NZ620202B2 - Pharmaceutical composition of oxidised avidin suitable for inhalation - Google Patents
Pharmaceutical composition of oxidised avidin suitable for inhalation Download PDFInfo
- Publication number
- NZ620202B2 NZ620202B2 NZ620202A NZ62020212A NZ620202B2 NZ 620202 B2 NZ620202 B2 NZ 620202B2 NZ 620202 A NZ620202 A NZ 620202A NZ 62020212 A NZ62020212 A NZ 62020212A NZ 620202 B2 NZ620202 B2 NZ 620202B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- lung
- biotinylated
- agent
- avidin
- inhalation
- Prior art date
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
- A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
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- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/041—Heterocyclic compounds
- A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
- A61K51/0453—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
oxidized avidin is suitable for use as The use of an oxidized avidin in the manufacture of a lung conditioning agent for the treatment of lung cancer diseases, lung inflammation diseases selected from the group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha-1- antitrypsin deficiency is disclosed where the lung conditioning agent is for administration by inhalation. The inhalation of the lung conditioning agent can be followed by administration of a biotinylated therapeutic agent A pharmaceutical inhalable formulation for nebulization comprises a) oxidized avidin in a first vial in a concentration from 0.005% to about 0.5% (w/v), b) a sterile buffer solution comprises sodium acetate at pH comprised between 5.0 and 6.9 in a second vial and optionally c) a non-ionic agent selected from the group comprising mannitol, glycerol, glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol, polyethylene- glycol, ethanol and isopropanol. a lung conditioning agent through inhalation such as for substantially wholly conditioning the lung. The oxidized avidin can be used in combination with a biotinylated therapeutic agent such as a radioactive agent, monoclonal antibodies, cytokines, chemokines, enzymes, chemotherapeutics, viral or plasmid vectors and cells. A pharmaceutical composition containing the oxidised avidin is useful in treating lung cancer diseases and lung inflammation diseases selected from the group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha- 1-antitrypsin deficiency. deficiency is disclosed where the lung conditioning agent is for administration by inhalation. The inhalation of the lung conditioning agent can be followed by administration of a biotinylated therapeutic agent A pharmaceutical inhalable formulation for nebulization comprises a) oxidized avidin in a first vial in a concentration from 0.005% to about 0.5% (w/v), b) a sterile buffer solution comprises sodium acetate at pH comprised between 5.0 and 6.9 in a second vial and optionally c) a non-ionic agent selected from the group comprising mannitol, glycerol, glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol, polyethylene- glycol, ethanol and isopropanol. a lung conditioning agent through inhalation such as for substantially wholly conditioning the lung. The oxidized avidin can be used in combination with a biotinylated therapeutic agent such as a radioactive agent, monoclonal antibodies, cytokines, chemokines, enzymes, chemotherapeutics, viral or plasmid vectors and cells. A pharmaceutical composition containing the oxidised avidin is useful in treating lung cancer diseases and lung inflammation diseases selected from the group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha- 1-antitrypsin deficiency.
Description
TITLE
Pharmaceutical composition of oxidised avidin suitable for inhalation
FIELD OF INVENTION
The invention described herein generally relates to a new pharmaceutical
formulation of an oxidized avidin for use by inhalation. Also described is a
formulation of biotinylated therapeutic agent/oxidized avidin complex and
biotinylated therapeutics for targeting preconditioned lungs of mammals affected by
inoperable and/or diffuse diseases.
BACKGROUND OF THE INVENTION
Inhalation of nebulised therapeutics has become a frequent drug delivery method
for the treatment of lung diseases such as asthma or lung infections and other
respiratory diseases. However, such mode of administration usually requires
repeating the treatment several times per day, which however, may not be always
compliant with patient’s health conditions depending on the disease he suffers from
or the grade of severity of the disease itself. Frequent inhalations of therapeutic
agents also constitutes a severe life-limiting stress. Continuous nebulisation
therapy of b2 agonists has proved to be a useful alternative therapy for patients
suffering from severe asthma (Raabe O.G., et al., Ann. Allergy Asthma Immunol.,
1998, 80, 499). However, even in this case, the therapy is very time consuming and
renders the patient’s life uncomfortable.
