which a polymer molecule has been allached substantially only (i.e., > 95%) in a single location). More specifically if polyethylene glycol is used, the present invention also encompasses use of pegylated GDNF protein orvariant lacking possibly antigenic linking groups and having the polyethylene glycol molecule directly coupled to the GDNF protein or variant.
Thus it is conle",plaled that GDNF protein products to be used in acco,dance with the present invention may include pegylated GDNF protein or variants wllerei n the PEG group(s) is (are) attached via acyl or alkyl groups.
As disc~ssed above, such products may be mono-pegylated or poly-pegylated (e.g., containing 2-6 and preferably 2-5 PEG groups). The PEG groups are generally attached to the protein at the oL- or E-amino groups of amino acids but it is also conler"~,laled that the PEG groups could be attached to any amino group attached 10 the protein which is suflicien11y reactive to become allacl,ed to a PEG
group under suitable reaction conditions.
The polymer molecules used in both the acylation and alkylation ~,,oal;l,es may be selected from among water soluble polymers as described above. The polymer selected should be modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation preft:rdbly so CA 022361~7 1998-0~-21 Wo 97/19694 Pcrluss6/l8806 that the degree of polymerization may be controlled as provided for in the present .,.ell,ods. An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S. Patent 5,252,714). The polymer may be 5 ~ranched or unbranched. For the acylation reactions, the polymer(s) selEc1ed should have a single reactive ester group. For the present reductive alkylation,the polymer(s) selected should have a single reactive aldehyde group. Generally.the water soluble polymer will not be selected from naturally-occurring glycosyl residues since these are usually made more conveniently by mammalian 10 recombinant expression systems. The polymer may be of any molecular weight, and may be branched or ul)brdncl)ed.
A particularly preferred water-soluble polymer for use herein is polyethylene glycol. As used herein, polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other pr.l~ ,s, such as 5 mono-(C1 -C10) alkoxy- or aryloxy-polyethylene glycol.
In general, chemical derivatization may be performed under any suitable condition used to reacl a biologically active substance with an activated polymer molecule. M_lhods for p(epa-i"g pegylated GDNF protein or variant will generally comprise the steps of (a) reacting a GDNF protein or variant with 20 polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the protein becomes allached to one or more PEG
groups, and (b) obtaining the reaction product(s). In general, the optimal reaction conclilions for the acylation reactions will be determined case-by-casebased on known parameters and the desired result. For example, the larger the 25 ratio of PEG:protein, the greater the percenlage of poly-pegylated product.
Reductive alkylation to produce a substantially homogeneous popu~~~ion of mono-polymer/GDNF protein (or variant) conjugate molecule will generally co,.,~ri~e the steps of: (a) reacting a GDNF protein or variant with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable to permit 30 sel~ctiJc ",oJi~icaticn of the a-amino group at the amino terminus of said GDNF
protein or variant; and (b) obtaining the reaction product(s).
For a substantially homogeneous population of mono-polymer/GDNF
protein ~or variant) conjugate molecules, the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble 35 polymer moiety to the N-terminus of GDNF protein or variant. Such reaction conditions generally provide for pKa differences between the Iysine amino groupsand the ~-amino group at the N-terminus (the pKa being the pH at which 50% of CA 022361~7 1998-0~-21 WO 97/lg6g4 PCT/US96/18806 the amino groups are protonated and 50% are not). The pH also affects the ratio of polymer to protein to be used. In general. if the pH is lower, a larger excess of polymer to protein will be desired (i.e., the less reactive the N-terminal ~-amino group, the more polymer needed to achieve optimal conditions). If the 5 pH is higher, the polymer:protein ratio need not be as large (i.e., more reactive groups are available, so fewer polymer molecules are needed). For purposes of the present invention, the pH will generally fall within the range of 3-9, preferably 3-6.
Another important consideration is the molecular weight of the polymer.
10 In general, the higher the molecular weight of the polymer, the fewer polymermolecules may be attached to the protein. Similarly, b,~nching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer:protein ratio. In general, for the pegylation reactions contemplated 15 herein, the preler,ed average molecular weight is about 2 kDa to about 100 kDa.
The prefer,t:d average molecular weight is about 5 kDa to about 50 kDa, particularly preferably about 12 kDa to about 25 kDa. The ratio of water-soluble polymer to GDNF protein or variant will generally range from 1:1 to 100:1, preferably (for polypegylation) 1:1 to 20:1 and (for monopegylation) 1:1 to 5:1.
Using the conditions indicated above, reductive alkylation will provide for selective attachment of the polymer to any GDNF protein or variant having an a-amino group at the amino terminus, and provide for a substantially homogenous preparation of monopolymer/GDNF protein (or variant) conjugate.
25 The term "".onopolymer/GDNF protein (or variant) conjugate" is used here to mean a composition co""~ri~ed of a single polymer molecule allached to a " ~lecu'e of GDNF protein or GDNF variant protein. The monopolymer/GDNF
protein ~or variant) conjugate preferably will have a polymer molecule located at the N-terminus, but not on Iysine amino side groups. The preparation will 30 preferably be greater than 90% monopolymer/GDNF protein (or variant) conjugate, and more preferably greater than 95% monopolymer/GDNF protein (or variant) con,ugate. with the remainder of observable molecules being unreacted (i.e., protein lacking the polymer moiety).
For the present reductive alkylation, the reducing agent should be stable 35 in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. F~refer,ad reducing agents may be 5el~Ctel:l from sodium borohydride. sodium cyanoborohydride, dimethylamine CA 022361~7 1998-0~-21 WO 97tl96g4 PCT/US96/18806 borane, trimethylamine borane and pyridine borane. A particularly preferred reducing agent is sodium cyanoborohydride. Other reaction parameters, such as solYent, reaction times. temperatures, etc., and means of purification of products, can be determined case-by-case based on the published information relating to derivatization of proteins with water soluble polymers (see the publications cited herein).
G t~DNF Protein Product Ph~rma~euti~l Cornpositions GDNF protein product pharmaceutical co",posi~;ons typically include a therapeutically effective amount of a GDNF protein product in admixture with oneor more pharmaceutically and physiologically acceptable formulation materials.
Suitable formulation materials include, but are not limited to, antioxidants, preserva1iYes, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents. excipients and/or pharmaceutical adjuvants. For example, a suitable vehicle may be water for injection, physiological saline solution, or artificialCSF, possi~,ly supplemented with other materials common in cG",posilions for parenteral adm;r,;st,alion. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
The primary solvent in a vehicle may be either aqueous or non-aqueous in nature. In addition, the vehicle may contain other pharmaceutically-acce~Jtable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
Similarly, the vehicle may contain still other pharmaceutically-accept~
excipients for ",o lifying or maintaining the rate of release of GDNF protein product, or for promoting the absorption or penetration of GDNF protein product across the me,.ll"dnes of the eye. Such excipients are those sulJalances usuallyand cuslo",a,ily employed to formulate dosages for parenteral adl"inis~ralion ineither unit dose or multi-dose form.
Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or Iyophilized powder. Such formulations may be stored either in a ready to use form or in a form, e.g., Iyophilized, requiring reconstitution prior to administration .
~5 The optimal pharmaceutical formulations will be determined by one skilled in the art depending upon considerations such as the route of aJI";.~ial~alion and desired dosage. See for example, Remlngton's Pharmaceutical CA 022361~7 1998-0~-21 Wo 97/19694 PCr/uss6/l88o6 Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vjl~o clearance of the present GDNF proteins, variants and derivatives.
Other effective administration forms, such as parenteral slow-release formulations, inhalant mists, or orally active formulations are also envisioned.For example, in a sustained release formulation, the GDNF protein product may be bound to or incorporated into particulate preparalions of polymeric compounds (such as polylactic acid, polyglycolic acid, etc.) or li~.oso",es.
Hylauronic acid may also be used, and this may have the effect of p,u,l,oting suslained duration in the circulation. The GDNF protein product pharmaceutical composition also may be formulated for parenteral administration, e.g., by intraocular infusion or injection, and may also include slow-release or sustained circulation formulations. Such parenterally administered therapeutic compositions are typically in the form of a pyrogen-free, parenterally a~ept~le aqueous solution co."prising the GDNF protein product in a pha""aceutically ~ceptAble vehicle. One preferred vehicle is sterile distilled water.
It is also contemplated that certain formulations containing GDNF protein product are to be administered orally. GDNF protein product which is administered in this fashion may be encapsulated and may be formulated with or without those carriers customarily used in the compounding of solid dosage forms. The capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is ".~xl",i~ed and pre-systemic degladalion is mi"i",ked. Additional excipients may be included to facilitate absorption of GDNF protein product. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, susper,ding agents, tablet disintegrating agents, and binders may also be employed.
The formulation of topical ophthalmic preparations, including ophthalmic solutions, suspensions and ointments is well known to those skilled in the art (see Re",ington~s Pharmaceutical Sciences, 18th Edition, Chapter 86, pages 1581-1592 Mack Publishing Company, 1990). Other modes of a~"inislrd~-on are available, including intracameral injections (which may be made directly inlo the anterior chamber or directly into the vitreous chamber), subconjunctival injections and retrobulbar injections, and methods and means CA 022361~7 1998-0~-21 - 2~ -for producing ophthalmic preparations suitable for such modes of ad~ ,tration are also well known.
As used in this application, Uextraocular'' refers to the ocular surface and the (extemal) space between the eyeball and the eyelid. Examples of extraocular regions include the eyelid fornix or cul-de-sac, the conjunctival surface and the comeal surface. This location is extemal to all ocular tissue and an invasive procedure is not required to access this region. Examples of extraocular systemsinclude inserts and "topically" applied drops, gels or ointments which may be used to deliver therapeutic material to these regions. Extraocular devices are 1 0 generally easily removable, even by the patient.