Serious lung diseases such as lung cancer or cystic fibrosis are anyway still largely
treated by systemic therapies which are unfortunately associated to significant side
effects.
Lung’s anatomy and physiology are well adapted to process exogenous nebulised
substances for clearance in order to protect the organ. It is also recognized that also
in the case of volunteer treatment exposure through inhalation of therapeutic
agents, the latter are fast cleared therefore influencing negatively and thereby
limiting the benefit of inhalation therapy.
It is further well known that enzymes aimed at detoxifying organs from external
aggression are also present in the lung. Some of those which play an important role
in the lung belong to the super family of proteins (i.e., AKR). AKR and short-chain
dehydrogenases/reductases (i.e., SDRs) are the main enzymes that catalyze
oxidation-reduction reactions involving a xenobiotic carbonyl. Among SDR
superfamily, carbonyl reductases (i.e., CBRs) exhibit broad substrate specificity for
carbonyl bearing xenobiotics (Matsunaga T., et al., Drug Metab. Pharmacokinet.,
2006, 21, 1, 1).
Attempts of local delivery of chemotherapeutics by aerosol have been recently
reported in pre-clinical models of lung cancer showing reduced toxicity compared to
systemic administration (Fulzele, S.V., et al., J. Pharm. Pharmacol., 2006, 58, 3,
327).
A dry powder formulation for inhalation of temozolomide has recently been
reported. The latter required a particle size reduction to enable a 51% release of the
administered dose (Wauthoz N., et al., Pharm. Res., 2011, 28, 762), and also the
presence of biocompatible and biodegradable phospholipids as surfactants to
stabilise the aqueous temozolomide suspension (Wauthoz N., et al., Eur. J. Pharm.
Sci., 2010, 39, 402).
A Phase I clinical trial involving cisplatin was conducted positively in order to
investigate the safety and pharmacokinetics of aerosolized Sustained Release Lipid
Inhalation Targeting (SLIT) cisplatin in patients with lung carcinoma. However,
still quite a lot of side effects (i.e., nausea, vomiting, dyspnea, fatigue and
hoarseness) were encountered in this study (Wittgen B.P.H., et al., Clin. Cancer
Res., 2007, 13, 2414).
Nebulisation of 5-fluorouracil in dogs demonstrated that the drug could reach very
high concentrations mainly in the trachea, to a less extent but still important into
bronchi and oesophagus and a reduced concentration (i.e., one fiftieth of the one
encountered in the trachea) into the lymph nodes at the bronchial level (Tatsumura
T., et al., Br. J. Cancer, 1993, 68, 1146).
Nevertheless, diffusion of these aerosolized small chemical drugs to the blood is still
a relevant issue together with the need of repeated administration due to the short
lung half life of these molecules.
Aerosol gene delivery is another application long pursued for the targeted therapy
of lung diseases. After the cloning of the cystic fibrosis gene, there was great
interest in the non-invasive delivery of genes directly to the pulmonary surfaces by
aerosol. This approach could have application to inoperable pulmonary cancers as
well and most early efforts focused mainly on the use of nonviral vectors, primarily
cationic lipids and other formulation excipients (Densmore C.L., et al., J. Gene
Med., 1999, 1, 4, 251; Densmore C.L., et al., Mol. Ther., 2000, 1, 2, 180).
Unfortunately, nebulisation shear forces, and inefficient pulmonary uptake and
residence of aerosolized protein therapeutics coupled to low expression of genetic
vectors have generally resulted in a poor therapeutic effect (Schwarz, L. A., et al.,
Hum. Gene Ther., 1996, 7, 731). Consequently, the interest in lung delivery of
protein-containing biologically active derivatives by aerosol has diminished in
recent years.
If inhalation of therapeutic proteins had been sought as an attractive solution for
targeted therapy of the lung, the formulation of protein to be nebulised still remains
challenging. Indeed, in order for the aerosolized substance to penetrate the lungs in-
depth, formulation requiring scrupulous selection of additives and particle size (i.e.,
up to 3 mm) have to be adjusted meticulously (Choi W.S., et al., Proc. Natl. Acad.