The fo"~ ;ng patents disclose extraocular systems which are used to nisler drugs to the extraocular regions. Higuchi et al. disclQses in U.S.
Patent Numbers 3,981,303, 3,986,510 and 3,995,635, a biodegradable ocular insert which contains a drug. The insert can be made in different shapes for 1 5 retention in the cul-de-sac of the eyeball, the extraocular space between 1he eyeball and the eyelid. Several common biocompatible polymers are d;sclosed as suitable for use in ~db,icat;ng this device. These polymers include zinc alginate, poly (lactic acid), poly (vinyl alcohol), poly (anhydrides) and poly (glycolic acid). The patents also describe membrane coated devices with reduced permeation to the drug and hollow chambers holding the drug formulation.
Theeuwes, U.S. Patent Number 4,217,898, discloses microporous reservoirs which are used for controlled drug delivery. These devices are placedextraocularly in the ocular cul-de-sac. Among the polymer systems of interest include poly (vinylchloride)-co-poly (vinyl acetate) copolymers. Kautman 2~ discloses in U.S. Patent Numbers 4,865,846 and 4,882,150 an ophthalmic drug delivery system which con~a;"s at least one bio-erodibte material or ointment carrier for the conjunctival sac. The patent discloses polymer systems, such as,poly (lactide), poly (glycolide), poly (vinyl alcohol) and cross linked collagen, as suitable delivery systems.
In the presently described use of GDNF protein product for the treatment of retinal disease or injury it is also advantageous that a topically applied ophthalmic formulation include an agent to promote the penetration or transport of the therapeutic agent Into the eye. Such agents are known in the art. For example. Ke et al., U.S. 5,221,696 ~i,close the use of materials to enhance the penetration of ophthalmic preparations through the cornea.
Intraocular systems are those systems which are sui~able for use in any tissue compartment within, between or around the tissue layers of the eye itself.
CA 022361~7 1998-0~-21 These locations include subconjunctival (under the ocular rnucous membrane adjacent to the eyeball), orbital (behind the eyeball), and intracameral ~withinthe chambers of the eyeball itself). In contrast to extraocular systems, an invasive procedure consisting of injection or implantation is required to access5 these regions.
The fol'~ g patents disclose intraocular devices. Wong, U.S. Patent Number 4,853,224, discloses microencapsulated drugs for introduction into the chamber of the eye. Polymers which are used in this system include polyesters and polyethers. Lee, U.S. Patent Number 4,863,457, discloses a biodeg,ddable 10 device which is surgically implanted intraocularly for the sustained release of therapeutic agents. The device is designed for surgical i",,i~lantalion under the conjunctiva (mucous membrane of the eyeball). Krezancaki, U.S. Patent Number 4,188,373, discloses a pharmaceutical vehicie which gels at human body temperature. This vehicle is an aqueous suspension of the drug and gums or 15 cellulose derived synthetic derivatives. Haslam et al. discloses in U.S. Patent Numbers 4,474,751 and 4,474,752 a polymer-drug system which is liquid at room temperature and gels at body temperature. Suit~ble polymers used in this system include polyoxyethylene and polyoxy propylene. Davis et al. disclose in U.S. 5,384,333 a biodegradable injectable drug delivery polymer which 20 provides long term drug release. The drug co"lposition is made up of a pharmaceutically active agent in a biodegradable polymer matrix, where the polymer matrix is a solid at temperatures in the range 20~ to 37~C and is flowable at temperatures in the range 38~ to 52~C. The drug delivery polymer is not limited to the delivery of soluble or liquid drug formulations. For example,25 the polymer can be used as a matrix for stabilizing and retaining at the site of injection drug-containing microspheres, liposomes or other particulate-bound drugs.
A particularly suitable vehicle for intraocular injection is sterile distilled water in which the GDNF protein product is formulated as a sterile, 30 isotonic solution, properly preserved. Yet another ophthalmic preparation mayinvolve the formulation of the GDNF protein product with an agent, such as injectable microspheres or liposomes, that provides for the slow or sustained release of the protein which may then be delivered as a depot injection. Other suitable means for the intraocular introduction of GDNF protein product 35 includes, i",planlable drug delivery devices or which contain the GDNF protein product.
CA 022361~7 1998-0~-21 Wo 97/19694 Pcr/uss6/l8806 The ophthalmic preparations of the present invention, particularly topical prepa~alions, may include other components, for example ophthalmically acce~t~ble preservatives, tonicity agents, cosolvents, wetting agenls, complexing agents, buffering agents, antimicrobials, antioxidants and surfactants, as are 5 well known in the art. For example, suitable ~onicity enhancing agents includealkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol and the like. Sufficient tonicity enhancing agent is advantageously added so that the formulation to be instilled into the eye is hypotonic or subalar,lially isotonic. Suitable preservatives include, but are not limited to, benzalkonium 10 chloride, thimerosal, phenethyl alcohol, methylparaben, propyl,u~ldben, chlo,l,exi~i.le, sorbic acid and the like. Hydrogen peroxide may also be used aspreservative. Suitable cosolvents include, but are not limited to, glycerin, propylene glycol and polyethylene glycol. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta -cyclodextrin or hydroxypropyl- beta -15 cyclcdextrin. Suitable surfactants or wetting agents include, but are not limitedto, sorbitan esters, polysorbates such as polysorbate 80, troilletl,&l"ine, lecithin, cholesterol, tyloxapol and the like. The buffers can be conventional buffers such as borate, citrate, phosphate, bicarbonate, or Tris-HCI.
The formulation components are prese!nt in concenllalions that are 20 ~cep~Allle to the extraocular or intraocular site of administration. For example, buffers are used to maintain the composition at physiological pH or at slightly lower pH, typically within a pH range of from about 5 to about 8.
Additional formulation components may incllJde materials which provide for the prolonged ocular residence of the extraocularly administered therapeutic25 agent so as to maximize the topical contact and promote absorbtion. Sui'?~blQmaterials include polymers or gel forming materials which provide for increased v;3cosily of the ophthalmic preparation. Chitosan is a particularly suitable material as an ocular release-rate controlling agent in sustained release liquidophthalmic drug formulations (see U.S. 5,422,116, Yen, et. al.) The suitability 30 of the formulations of the instant invention for controlled release (e.g., sustained and prolonged delivery) of an ophthalmic treating agenl in the eye can be clete,l"ined by various procedures known in the art, e.g., as described in Journal of Controlled Release, 6:367-373, 19~7, as well as variations thereof.
Yet another ophthalmic preparation may involve an effective quantity of 35 GDNF protein product in a mixture with non-toxic ophthalmically acce~ ;P~!e excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other appropriate vehicle, ophthalmic solutions can CA 022361~7 1998-0~-21 WO 97/196g4 Pcr/us96/18806 be prepared in unil dose form. Suitable excipienls include, but are nol limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lul,licaling agents such as magnesium stearale, stearic acid. or talc D. ~.lnlinisl~ation/Delivery of (~DNF Protein Product The GDNF protein product may be administered parenterally via a subcutaneous, inlramuscular, intravenous, transpulmonary, transdermal, intrathecal or illl,acerebral route. For the treatment of ophthalmic conditions,10 the GDNF protein product may also be advantageously administered extraocularly or intraocularly, as described above, by topical a~l.calion, inserts, injection,i"lpla"ls, cell therapy or gene Iherapy. For example, slow-releasing i",plan~
containing the neurotrophic factor embedded in a biodegradable polymer matrix can deliver GDNF protein product. GDNF protein product may be admi";slered 15 exlfacerebrally in a form that has been modified chemically or packaged so that it passes the blood-brain barrier, or il may be administered in connection with one or more agents capable of promoting penetration of GDNF protein product across the barrier. Similarly, the GDNF protein product may be administered intraocularly, or it may be administered extraocularly in connection with one or20 more agents capable of promoting penetration or transport of GDNF protein product across the me,llbranes of the eye. The frequency of dosing will depend on the pha""acokinetic parameters of the GDNF protein product as formulated, and the route of administration.
The specific dose may be calculated according to considerdlions of body 25 weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in theart and is within the ambit of tasks routinely pe,lc,r",ed, especially in light of the dosage inforrnation and assays r~isclosed herein. Approp, iale dos~es may be30 asce,lailled through use of the estatjl;shed assays for determining dos~ges utilized in conjunction with appropriate dose-response data. According to the currently pfe~er,ed en-bodi."ents of the present invention, the GDNF protein product is most advantageously a tm;.~;slered intraocutarly at a dose between about 0.001 mg/day and 10 mg/day, and preferably at a dose between about 35 0.01 mg/day and 1 mg/day, and most preferably at a dose between about 0.1 mg/day and 0.5 mg/day. It will be appreciated by those skilled in the art that the dosage used in intraocularly administered formulations will be CA 022361~7 1998-0~-21 rninuscule as compared lo that used in a systemic injection or oral administration .
The final dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician 5 conside,il,g various factors which modify the action of drugs e.g. the age, condition, body weight sex and diet of the patient the severity of any infectiontime of administration and other clinical factors. As studies are conducted further infor",dlion will emerge rega,ding the appropriate dosage levels for thetreatment of various diseases and conditions.
It is envisioned that the continuous administration or sustained delivery of GDNF may be advantageous for a given treatment. While continuous a~l"in;~l,dlion may be accomplished via a mechanical means such as with an infusion pump, it is contemplated that other modes of continuous or near continuous adl"i.);sllalion may be p(acliced. For example chemical derivatization or encapsulation may result in sustained release forms of the protein which have the effect of continuous presence in predictable amounts based on a determined dosage regimen. Thus GDNF protein products include proteins derivatized or otherwise formulated to effectuate such conlinuous administration .