Sci., 98, 20, 11103). Furthermore, the quaternary, but also the secondary and
tertiary structure of the protein can be altered by the nebulisation process. To
overcome this drawback, Arakawa T., et al. disclosed the use of polyethylene glycol
and/or a surfactant to preserve said structural conformation prior to nebulisation
(WO199503034).
Variously grafted nanoparticles designed for aerosol administration to target the
lungs and which can entrapped different anti-cancer drugs have also been described
lately, but were reported to present some inflammatory disadvantages (Dailey L.A.,
et al., Toxicol. Appl. Pharmacol., 2006, 215, 1, 100).
A polymer-based nanoparticle delivery system for inhalation has been disclosed
recently (WO2009121631), wherein particles were administered by endotracheal
instillation to mice.
Borlak J., et al. (EP2106806), described an improved drug delivery system to the
lung consisting of:
a polymer-based nanoparticle,
a maleimide-based molecular linker,
a targeting agent such as an antibody, a low molecular weight compound or
a protein (preferably covalently linked to the linker),
a drug.
Such nanoparticles had a mean size of 150 to 180 nm. According to the inventors,
such delivery system took advantage of the molecular linker having a lipophilic
portion which non-covalently anchors to the particle's polymeric matrix and a
second portion comprising a maleimide compound to which it is possible to bind a
targeting agent. Still according to the inventors, the targeting agent could be a
member of a binding couple such as avidin-biotin. However, the specific
embodiment involving avidin-biotin was not described in an enabling manner in
this document.
Ethanol based formulations have been previously reported to be suitable for
nebulisation of proteins with biological activity including enzymes (Choi W.S., et al.,
Proc. Natl. Acad. Sci., 2001, 98, 20, 11103). However, very limited exposure to
inhaled ethanol is disclosed (i.e. 10 minutes) since longer inhalation period can
provoke inflammatory side effects.
Therefore and as alluded herein-above, the physical stresses inherent to
nebulisation, coupled to the formation of a large area of air-water interface, can
destabilize the structure of many proteins.
111 99m
[ In]-avidin and [ Tc]-biotin-liposomes administration through the pleural route
has been disclosed and proved to be a better route to target lung mediastinal nodes
than intraperitoneal route (Medina L.A., et al., Nucl. Med. Biol., 2004, 31, 1, 41).
However, injection in the pleural cavity implies an invasive procedure and is not
adequate to obtain homogenous distribution in the lung tissue. In fact, the intention
of Medina’s work was to target mediastinal lymph nodes. Moreover, the
radiolabelled biotin needed to be formulated in liposome to improve
pharmacokinetic and pharmacodynamic features.
Therefore, there is a strong medical need in providing specific way of administration
to directly deliver to the diseased lung an efficacious amount of therapeutic agent in
order to avoid the bottlenecks associated with:
o oral delivery (e.g., drug permeability issues, first-pass effect); and/or
o other systemic drug delivery routes which can be correlated to severe if
not compromising toxic side effects and/or high clearance issues; and/or
o instability of the therapeutic according to its administration route
and/or metabolic process; and/or
o multiple administrations of the therapeutic every day
It has now been surprisingly found that The oxidized avidin, administered by
inhalation, link to the surface of lung epithelial cells uniformly down to the alveoli,
and that it does not surprisingly bind to the respiratory upper track (e.g., trachea).
This finding was completely unexpected since The oxidized avidin did not bind to
tissue surfaces like skin, eye or bladder unless a damage was procured to said
surfaces. It was also surprising that such mode of administration preserves the
chemical integrity of the protein.
DESCRIPTION OF THE INVENTION
In one aspect the invention relates to the use of an oxidized avidin in the
manufacture of a lung conditioning agent for the treatment of lung cancer diseases,
lung inflammation diseases selected from the group comprising COPD, asthma,
lung alveolitis and cystic fibrosis and alpha antitrypsin deficiency, wherein the
lung conditioning agent is for administration by inhalation.
In another aspect, the invention relates to the use of an oxidized avidin and a
biotinylated therapeutic agent in the manufacture of a medicament for the
treatment of lung cancer diseases, lung inflammation diseases selected from the
group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha
antitrypsin deficiency, wherein the medicament is formulated for administration of
that biotinylated therapeutic agent after the oxidized avidin, and wherein the
oxidized avidin is formulated for administration by inhalation.