GDNF protein product cell therapy e.g. intraocular i",planlai:cn of cells producing GDNF protein product is also conte",~lated. This embodiment wouW
involve i""~lanting into patients cells aF~!e of synthesizing and secrelil-g a biologically active form of GDNF protein product. Such GDNF protein product-producing cells may be cells that are natural producers of GDNF protein product (analogous to B49 glioblastoma cells) or may be reco",b nanl cells whose ability to produce GDNF protein product has been augmented by l.ansf~".-ation with a gene encoding the desired GDNF protein product in a vector suitable for promoting its e,~,uression and secretion. In order to minimize a po1ential immuncl~cal reaction in patients being admi~islered GDNF protein product of a foreign species it is preler,~d that the natural cells producing GDNF
protein product be of human origin and produce human GDNF protein product.
Likewise it is prefer-ed that the recombinant cells producing GDNF protein product be l,anslor",ed with an expression vector containing a gene encoding a human GDNF protein product. Ill,planled cells may be encarsu~ted to avoid infil~,dlion of surrounding tissue. Human or non-human animal cells may be implanted in patients in biocompatible semipermeable polymeric enclosures or ",~",br;~l,es that allow release of GDNF protein product but lhat prevent CA 022361~7 1998-0~-21 wo 97/19694 Pcrluss6/l88o6 destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue. Such an implant, for example. may be attached to the sclera to produce and release GDNF protein product directly intothe vitreous humor.
It is also contemplated that the patient's own cells may be l.dn~forl"ed ex t~ivo to produce GDNF protein product and would be directly implanted without enoal~su~ Qn. The cells would be transformed with an ap~rupriale vector and ~,ar,~ lanled back into the patient's retina where they would produce and release the desired GDNF protein or GDNF protein variant.
Photoreceptor cell l,dnsplantation studies designed to replace defective or lost cells due to retinal disease or damage have been performed successfully in animal models of retinal degeneration (Silverman and Hughes, Invest.
Ophthalmoi. Vis. Sci.. 30:1684-1690, 1989; Gouras et al., Neuro-Oph~haimol., 10:165-t76, 1990). It is contemplated that photoreceptor cells may be 15 obtained from donor eyes and maintained in culture as desc~ ed herein. The cells would then be used as a source of purified photoreceplor~ to be t,ansplanted via the subretinal space into the retina of patients suffering from retinal disease or da",age. These patients will be treated with immunosuppressive therapies to eliminate immunological responses and rejection of the grafted cells. The ex vivo 20 donor retinas will be cultured in the presence of GDNF, in order to enhance their growth and survival. The patients that will receive photorecep:or cell ~ansplants will be treated with intravitreal injections of GDNF, that will be needed to promote the survival and the maturation of the grafted photo.eceptor~.GDNF protein product in vivo gene therapy is also envisioned. by 25 introducing the gene coding for GDNF protein product into targeted cells via local injection of a nucleic acid construct or other d~.propriate delivery vectors.
(Hefti, J. Neurobiol., 25:1418-1435, 1994). For example, a nucieic acid sequence encoding a GDNF protein product may be contained in an adeno-~csoc:~led virus vector or adenovirus vector for delivery to the retinal 3û cells. Alternative viral vectors inc1ude, but are not limited to, retrovirus,herpes simplex virus and papilloma virus vectors. Physical transfer, either in vivo or ex vivo as appropriate, may also be achieved by liposome-mediated transfer, direct injection (naked DNA), receptor-mediated transfer (ligand-DNA
complex), electroporation, calcium phosphate precipitation or microparticle 35 bG,.IL,afd",ent ~gene gun).
The methodology for the membrane encarsulation of living cells is familiar to those of ordinary skill in the art, and the preparation of the CA 022361~7 1998-0~-21 encP~rsu~ated cells and their implanlation in patients may be accomplished without undue experimentation. See, e.g., U.S. Patent Numbers 4,892,538, 5,011,472, and 5,106,627. each of which is specifically incorporated herein by reference. A system for enc~rsul~ting living cells is described in PCT
Applioalion WO 91110425 of Aebischer et al., specifically incorporated herein by reference. See also, PCT A~pllc2 -~n WO 91~10470 of Aebischer et al., Winn et al.. Expsr. NeuroJ., 113:322-329, 1991, Aebischer et al., Exper. Neurol., 111:26~-275, 1991; Tresco et al., ASAIO, 38:17-23, 1992, each of which is sp~ifically incorporated herein by reference. Additional implantable devices are described in WO 93121902 (International Application No.
PCT/US93103850) which is incorporated herein by reference. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible particles or beads and depot injections, are also known to those skilled in the art.
It should be noted that the GDNF protein product formulations desc,iLed herein may be used for veterinary as well as human applicalions and that the term "patient" should not be construed in a limiting manner. In the case of veterinary a~ F'i~tians, the dosage ranges should be the same as speci~ied above.
Other aspects and advantages of the present invention will be under~lood upon considerat,on of the ~oll~ ,;ng illustrative examples. The examples addressthe effect of GDNF protein product on both normal and mutated retinal neurons.
In addition. the examples set forth unique techniques for culturing retinal cells.
EXAMPLES
MATFP'AI .S AND MErHODS
The materials used in the following Examples were obtained as follows.
Cell Culture Mer~i~
High glucose Dulbecco's Modified Eagle's Medium (DMEM; #11965-092), Ham's F12 medium (F12; #11765-021), Leibovitz~s L15 medium without sodium bicarbonate (#41300-039); B27 medium supplement (#17504-010), penicillinlstreptomycin (#15070-014), L-glutamine (#25030-016), Dulbecco's phosphate-buffered saline (D-PBS; #14190-052), Hank's balanced salt solution with calcium and magnesium salts (HBSS;
CA 022361~7 1998-0~-21 #24020-026), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; #15630-015~, mouse laminin (#23017-015), bovine serum albumin and fractionV (#110-18-017) were all from GIBCO/BRL, Grand Island, NY. Heat-inactivated horse serum was from HyClone, Logan, Utah. Poly-L-ornithine hydrobromide (P-3655), bovine insulin (1-5500), hùman transferrin (T-2252), putrescine (P-6024), progesterone (P-6149) and sodium selenite (S-9133) were all from Sigma Chemical Company, Saint-Louis, MO. Papain, deoxyribonuclease I (DNAase) and ovalbumin (Papain ~issociation system) were from Worthington Biochemicals, Freehold, NJ. Falcon sterile g6-well microplates (#3072), tissue culture plastic ware and polypropylene centrifuge tubes were from Beckton-Dickinson, Oxnard, CA. Nunc Lab-Tek tissue culture chamber coverglasses (#136439) were from Baxter, Irvine, CA.
Nitex 20 llm nylon mesh (#460) was from Tetko, Elmsford, NY. The 4"
dissecting forceps and 4" dissecting scissors were from Roboz Surgical, Washington, DC.
Antibodies. Radioicotopes and Rel~ted Re~n~
Polyclonal rabbit antibody and mouse monoclonal rho4D2 anti-bovine rhodopsin antibodies were from the University of British Columbia, Vancouver, Canada. The polyclonal rabbit antibody was directed to the following synthetic peptide sequence of the rod-specific protein arreslin: Val-Phe-Glu-Glu-Phe-Ala-Arg-Gln-Asn-Leu-Lys-Cys (SEQ ID NO:2). Biotinylated horse anti-mouse IgG, biotinylated goat anti-rabbit IgG, peroxidase-conjugated avidin/biotin con~plex and Texas Red-conjugated streptavidin (ABC Elite; kit PK-6100) were from Vector Laboratories, Burlingame, CA. Fluorescein isothiocyanate con,ug~ted rabbit anti-mouse immunoglobulins were from Dako Corporation (Carpinteria, CA). 3',3'-diaminobenzidine was from Cappel Laboratories, West Chester, PA. Superblock blocking buffer in PBS (#37515) was from Pierce, Rockford, IL. Triton X-100 (X100), Nonidet P-40 (N6507) and hydrogen peroxide (30%, v/v; H1009) were from Sigma. L-13,4-3H]-Glutamic acid (NET-490; 40-80 CiJmmol) was from New England Nuclear, Boston, MA.
Optiphase Supermix scintillation cocktail was from Wallac, Turku, Finland.
White ViewPlate-96 microplates (#600~182) were from Packard Instruments Corporation, Meriden, CT. All other reagents were obtained from Sigma Chemical Company (Saint-Louis, MO), unless otherwise specified.
CA 022361~7 1998-0~-21 WO g7/196g4 PCr/USg6/18806 - 3~ -Prep~r~tion of Media A basal medium was prepared as a 1:1 mixture of DMEM and F12 medium, and was supplemented with B27 medium supplement added as a 50-fold concentrated stock solution. The B27 medium supplement consials of biotin, L-5 carnitine, corticosterone, ethanolamine, D(+)-galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, selenium, T3 (triodo-l-thyronine, DL-alpha-tocopherol (vitamin E), DL-alpha-tocopherol acetate, bovine serum albumin, cat~l~ce~ insulin, superoxide dismutase and transferrin. L-glutamine was added at a final concentldl:on of about 2 mM, penicillin at about 100 IUII, and streptomycin at about 100 mg/l.
Heat-inactivated horse serum was added to a final concer,lralion of about 2.5 percent, D-glucose was added to a final concentration of about 5 9/l, HEPES
buffering agent was added to a final concentration of about 20 mM, bovine insulin was added to a final concentration of about 2.5 mglml, and human ~,ansler-in 15 was added to a final concehl-alion of about 0.1 mg/ml. After mixing, the pH was adjusted to about 7.3 and the medium was kept at 4~C. The media were p(epare~l fresh just before use in order to minimize inter-experimental variations.
Plastic pipettes and containers were used throughout to minimize protein adsorption.
,DNF Protein Product Solutions Purified human recombinant GDNF protein products were prepared as 1 mg/ml solutions in D-PBS (phosphate buffered saline prepared with distilled water) conlail~ing five percent bovine serum albumin. The solutions were stored 25 at -85~C in aliquots. Serial dilutions were prepared in 96-well microplates.
Ten microliters of ten-fold concentrated GDNF protein product solutions were added to cell cultures containing culture medium (90 1l1). Control cultures received D-PBS with 5 percent albumin (10 ~11). The GDNF protein product treatments were initiated one hour after cells were seeded and, in some 30 i"~lances, repeated every other day.
Cultllre Substratum To encourage optimal attachment of photoreceptors on substratum, outer segment outgrowth and neurite outgroJ/Ih, the microtiter plate surfaces (the 35 culture substratum) were modified by sequentiat coating with poly-L-ornithinefbl'~ ed by laminin in accordance with the following procedure. The plate surfaces were completely covered with a 0.1 mglml sterile solution of CA 022361~7 1998-0~-21 polyornithine in 0.1 M boric acid (pH 8.4) for at least one hovr at room temperature, followed by a sterile wash with Super-O water. The water wash was then aspirated and a 1 ,ugtml solution of mouse laminin in PBS was added andinc~ ed at 37~C for two hours. These procedures were conducted just before using the plates in order to ensure reproducibility of the results.
Prer~r~tion of Chick and Mouse Photorece~tor Cultllres Seventeen-day-old White Leghorn chick embryos and 5-day-old C57BI16 mouse pups (obtained from Jackson Laboratories, Bar Harbor, Maine) were killed by decapitation and the eyes were dissected sterilely into L15 medium (without sodium bicarbonate). A maximum of 24 eyes were processed per ex, eri",ent. The eyes were hemisected, and the lens and vitreous were removed.
The neural retinas were carefully removed and dissected free of the pigment epithelium, cut into small (about 1 s~uare mm or less) fragments and placed intoice-cold D-PBS. The cells were collected, and then transferred into 10 ml dissoc;~lion medium (120 units papain and 2000 units DNAase in HBSS). The cells were incubated for 45 minutes at about 37~C on a rotary pldlkJr", shaker at about 200 rpm. The cells were then dispersed by tritùration through fire-polished Pasteur pipettes, sieved through a 20 llm Nitex nylon mesh to discard undissoci~ted tissue, and centrifuged for five minutes at 200 x 9 using an IE
clinical centrifuge. The resulting cell pellet was resuspended into HBSS
containing ovalbumin and about 500 units DNAase, layered on top of a 4 percent ovalbumin solution (in HBSS) and centrifuged for about 10 minutes at 500 x 9.
The final pellet was resuspended in complete culture medium (see above), adjusted to about 15,000 cells/ml, and seeded in 90 1l1 aliquots into the 6 mm-wells of 96-well microplates previously coated with polyornithine and laminin.
Attachment of cells occurred rapidly, and the plating efficiency was about 75 percen{.
Cultures of photoreceptors from adult mouse retina Cultures of adult photoreceptors were obtained by seeding dissociated retinal cells from post-natal day 18 to 39 mice on top of pre-established monolayers of post-natal day 5 mouse retinal glial cells or rat retina pigment epithelium cells. Dissociation procedures and culture media were the same as described above. Cultures of retinal glial cells and pigmenl epithelium cells were established in tissue culture flasks (225 crn2 Costar flasks) and grown until confluence was reached. The cells were then detached by a short (about two CA 022361~7 1998-0~-21 Wo 97/19694 Pcr/uss6/18806 minute) incubation with 0.1% trypsin and plated in 96-well microplates or 16-well glass coverslip chambers. Dissociated adult retinal cells were added after about 3 to 5 days.
Cultures of photoreceptors from rd/rd mouse retina Rd/rd C57BI16 mice (obtained from Jackson Laboratories, Bar Harbor, Maine) have an inherited photoreceptor degeneration resulting from the ex~J,ession of a mutation in the beta subunit of phos,uhodiesterase tan enzyme locz~;~e~ in the outer segments and involved in the photol,dnsduction processes~.
These mice provide a useful model to study the role of trophic factors on lesioned pholoreceFt,rs. Photoreceptor death in rd/rd mice peaks at around 10 days after birth. Cultures of rd/rd photoreceptors were established from 5-day-old mice and maintained in cultures for eight days, covering ~he period of maximal photoreceptor death. The dissociated retinal cells were seeded on top of a pre-established monolayer of retinal glial cells (see above) at a density of about 10,000 cells per 6-mm well and were maintained in the culture medium desc-ibed above.
Immunohistochemistry of Photoreceptors To characterize mouse photoreceptors, an indirect immunoperoxidase method described by Louis et al. (J. Pharmacol. Exp. Therap., 262:1274-1283, 1992; Science, 259:689-692, 1993) was used, with slight modifications as follows. Cul~ures of photoreceptors were fixed for about 30 minutes at room te",perall~re with 4 percent paraformaldehyde in D-PBS, pH 7.4, followed by three washes in D-PBS (200 111 per 6-mm well). The fixed cultures were then incub~ted in Superblock blocking buffer in PBS, containing one percent Nonidet P-40 to increase the penetration of the antibodies. The anti--l,odopsi"
antibodies (rabbit and mouse) were then applied at a dilution of between 1:1000-1:4000 in the same buffer, and the cultures were incubated for one hour at 37~C on a rotary shaker. After three washes with D-PBS, the photorecPF~,r-bound antibodies were detected using goat-anti-rabbit or horse-anti-mouse biotinylated IgG (Vectastain kit from Vector Laboratories, Burlingame, CA) at about a 1:500 dilution: these secondary an~iL~d!~s were inc~b~ted with the cells for about one hour at 37~C, the cells were then washeclthree times with D-PBS. The secondary antibodies were then labeled with an avidin-biotin-peroxidase complex diluted at 1:500, and the cells were incubated for about 45 minutes at 37~C. After three more washes with D-PBS, the labeled CA 022361~7 1998-0~-21 cell cultures were reacted for 5-20 minutes in a solution of 0.1 M Tris-HCI, pH
7.4, containing 0.04% 3',3'-diaminobenzidine-(HCI)4, 0.06 percent NiCI2 and 0.02 percent hydrogen peroxide.
For double staining experiments, the cultures were grown on glass 5 coverslip chambers. After paraformaldehyde fixation, permeah~ tion and blocking of the non-specific sites (as described above), the cultures were incubated with rabbit anti-arrestin and mouse anti-rhodopsin antibodies.
Arrestin was revealed by further incubation with biotinylated goat anti-rabbit IgG, hllowed by Texas-Red oon,ug~ted streptavidin (1:200 dilution~. nhodopsil) 10 was revealed by further incubation with fluorescein isothiocyanate-conjug~tedrabbit anti-mouse IgG. Fluorescence was visu~lized under epifluorescence, using the appropriate filter combinations for Texas Red and fluorescein.
Determininq Photoreceptor Surviv~l Mouse photoreceptor cultures were fixed, processed and immunostained as described above, and the photoreceptor cultures were then examined with bright-light optics st 200X magnification. The number of stained neurons was counted in one diametrical 1 X 6 mm strip, representing about 20 percent of the total surface area of a 6 mm-well. Viable photoreceptors were char~cteri~ed as having a regularly-shaped cell body, with a usually short axon-like process.
Photoreceptors showing signs of degeneration, such as having irregular, vacuolated perikarya or fragmented neurites, were excluded from the counts (most of the degenerating photoreceptors, however, detached from the culture substratum). Cell numbers were expressed either as cells/6-mm well or as the fold-change relative to control cell density.
Neurite An~b~sis M~r~ho",et,ic analysis of neurite (i.e., the process at the photoreceptor cell body) development was performed using 6-day-old cultures of mouse retina.
Cultures con~ainin~ about 10,û00 neurons per 6-mm well were immunostained for arrestin and examined with brightfield optics. Photographs of randornly chosen fields of photoreceptors in control and treated 6-mm well cullures were taken with an Optronics video-camera and enlarged to a final magr, 'ioalion of approximately 800-fold. Neuritic size was determined by measuring the length of the neurites of each photoreceptor with a stylus coupled to a SummaSketchll digitizing tablet (Summagraphics Corporation, Houston, TX), utilizing a CA 022361~7 1998-0~-21 Wo 97/19694 PCT/US96/18806 digitlzing program (MacMeasure 1.9t and a Maclntosh Centris 650 personal computer.
pF.C~I 11 TS
Example 1 Promotion of rod photoreceptor survival and development in cultures of post-natal mouse retina.
Cultures of mouse retinas were used to demonstrate the effect of GDNF
protein product on photoreceptor survival. The photoreceptor cultures were established by seeding dissociated retinal cells into polyornithine-laminin-coated microplates at a density of about 12,500 per 6-mm well in DMEM/F12 supplemented with B27 medium supplemenl, 2.5% heat-inactivated horse 15 serum, D-glucose, HEPES, insulin and transferrin. Photoreceptors were idenlilied by the presence of arrestin (a rod-specific antigen) and Ihodopsii, (the rod-specific visual pigment) immunoreactivities.
After 6 days in vi~ro the cells were fixed with 4% paraformaldehyde, and photoreceptors in the cultures were immunostained using arrestin, a marker 20 that identifies mammalian rod photoreceptors. After immunoslai";.,g, as described above, phase-contrast micrography of a field selected for the presenceof d;f~erenl retinal cell types revealed photoreceptors (identifiable as small cells covered by a brown reaction materiai after immunostaining for arrestin), neurons and Mueller glial cells. Bright-field examination of a defined field 25 demonstrated that the anti-arrestin antiserum, raised in rabbit against an arrestin-specific synthetic peptide, exclusively bound to rod photoreceptors anddid not bind to other retinal neurons, or Mueller glial cells.