In another aspect, the invention provides a pharmaceutical inhalable formulation
for nebulization comprising:
a) oxidized avidin in a first vial in a concentration from 0.005% to about 0.5%
(w/v),
b) a sterile buffer solution comprises sodium acetate at pH comprised between 5.0
and 6.9 in a second vial and optionally
c) a non-ionic agent selected from the group comprising mannitol, glycerol, glucose,
lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol, polyethylene-
glycol, ethanol and isopropanol.
Certain statements that appear below are broader than what appears in the
statements of the invention above. These statements are provided in the interests
of providing the reader with a better understanding of the invention and its
practice. The reader is directed to the accompanying claim set which defines the
scope of the invention.
Described is a pharmaceutical inhalable composition comprising an oxidized avidin
or a biotinylated therapeutic agent/oxidized avidin complex, for targeted delivery of
biotinylated therapeutics by inhalation to the lung. Also described is the aerosol
delivery of an oxidized avidin as a tool for conditioning the lung to accept a stable
linkage of biotinylated therapeutics useful for the treatment of lung cancer, asthma,
tuberculosis, Chronic Obstructive Pulmonary Disease (COPD), lung alveolitis, cystic
fibrosis and alphaantitrypsin deficiency.
Biotinylation of proteins, cells and nucleic acid is a biochemical procedure well
known in the art. It can be performed by a number of different commercially
available reagents reacting with different groups (i.e. primary and second amines,
sulphydryl and carboxyl groups, etc). Biotinylation reagents are designed to be very
flexible (long spacers to reduce steric hindrance) or to be releasable (to free active
moieties) and have been extensively used for targeting functional biotinylated
moieties to proteins of the avidin family. In particular, protein biotinylation has
been extensively applied to monoclonal antibodies and other functional proteins
(Bayer E.A., et al., Methods Enzymol, 1990b, 184, 138).
Suitable biotinylated anti-cancer therapeutics are for example chosen from the
group comprising IgG1 monoclonal antibody that binds specifically to the epidermal
growth factor receptor (EGFR) such as biotinylated cetuximab (Hama Y., et al.,
Cancer Res., 2007, 67, 3809). Further biotinylated anti-cancer therapeutics are for
example biotinylated adducts of anti c-Met antibody (Stella G.M., et al., Expert.
Opin. Investig. Drugs, 2010, 19, 11, 1381); anti-HGF (Okamoto W., et al., Mol.
Cancer Ther., 2010, 9, 10, 2785); anti-CTLA4 (Di Giacomo A.M., Cancer Immunol.
Immunother., 2009, 58, 8, 1297); anti-VEGF (Ferrara N., et al., Biochem. Biophys.
Res. Commun., 2005, 333, 2, 328); anti-EpCAM (Kurtz J.E., et al, Expert. Opin.
Biol. Ther., 2010, 10, 6, 951); anti-HER2 monoclonal antibodies (Smith B.L., et al.,
Br. J. Cancer, 2004, 91, 6, 1190); TNF (Balkwill F., Nat. Rev. Cancer, 2009, 9, 5,
361), TRAIL (Kim T.H., et al., Bioconjug. Chem., 2011, ); or other cytokines like IL-2
(Herberman R., Cancer Invest., 1989, 7, 5, 515; Koten J.W., et al., Cytokine, 2003,
24, 3, 57); G-CSF (Cavalloni G., et al., Anticancer Drugs, 2008, 19, 7, 689); GM-CSF
(He Q., et al., Cancer Immunol. Immunother., 2011, 60, 5, 715); IL-12 (Penichet
M.L., et al., J. Immunol. Methods, 2001, 248, 1-2, 91; Adris S., et al., Cancer Res.,
2000, 60, 23, 6696); gamma interferon (Weiner L.M., Mol.Biother., 1991, 3, 4, 186).