Based on arrestin-immunoreactivity, it was determined that about 90 percent of the cells in the cultures were photoreceptors. The remaining cells 30 were large multipolar and smaller unipolar NSE-positive neurons. The cells were then immunostained for rhodopsin. About 50% of the photor~ceFt~s expressed the rod visual pigment rhodopsin, as determined by immunostaining with lhe mouse monoclonal anti-rhodopsin anlil,ody. Photoreceptors appeared as rounded cells with a small cell body diameter, one or two neurites and, in some 35 cases, a short vertical process that ,ep~eser,l~ the connecting cilium. At this level of resolution, there was no evidence of outer segment formation.
CA 022361~7 1998-0~-21 Wo 97/19694 PCr/uss6ll88o6 Post-natal day 6 mouse retina cultures were then evaluated for the effect of GDNF protein product administration on photoreceptor survival. Cultures of photoreceptors (10,00016-mm well) were treated with human recombinant GDNF protein product (ten-fold serial dilutions ranging from 10 nglml to 1 5 pg/ml). The cultures were fixed after six days and immunostained for arrestin.Photoreceptor survival was determined by counting the number of arrestin-positive cells per 6 sq. mm fields (representing about 21% of the total surface area of a 6-mm well).
In cultures that were not treated with GDNF protein product, the number 10 of photoreceptors declined steadily over time to reach about 25 percent of the initial number after six days in culture. Treatment of the cultures with E. coliexpressed recombinant human GDNF protein product resulted in an about two-fold increase in the number of viable arrestin-positive photoreceptors after sixdays in culture (See Figure 1; each value is the mean + s.d. of three cultures.)15 The effect of GDNF pro~ein product was maximal al aboul 200 pg/ml, with an ED50 of about 30 pg/ml.
In addition to promoting photoreceptor survival, the addition of the GDNF
protein product also stimulated the extension of their axon-like prucess (further ~ er-ed to as neurite), thereby demonstrating an effect on the ",G",ho'ogical 20 development of the photoreceptors. Cultures of photoreceptors were incl-h~ted for six days with or without recoi"binant human GDNF protein product (1 nglml). The cultures were then immunostained for arrestin. About 715 photoreceptors from two independent control cultures and 710 photoreceptors from two independent GDNF protein product-treated cultures were photographed 25 and analyzed for neurite lengths. The effect of GDNF protein product admi"ial,alion on neurite outgrowth was quantified by measuring neurite lengths of the photoreceptors. Figure 2 depicts the promotion of photorsceptor neurite outg.~ tl, by GDNF protein product. The data are expressed as a cumulative frequency distribution plot of the neurite lengths. The percentage of 30 photoreceplors (ordinate) with neurites longer than a given length in micrometers (abscissa) is plotted. The addition of GDNF protein product shifted the distribution of neurite lengths to higher values compared with untreated cultures. Some photoreceptors in the GDNF protein product-treated cultures displayed neurites about 180 llm in length, whereas the longest neurites 35 observed in untreated cultures were 100 llm in length. The mean neurite length of photoreceptors in GDNF protein product-treated cultures was 68 llm, co"",afed to 27 llm in control cultures.
CA 02236l~7 l998-0~-2l Pho1Oreceptors utilize glutamate as their neurotransmitter to signal to second order neurons. In cultures consisting of ~g0% photoreceplor~, the degree of glutamate uptake by the cells indicates the number and activity of high-5 affinity glutamate reuptake transporter sites present on photoreceptors andthereby reflects their functional differentiation. The stimulation of glutamate uptake by GDNF protein product admin;slf~tion was evaluated to assess its effects on photo,l:ceptor functional differentiation. Cultures were grown as described above and were either untreated or treated with recombinant human GDNF
10 protein product for six days. Cultures were then processed for ~3Hl-glutamateuptake (50 nM; 1.5 million dpm/ml; one hour incubation at 37~C) in accordance with the following procedure.
m~te Upt~ke As~y: Glutamate uptake was determined in cultures of photoreceptors from 5-day-old mouse pups that had been established in 96-well 15 ~;c~upla~es The cultures were washed with about 100 1ll of pre-warmed uptake buffer which consists of a modified Krebs-Ringer solution, pH 7.4 containing about 120 mM NaCI, 4,7 mM KCI, 1.8 mM CaCI2, 1.2 mM MgSO4, 32 mM
NaHPO4, 1.3 mM EDTA, and 5.6 mM D-glucose. The cells were then preincub~ted at 37~C for about 10 minutes in uptake buffer. Tritiated L-20 glutamate (about 60 Ci/mmol) was then added to the cultures at a conce~ alionof about 50 nM in 75 1ll of uptake buffer and the cultures were incubated for about 60 minutes at 37~C. The uptake was a,(esled by aspiral;on of the incubation medium followed by three rapid washes with about 120 1ll of ice-cold uptake buffer. The cells were then Iysed by addition of 200 ~11 of Optiphase 25 Supermix scintillation cocktail (Wallac), and radioactivity was determined byscintillation spectrometry using a Wallac MicrobetaPlus 96-well microplate counter. The results are expressed either as dpm/6-mm well or as the fold-change relative to control cultures.
The GDNF protein product was found to stimulate glutamate uptake in a 30 dose-dependent fashion. with maximal activity reached at about 200 pg/ml and an ED50 of about 2~ pglml. The results are illustrated in Figure 3. Each data point is the mean + s.d. of 3 wells from a representative experiment. Similar results were obtained in two independent experiments. The results demonstrate that in addition to promoting the survival and morphological development of 35 photoreceptors, GDNF enhances the maturalion of neurotransmission-related functions, such as glutamate uptake, that are critical to the visual transduction p-~cess.
CA 022361~7 1998-0~-21 Example 2 Promotion of rod photoreceptor survival and regeneration in cultures of adult mouse retina.
Photoreceptor development is complete at about three weeks after birth.
By this time, photoreceptors have developed functional outer segments that concentrate the cellular machinery necessary for phototransduction, including the visual pigments. Mature rat photoreceptors were dissociated from 18- and 10 39-day old retinas and maintained in culture for over a week. The neurons were seeded (at a density of about 2,500/6-mm well ) on top of a pre-existing monolayer of retinal glial cells. Glial cells encourage adhesion of dissoci~ed photoreceptors and provide them with nutrients and factors essential for their development. Adult photoreceptors co-cultured with retinal glial cells were 15 identified by double-immunostaining for arrestin and rhodopsin using lhe antibodies and immunostaining techniques described above.
Cultures were treated with recombinant human GDNF protein product (0.1, 1 or 10 ng/ml). The cells were fixed after seven days and immunostained for arrestin. Photoreceptor survival was determined by counting the number of 20 arrestin-positive neurons per 6-mm well. The number of rod photoreceptors in cultures of both 18- and 39-day old retinas was about 3.5-fold higher in cultures treated with GDNF protein product (see Figure 4; each value is the mean + s.d. of 2-3 cultures). Maximal support was found with GDNF protein product concentrations of about 3ûO pg/ml, with an ED50 of about 40 pg/ml.
25 These results illustrate the changes in photoreceptor number, and thus, the promotion of photoreceptor survival in response to treatment with GDNF protein product.
In a further study, dissociated retina cells were seeded on top of a pre-established monolayer of mouse retina glial cells (1000 retina cells/6-mm 30 well) and treated with recombinant human GDNF protein product (1 or 10 nglml). The cultures were fixed after seven days and immunostained for arrestin. In addition to promoting photoreceptor survival, it was found that GDNF protein product strongly enhanced morphological development of the photoreceptors as demonstrated by the outg,~ of their axonal p,ucesses and, 35 in some instances, the outgrowth of a short apical process reminiscent of an immature outer segment. These cultures originated from adult retinas in which the photoreceptors were fully developed. Since the photoreceptors lost their CA 022361~7 1998-0~-21 WO 97/19694 PCI~/US96/18806 processes during the dissociation procedure, the current data de",on~l,ate the ability of GDNF protein product to promote the regeneration of photoreceptors, and in particular promote the development of their axonal processes and outer segl"enls which are critical to the visual process. These results indicate that the 5 ad~";nislra~ion of a GDNF protein product may be a useful therapy for condi~ions in which vision is lost due to the degeneration of photoreceptors, such as senile macular degeneration, inherited retinal degenerations and other retinal dystrophies .
1 0 Example 3 Promotion of rod photoreceptor survival in cultures of retina from mice with inherited retinal degeneration (rd/rd).
Rd/rd mice carry a mutation in the beta-subunit of phosphodiesterase (an enzyme locali~ed in the outer segments and involved in the pholol,ansductionprocesses), which results in its malfunction and causes early-onset photoreceptor degeneration and the fulminant death of photoreceplor~. Mutations similar to rd/rd are found in humans and are responsible for a subset of retinitis pi~mentosa cases. Photoreceptor death in rd~rd mice peaks at around 10 days after birth. These mutant mice provide a useful model for studying the effects of GDNF protein product on the survival of rd/rd photoreceptors.
Cultures of rd~rd photoreceptors were established from 5-day-old mice and maintained in cultures for seven days, a period covering the occurrence of maximal photoreceptor death. Due to their inherent vulnerability, the dissoci~ed rd/rd photoreceptors were seeded ~at a density of about 2,500/6-mm well) on top of a pre-established monolayer of retinal glial cells (as descril,ed aboYe). Cultures of rd/rd retinas were compared to cultures of cells from normal (wild-type) mice retinas obtained from animals of the same age and processed in the same way. Cultures were treated with recombinant human GDNF
protein product (1 ng/ml~, fixed after seven days and immunostained for arrestin. Photoreceptor survival was determined by counting the number of arrestin-positive neurons per 6-mm well.
The addition of GDNF protein product caused a modest ~about 15%) but significant increase in photoreceptor number after seven days culture in vitro (see Figure 5; each value is the mean + s.d. of 3-4 cultures). In contrast, and in spile of the glial cells support, when GDNF protein product was not added thenumber of photoreceptors in cultures of rd/rd mice dropped sharply to reach CA 022361~7 1998-0~-21 wo 97/19694 PcrtUS96/18806 about 40% of wild-type photoreceptors after seven days. In the presence of GDNF protein product (1 ng/ml), the number of surviving rd/rd photoreceptors was increased by about 2.5-fold, reaching the survival levels seen in cultures of untreated wild-type photoreceptors. These data demonstrate 5 that GDNF protein product treatment made the mutant photoreceptors more resialant to the stress imposed upon them by the rd/rd mutation. This indicates that the a.ll"in;slrdlion of GDNF protein product may be useful for the treatment of inherited retinal degenerations, such as retinitis pigmentosa.
1 0 Example 4 Promotion of cone photoreceptor survival and outer segment development in cultures of embryonic chick retina.
The development of the chick visual system is much more p~ecociolJs than in rodents. Photoreceptor outer segment outgrowth starts at about 11-12 days of ~gest~t;onal age, and at birth the photoreceptors are fully doveloped.
Therefore, the effect of GDNF protein product administration on pholoreceptor survival and regeneration can be studied in cultures of embryonic chick retinas.Cultures of embryonic day 17 chick retina cells were grown in 96-well microplates, as described above, and fixed with 4% paraformaldehyde after six days in vitro. The cultures were found to contain about 60 percent photoreceptorcells and 40 percent large multipolar neurons. Phase-contrast micrographs of a representative field of the controt cultures revealed the two major types of retinal cells present in the cullure: cone photoreceptors, idenliliable by the presence of a lipid droplet in the apical part of the cell soma, and retinal neurons. The photoreceptor cells were identified by oval cell bodies that were occupied almost exclusively by the nucleus, a shorl inner segment with a small lipid droplet, a single short, unbranched neurite emerging from a point op~.ositc to the lipid droplet, and a short distal cilium. These features are characteristic of cones. Anti-rhodopsin immunostaining was performed, as described above, and bright-field micrography of a 6-day-old culture revealed the presence of rod photoreceptors. It was determined that about 20% of the photoreceptors were rods. The remaining 80% of the photoreceptors were cone photoreceptors, that do not contain rhodopsin.
Figure 6 depicts the effect of GC)NF protein product on photoreceptor survival in cultures of embryonic day 17 chick retina. Cultures of dissociated retina cells (plated at a density of about 10,000t6-mm well) were treated with CA 022361~7 1998-0~-21 recombinant human GDNF prolein product (ten-fold serial dilutions ranging from 10 nglml to 1 pglml). The cultures were fixed after six days with 4%
paraformaldehyde and observed under phase-contrast optics. Cone photoreceptors were identified by the presence of a phase-bright lipid droplet.
5 The lipid droplet marks the junction between the inner and outer segments.
Photoreceptor survival was determined by counting the number of cones per 6 sq. mm ~ia",elrical strips (representing about 21% of the total area of a 6-mm well). Each value is lhe mean + s.d. of 3 cultures. The number of cones found inthe chick retina cultures was about two-fold higher in the BDNF protein 10 product-treated cultures than in untreated cultures. The maximal GDNF protein product effect was observed at about 200 pg/ml, with an ED50 of about 50 pg/ml.
Photoreceptor cell morphology was evaluated by phase-contrast micrography, and GDNF protein product was found to promote the development of 15 both the inner segments and outer segments and of the axonal process. In contrast to untreated cultures, cultures treated with GDNF protein product (1 ng/ml for seven days) contained a large proportion of cone photoreceptors which a~eared as highly elongated, polarized, compa.l",entalized cells. These cones had an elongated inner segment that was in some cases connected to a tri-dimensional.
20 phase-bright structure characteristic of an outer segment. Other cones developed a thick, long and branched neurite. In some instances, double cones extending two outer segments (typical of avian relinas) were observed in GDNF
protein product-treated cultures. In addition to the cell survival/proliferationeffects, the effect of GDNF protein product administration on outer segment 25 development in the chick cultures demonstrates its ability to promote the regeneration of outer segments damaged by the dissociation procedure. This in turn indicates that GDNF protein product administration would also useful in thetreatment of retinal dystrophies, in addition to inherited retinal degenerative conditions and retinopathies.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of the presently preferred embodiments thereof.
Consequently, the only limitations which should be placed upon the scope of the 35 present invention are those which appear in the appended claims.
W O 97/19694 PCT~US96/18806 SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT: Jean-Claude Louis (ii) TITLE OF INVENTION: METHODS FOR TREATING PHOTORECEPTORS
USING GLIAL CELL LINE-DERIVED NEUROTROPHIC
FACTOR (GDNF~ PROTEIN PRODUCT
(iii~ NUMBER OF SEQUENCES: 2 ~iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: AMGEN INC.
(B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D~ STATE: California ~E) COUNTRY: United States of America (F) ZIP: 91320 (v~ COMPUTER READABLE FORM:
(A~ MEDIUM TYPE: Floppy disk ~B) COMPUTER: IBM PC compatible ~C) OPERATING SYSTEM: PC-DOS~MS-DOS
~D) SOFTWARE: PatentIn Release #1.0 Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
~C) CLASSIFICATION:
~viii) ATTORNEY~AGENT INFORMATION:
~AJ NAME: Curry Daniel R.
~B~ REGISTRATION NUMBER: 32 727 (C~ REFERENCE~DOCKET NUMBER: A-363 ~ix) TELECOMMUNICATION INFORMATION:
tA) TELEPHONE: 805-447-8102 ~B~ TELEFAX: 805-499-8011 ~C~ TELEX:
~2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 134 amino acid residues (B) TYPE: amino acid ~D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: inferred amino acid sequence for mature human GDNF
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ser Pro Asp Lys Gln Met Ala Val Leu Pro Arg Arg Glu Arg Asn Arg ~ln Ala Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile His Leu Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr A~p ~ys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp Lys ~5 90 95 ~al Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser ~he Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser Ala Lys Arg Cys Gly Cys Ile (2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: synthetic peptide sequence of the rod-specific protein arrestin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Val Phe Glu Glu Phe Ala Arg Gln Asn Leu Lys Cys
For double staining experiments, the cultures were grown on glass 5 coverslip chambers. After paraformaldehyde fixation, permeah~ tion and blocking of the non-specific sites (as described above), the cultures were incubated with rabbit anti-arrestin and mouse anti-rhodopsin antibodies.
Arrestin was revealed by further incubation with biotinylated goat anti-rabbit IgG, hllowed by Texas-Red oon,ug~ted streptavidin (1:200 dilution~. nhodopsil) 10 was revealed by further incubation with fluorescein isothiocyanate-conjug~tedrabbit anti-mouse IgG. Fluorescence was visu~lized under epifluorescence, using the appropriate filter combinations for Texas Red and fluorescein.
Determininq Photoreceptor Surviv~l Mouse photoreceptor cultures were fixed, processed and immunostained as described above, and the photoreceptor cultures were then examined with bright-light optics st 200X magnification. The number of stained neurons was counted in one diametrical 1 X 6 mm strip, representing about 20 percent of the total surface area of a 6 mm-well. Viable photoreceptors were char~cteri~ed as having a regularly-shaped cell body, with a usually short axon-like process.
Photoreceptors showing signs of degeneration, such as having irregular, vacuolated perikarya or fragmented neurites, were excluded from the counts (most of the degenerating photoreceptors, however, detached from the culture substratum). Cell numbers were expressed either as cells/6-mm well or as the fold-change relative to control cell density.
Neurite An~b~sis M~r~ho",et,ic analysis of neurite (i.e., the process at the photoreceptor cell body) development was performed using 6-day-old cultures of mouse retina.
Cultures con~ainin~ about 10,û00 neurons per 6-mm well were immunostained for arrestin and examined with brightfield optics. Photographs of randornly chosen fields of photoreceptors in control and treated 6-mm well cullures were taken with an Optronics video-camera and enlarged to a final magr, 'ioalion of approximately 800-fold. Neuritic size was determined by measuring the length of the neurites of each photoreceptor with a stylus coupled to a SummaSketchll digitizing tablet (Summagraphics Corporation, Houston, TX), utilizing a CA 022361~7 1998-0~-21 Wo 97/19694 PCT/US96/18806 digitlzing program (MacMeasure 1.9t and a Maclntosh Centris 650 personal computer.
pF.C~I 11 TS
Example 1 Promotion of rod photoreceptor survival and development in cultures of post-natal mouse retina.
Cultures of mouse retinas were used to demonstrate the effect of GDNF
protein product on photoreceptor survival. The photoreceptor cultures were established by seeding dissociated retinal cells into polyornithine-laminin-coated microplates at a density of about 12,500 per 6-mm well in DMEM/F12 supplemented with B27 medium supplemenl, 2.5% heat-inactivated horse 15 serum, D-glucose, HEPES, insulin and transferrin. Photoreceptors were idenlilied by the presence of arrestin (a rod-specific antigen) and Ihodopsii, (the rod-specific visual pigment) immunoreactivities.
After 6 days in vi~ro the cells were fixed with 4% paraformaldehyde, and photoreceptors in the cultures were immunostained using arrestin, a marker 20 that identifies mammalian rod photoreceptors. After immunoslai";.,g, as described above, phase-contrast micrography of a field selected for the presenceof d;f~erenl retinal cell types revealed photoreceptors (identifiable as small cells covered by a brown reaction materiai after immunostaining for arrestin), neurons and Mueller glial cells. Bright-field examination of a defined field 25 demonstrated that the anti-arrestin antiserum, raised in rabbit against an arrestin-specific synthetic peptide, exclusively bound to rod photoreceptors anddid not bind to other retinal neurons, or Mueller glial cells.
Based on arrestin-immunoreactivity, it was determined that about 90 percent of the cells in the cultures were photoreceptors. The remaining cells 30 were large multipolar and smaller unipolar NSE-positive neurons. The cells were then immunostained for rhodopsin. About 50% of the photor~ceFt~s expressed the rod visual pigment rhodopsin, as determined by immunostaining with lhe mouse monoclonal anti-rhodopsin anlil,ody. Photoreceptors appeared as rounded cells with a small cell body diameter, one or two neurites and, in some 35 cases, a short vertical process that ,ep~eser,l~ the connecting cilium. At this level of resolution, there was no evidence of outer segment formation.
CA 022361~7 1998-0~-21 Wo 97/19694 PCr/uss6ll88o6 Post-natal day 6 mouse retina cultures were then evaluated for the effect of GDNF protein product administration on photoreceptor survival. Cultures of photoreceptors (10,00016-mm well) were treated with human recombinant GDNF protein product (ten-fold serial dilutions ranging from 10 nglml to 1 5 pg/ml). The cultures were fixed after six days and immunostained for arrestin.Photoreceptor survival was determined by counting the number of arrestin-positive cells per 6 sq. mm fields (representing about 21% of the total surface area of a 6-mm well).
In cultures that were not treated with GDNF protein product, the number 10 of photoreceptors declined steadily over time to reach about 25 percent of the initial number after six days in culture. Treatment of the cultures with E. coliexpressed recombinant human GDNF protein product resulted in an about two-fold increase in the number of viable arrestin-positive photoreceptors after sixdays in culture (See Figure 1; each value is the mean + s.d. of three cultures.)15 The effect of GDNF pro~ein product was maximal al aboul 200 pg/ml, with an ED50 of about 30 pg/ml.
In addition to promoting photoreceptor survival, the addition of the GDNF
protein product also stimulated the extension of their axon-like prucess (further ~ er-ed to as neurite), thereby demonstrating an effect on the ",G",ho'ogical 20 development of the photoreceptors. Cultures of photoreceptors were incl-h~ted for six days with or without recoi"binant human GDNF protein product (1 nglml). The cultures were then immunostained for arrestin. About 715 photoreceptors from two independent control cultures and 710 photoreceptors from two independent GDNF protein product-treated cultures were photographed 25 and analyzed for neurite lengths. The effect of GDNF protein product admi"ial,alion on neurite outgrowth was quantified by measuring neurite lengths of the photoreceptors. Figure 2 depicts the promotion of photorsceptor neurite outg.~ tl, by GDNF protein product. The data are expressed as a cumulative frequency distribution plot of the neurite lengths. The percentage of 30 photoreceplors (ordinate) with neurites longer than a given length in micrometers (abscissa) is plotted. The addition of GDNF protein product shifted the distribution of neurite lengths to higher values compared with untreated cultures. Some photoreceptors in the GDNF protein product-treated cultures displayed neurites about 180 llm in length, whereas the longest neurites 35 observed in untreated cultures were 100 llm in length. The mean neurite length of photoreceptors in GDNF protein product-treated cultures was 68 llm, co"",afed to 27 llm in control cultures.
CA 02236l~7 l998-0~-2l Pho1Oreceptors utilize glutamate as their neurotransmitter to signal to second order neurons. In cultures consisting of ~g0% photoreceplor~, the degree of glutamate uptake by the cells indicates the number and activity of high-5 affinity glutamate reuptake transporter sites present on photoreceptors andthereby reflects their functional differentiation. The stimulation of glutamate uptake by GDNF protein product admin;slf~tion was evaluated to assess its effects on photo,l:ceptor functional differentiation. Cultures were grown as described above and were either untreated or treated with recombinant human GDNF
10 protein product for six days. Cultures were then processed for ~3Hl-glutamateuptake (50 nM; 1.5 million dpm/ml; one hour incubation at 37~C) in accordance with the following procedure.
m~te Upt~ke As~y: Glutamate uptake was determined in cultures of photoreceptors from 5-day-old mouse pups that had been established in 96-well 15 ~;c~upla~es The cultures were washed with about 100 1ll of pre-warmed uptake buffer which consists of a modified Krebs-Ringer solution, pH 7.4 containing about 120 mM NaCI, 4,7 mM KCI, 1.8 mM CaCI2, 1.2 mM MgSO4, 32 mM
NaHPO4, 1.3 mM EDTA, and 5.6 mM D-glucose. The cells were then preincub~ted at 37~C for about 10 minutes in uptake buffer. Tritiated L-20 glutamate (about 60 Ci/mmol) was then added to the cultures at a conce~ alionof about 50 nM in 75 1ll of uptake buffer and the cultures were incubated for about 60 minutes at 37~C. The uptake was a,(esled by aspiral;on of the incubation medium followed by three rapid washes with about 120 1ll of ice-cold uptake buffer. The cells were then Iysed by addition of 200 ~11 of Optiphase 25 Supermix scintillation cocktail (Wallac), and radioactivity was determined byscintillation spectrometry using a Wallac MicrobetaPlus 96-well microplate counter. The results are expressed either as dpm/6-mm well or as the fold-change relative to control cultures.
The GDNF protein product was found to stimulate glutamate uptake in a 30 dose-dependent fashion. with maximal activity reached at about 200 pg/ml and an ED50 of about 2~ pglml. The results are illustrated in Figure 3. Each data point is the mean + s.d. of 3 wells from a representative experiment. Similar results were obtained in two independent experiments. The results demonstrate that in addition to promoting the survival and morphological development of 35 photoreceptors, GDNF enhances the maturalion of neurotransmission-related functions, such as glutamate uptake, that are critical to the visual transduction p-~cess.
CA 022361~7 1998-0~-21 Example 2 Promotion of rod photoreceptor survival and regeneration in cultures of adult mouse retina.
Photoreceptor development is complete at about three weeks after birth.
By this time, photoreceptors have developed functional outer segments that concentrate the cellular machinery necessary for phototransduction, including the visual pigments. Mature rat photoreceptors were dissociated from 18- and 10 39-day old retinas and maintained in culture for over a week. The neurons were seeded (at a density of about 2,500/6-mm well ) on top of a pre-existing monolayer of retinal glial cells. Glial cells encourage adhesion of dissoci~ed photoreceptors and provide them with nutrients and factors essential for their development. Adult photoreceptors co-cultured with retinal glial cells were 15 identified by double-immunostaining for arrestin and rhodopsin using lhe antibodies and immunostaining techniques described above.
Cultures were treated with recombinant human GDNF protein product (0.1, 1 or 10 ng/ml). The cells were fixed after seven days and immunostained for arrestin. Photoreceptor survival was determined by counting the number of 20 arrestin-positive neurons per 6-mm well. The number of rod photoreceptors in cultures of both 18- and 39-day old retinas was about 3.5-fold higher in cultures treated with GDNF protein product (see Figure 4; each value is the mean + s.d. of 2-3 cultures). Maximal support was found with GDNF protein product concentrations of about 3ûO pg/ml, with an ED50 of about 40 pg/ml.
25 These results illustrate the changes in photoreceptor number, and thus, the promotion of photoreceptor survival in response to treatment with GDNF protein product.
In a further study, dissociated retina cells were seeded on top of a pre-established monolayer of mouse retina glial cells (1000 retina cells/6-mm 30 well) and treated with recombinant human GDNF protein product (1 or 10 nglml). The cultures were fixed after seven days and immunostained for arrestin. In addition to promoting photoreceptor survival, it was found that GDNF protein product strongly enhanced morphological development of the photoreceptors as demonstrated by the outg,~ of their axonal p,ucesses and, 35 in some instances, the outgrowth of a short apical process reminiscent of an immature outer segment. These cultures originated from adult retinas in which the photoreceptors were fully developed. Since the photoreceptors lost their CA 022361~7 1998-0~-21 WO 97/19694 PCI~/US96/18806 processes during the dissociation procedure, the current data de",on~l,ate the ability of GDNF protein product to promote the regeneration of photoreceptors, and in particular promote the development of their axonal processes and outer segl"enls which are critical to the visual process. These results indicate that the 5 ad~";nislra~ion of a GDNF protein product may be a useful therapy for condi~ions in which vision is lost due to the degeneration of photoreceptors, such as senile macular degeneration, inherited retinal degenerations and other retinal dystrophies .
1 0 Example 3 Promotion of rod photoreceptor survival in cultures of retina from mice with inherited retinal degeneration (rd/rd).
Rd/rd mice carry a mutation in the beta-subunit of phosphodiesterase (an enzyme locali~ed in the outer segments and involved in the pholol,ansductionprocesses), which results in its malfunction and causes early-onset photoreceptor degeneration and the fulminant death of photoreceplor~. Mutations similar to rd/rd are found in humans and are responsible for a subset of retinitis pi~mentosa cases. Photoreceptor death in rd~rd mice peaks at around 10 days after birth. These mutant mice provide a useful model for studying the effects of GDNF protein product on the survival of rd/rd photoreceptors.
Cultures of rd~rd photoreceptors were established from 5-day-old mice and maintained in cultures for seven days, a period covering the occurrence of maximal photoreceptor death. Due to their inherent vulnerability, the dissoci~ed rd/rd photoreceptors were seeded ~at a density of about 2,500/6-mm well) on top of a pre-established monolayer of retinal glial cells (as descril,ed aboYe). Cultures of rd/rd retinas were compared to cultures of cells from normal (wild-type) mice retinas obtained from animals of the same age and processed in the same way. Cultures were treated with recombinant human GDNF
protein product (1 ng/ml~, fixed after seven days and immunostained for arrestin. Photoreceptor survival was determined by counting the number of arrestin-positive neurons per 6-mm well.
The addition of GDNF protein product caused a modest ~about 15%) but significant increase in photoreceptor number after seven days culture in vitro (see Figure 5; each value is the mean + s.d. of 3-4 cultures). In contrast, and in spile of the glial cells support, when GDNF protein product was not added thenumber of photoreceptors in cultures of rd/rd mice dropped sharply to reach CA 022361~7 1998-0~-21 wo 97/19694 PcrtUS96/18806 about 40% of wild-type photoreceptors after seven days. In the presence of GDNF protein product (1 ng/ml), the number of surviving rd/rd photoreceptors was increased by about 2.5-fold, reaching the survival levels seen in cultures of untreated wild-type photoreceptors. These data demonstrate 5 that GDNF protein product treatment made the mutant photoreceptors more resialant to the stress imposed upon them by the rd/rd mutation. This indicates that the a.ll"in;slrdlion of GDNF protein product may be useful for the treatment of inherited retinal degenerations, such as retinitis pigmentosa.
1 0 Example 4 Promotion of cone photoreceptor survival and outer segment development in cultures of embryonic chick retina.
The development of the chick visual system is much more p~ecociolJs than in rodents. Photoreceptor outer segment outgrowth starts at about 11-12 days of ~gest~t;onal age, and at birth the photoreceptors are fully doveloped.
Therefore, the effect of GDNF protein product administration on pholoreceptor survival and regeneration can be studied in cultures of embryonic chick retinas.Cultures of embryonic day 17 chick retina cells were grown in 96-well microplates, as described above, and fixed with 4% paraformaldehyde after six days in vitro. The cultures were found to contain about 60 percent photoreceptorcells and 40 percent large multipolar neurons. Phase-contrast micrographs of a representative field of the controt cultures revealed the two major types of retinal cells present in the cullure: cone photoreceptors, idenliliable by the presence of a lipid droplet in the apical part of the cell soma, and retinal neurons. The photoreceptor cells were identified by oval cell bodies that were occupied almost exclusively by the nucleus, a shorl inner segment with a small lipid droplet, a single short, unbranched neurite emerging from a point op~.ositc to the lipid droplet, and a short distal cilium. These features are characteristic of cones. Anti-rhodopsin immunostaining was performed, as described above, and bright-field micrography of a 6-day-old culture revealed the presence of rod photoreceptors. It was determined that about 20% of the photoreceptors were rods. The remaining 80% of the photoreceptors were cone photoreceptors, that do not contain rhodopsin.
Figure 6 depicts the effect of GC)NF protein product on photoreceptor survival in cultures of embryonic day 17 chick retina. Cultures of dissociated retina cells (plated at a density of about 10,000t6-mm well) were treated with CA 022361~7 1998-0~-21 recombinant human GDNF prolein product (ten-fold serial dilutions ranging from 10 nglml to 1 pglml). The cultures were fixed after six days with 4%
paraformaldehyde and observed under phase-contrast optics. Cone photoreceptors were identified by the presence of a phase-bright lipid droplet.
5 The lipid droplet marks the junction between the inner and outer segments.
Photoreceptor survival was determined by counting the number of cones per 6 sq. mm ~ia",elrical strips (representing about 21% of the total area of a 6-mm well). Each value is lhe mean + s.d. of 3 cultures. The number of cones found inthe chick retina cultures was about two-fold higher in the BDNF protein 10 product-treated cultures than in untreated cultures. The maximal GDNF protein product effect was observed at about 200 pg/ml, with an ED50 of about 50 pg/ml.
Photoreceptor cell morphology was evaluated by phase-contrast micrography, and GDNF protein product was found to promote the development of 15 both the inner segments and outer segments and of the axonal process. In contrast to untreated cultures, cultures treated with GDNF protein product (1 ng/ml for seven days) contained a large proportion of cone photoreceptors which a~eared as highly elongated, polarized, compa.l",entalized cells. These cones had an elongated inner segment that was in some cases connected to a tri-dimensional.
20 phase-bright structure characteristic of an outer segment. Other cones developed a thick, long and branched neurite. In some instances, double cones extending two outer segments (typical of avian relinas) were observed in GDNF
protein product-treated cultures. In addition to the cell survival/proliferationeffects, the effect of GDNF protein product administration on outer segment 25 development in the chick cultures demonstrates its ability to promote the regeneration of outer segments damaged by the dissociation procedure. This in turn indicates that GDNF protein product administration would also useful in thetreatment of retinal dystrophies, in addition to inherited retinal degenerative conditions and retinopathies.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of the presently preferred embodiments thereof.
Consequently, the only limitations which should be placed upon the scope of the 35 present invention are those which appear in the appended claims.
W O 97/19694 PCT~US96/18806 SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT: Jean-Claude Louis (ii) TITLE OF INVENTION: METHODS FOR TREATING PHOTORECEPTORS
USING GLIAL CELL LINE-DERIVED NEUROTROPHIC
FACTOR (GDNF~ PROTEIN PRODUCT
(iii~ NUMBER OF SEQUENCES: 2 ~iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: AMGEN INC.
(B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D~ STATE: California ~E) COUNTRY: United States of America (F) ZIP: 91320 (v~ COMPUTER READABLE FORM:
(A~ MEDIUM TYPE: Floppy disk ~B) COMPUTER: IBM PC compatible ~C) OPERATING SYSTEM: PC-DOS~MS-DOS
~D) SOFTWARE: PatentIn Release #1.0 Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
~C) CLASSIFICATION:
~viii) ATTORNEY~AGENT INFORMATION:
~AJ NAME: Curry Daniel R.
~B~ REGISTRATION NUMBER: 32 727 (C~ REFERENCE~DOCKET NUMBER: A-363 ~ix) TELECOMMUNICATION INFORMATION:
tA) TELEPHONE: 805-447-8102 ~B~ TELEFAX: 805-499-8011 ~C~ TELEX:
~2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 134 amino acid residues (B) TYPE: amino acid ~D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: inferred amino acid sequence for mature human GDNF
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ser Pro Asp Lys Gln Met Ala Val Leu Pro Arg Arg Glu Arg Asn Arg ~ln Ala Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile His Leu Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr A~p ~ys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp Lys ~5 90 95 ~al Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu Ser ~he Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser Ala Lys Arg Cys Gly Cys Ile (2) INFORMATION FOR SEQ ID NO:2:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acid residues (B) TYPE: amino acid (D) TOPOLOGY: linear (ix) FEATURE:
(A) NAME/KEY: synthetic peptide sequence of the rod-specific protein arrestin (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Val Phe Glu Glu Phe Ala Arg Gln Asn Leu Lys Cys
Claims (12)
1. The use of a glial cell line-derived neurotrophic factor (GDNF) protein product for the manufacture of a pharmaceutical composition for the treatment ofinjury or degeneration of photoreceptors.
2. The use according to claim 1 wherein the injury or degeneration of the photoreceptors is associated with retinitis pigmentosa, Bardet-Biedl syndrome, Bassen-Kornzweig syndrome (abelalipoproteinemia), Best disease (vitelliform dystrophy), choroidemia, gyrate atrophy, congenital amaurosis, Refsum syndrome, Stargardt disease and Usher syndrome, age-related macular degeneration, diabetic retinopathy, peripheral vitreoretinopathies, photic retinopathies, surgery-induced retinopathies, viral retinopathies, ischemic retinopathies, retinal detachment or traumatic retinopathy
3. The use according to claims 1 or 2 wherein the pharmaceutical composition comprises a GDNF amino acid sequence set forth in SEQ ID NO:1, or a variant or derivative thereof.
4. The use according to claim 3 wherein the pharmaceutical composition comprises [Met-1]GDNF.
5. The use according to claims 1 or 2 wherein the pharmaceutical composition comprises GDNF attached to a water soluble polymer.
6. The use according to claims 1 or 2 wherein the pharmaceutical composition comprises a truncated human GDNF protein.
7. The use according to claims 1 or 2 wherein the pharmaceutical composition comprises cells which have been modified to produce and secrete the GDNF protein product.
8. The use according to claims 1 or 2 wherein the pharmaceutical composition further comprises an effective amount of a second therapeutic agent for treating retinal disease.
9. The use according to claim 8 wherein the second therapeutic agent is selected from brain derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, neurotrophin-6, insulin-like growth factor, ciliary neurotrophic factor, acidic and basic fibroblast growth factors, fibroblast growth factor-5, transforming growth factor-.beta., and ***e-amphetamine regulated transcript .
10. The use according to claims 1 or 2 wherein the pharmaceutical composition is formulated as an ocular insert, ocular injection or ocular implant.
11. A method for providing photoreceptor cells for implantation, comprising culturing dissociated photoreceptor cells in the presence of a glial cell line-derived neurotrophic factor (GDNF) protein product.
12. A composition comprising photoreceptor cells and a glial cell line-derived neurotrophic factor (GDNF) protein product in amounts to enhance the survival and allow the continued growth and maturation of said photoreceptor cells.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/564,833 US5641750A (en) | 1995-11-29 | 1995-11-29 | Methods for treating photoreceptors using glial cell line-derived neurotrophic factor (GDNF) protein product |
| US08/564,833 | 1995-11-29 | ||
| PCT/US1996/018806 WO1997019694A1 (en) | 1995-11-29 | 1996-11-22 | Methods for treating photoreceptors using glial cell line-derived neurotrophic factor (gdnf) protein product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2236157A1 true CA2236157A1 (en) | 1997-06-05 |
Family
ID=29406107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2236157 Abandoned CA2236157A1 (en) | 1995-11-29 | 1996-11-22 | Methods for treating photoreceptors using glial cell line-derived neurotrophic factor (gdnf) protein product |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2236157A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017121766A1 (en) | 2016-01-12 | 2017-07-20 | Kaleyde Pharmaceuticals Ag | Pharmaceutical formulations and their use for the treatment of retinitis pigmentosa |
-
1996
- 1996-11-22 CA CA 2236157 patent/CA2236157A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017121766A1 (en) | 2016-01-12 | 2017-07-20 | Kaleyde Pharmaceuticals Ag | Pharmaceutical formulations and their use for the treatment of retinitis pigmentosa |
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