Such therapeutics are known to block tumour cell replication, induce tumour cell
death and/or stimulate an anti-cancer immune response. Nevertheless, toxic side
effects are associated to their systemic use. Therefore, stable localization within the
lung of the biotinylated therapeutic agent upon:
o its aerosol delivery following oxidized avidin inhalation, or upon
o inhalation of a biotinylated therapeutic agent/oxidized avidin complex whenever
nebulisation is compatible with the integrity and biological function of either the
therapeutic agent itself or of the complex; or upon
o its systemic delivery (e.g., parenteral administration) following oxidized avidin
inhalation,
will allow to reduce their therapeutic dose and minimize systemic exposure thus
improving their therapeutic index. Also described is nebulised oxidized avidin for
use in lung targeting of biotinylated anti-cancer effector cells, viral or plasmid
vectors (D’Atri S., et al., Immunopharmacol., 1991, 21, 3, 199; Densmore C.L., Curr.
Cancer Drug Targets, 2003, 3, 4, 275).
Also described is the aerosol delivery of oxidized avidin as a useful tool for
conditioning the lung to accept a stable linkage of anti-inflammatory biotinylated
therapeutics. Suitable anti-inflammatory biotinylated therapeutics includes for
example biotinylated adducts of anti-TNF, anti-Tweak, anti-IL-6, anti-IL-23, anti
IL-17 monoclonal antibodies; IL-10 or other anti-inflammatory cytokines (Marchi
E., et al., Chest, 2011) or chemokines (Farberman M.M., et al., Am. J. Respir. Cell
Mol. Biol., 2010; Tauler J., et al., Curr. Opin. Pharmacol., 2009, 9, 4, 384; Hartl D.,
et al., Curr. Opin. Allergy Clin. Immunol., 2009, 9, 1, 60; Brennan S., et al., Eur.
Respir. J., 2009, 34, 3, 655) for the treatment of asthma or lung alveolitis or other
forms of lung chronic inflammation. Delivery of biotinylated enzymes like alpha
antitrypsin to the lungs via oxidized avidin nebulisation is also envisaged to cure
primary genetic deficiency (Brand P., et al., Eur. Respir. J., 2009, 34, 2, 354; Geller
D.E., et al., J. Aerosol Med. Pulm. Drug Deliv., 2010, 23 Suppl 1, S55) and Cystic
Fibrosis Transmembrane Conductance Regulator (CFTCR) protein to cure cystic
fibrosis (Sloane P.A., et al., Curr. Opin. Pulm. Med., 2010, 16, 6, 591; Frizzell R.A.,
Am. J. Respir. Crit. Care Med., 1995, 151, S54).
The avidin-biotin system has been known for many years as an exceptional tool for
qualitative and quantitative studies in interactions between small molecules and
biological receptors (Wilchek, M., Methods Enzymol., 1990, 184, 14).
Avidin is a glycoprotein of about 68 kDa present in the poultry egg white and
showing high affinity for the vitamin H biotin. Its dissociation constant (K ~10 M)
is the lowest known in nature (Green, N.M., Adv. Protein Chem., 1975, 29, 85;
Hytonen V.P., et al., Biochem. J., 2003, 372, Pt1, 219). It is composed of four
subunits of identical amino acid sequence, each of which can potentially bind one
molecule of biotin. Glycosylation accounts for about 10% of its molecular weight
with an average of four to five mannose and three N-acetylglucosamine residues per
subunit (Bruch R.C., et al., Biochemistry, 1982, 21, 21, 5334).
In WO2009016031 in the name of the Applicant, was reported for the first time a
chemically oxidized avidin named OXavidin , (herein named oxidized avidin),
HABA
that had higher permanence in injected tissues compared to wild type avidin as a
consequence of the formation of chemical linkages between the oxidized avidin
aldehyde groups and the tissue protein amino groups. Said oxidized avidin could be
administered directly into the diseased tissue either alone, in which case a second
step for delivering the biotinylated active therapeutic agent was necessary, or as a
complex with the therapeutic agent. It is important to note that in said document
the oxidized avidin was administered locally by means of injection rendering
impractical a homogeneous whole lung conditioning. This patent application opened
a new era by disclosing new solutions useful for the localized treatment of solid
tumour cancer and degenerative or genetic diseases. Tolerability of oxidized avidin
was also recently reported (Petronzelli F., et al., Basic Clin. Pharmacol. Toxicol.,
2011, 233).
However, even this new tool did not allow to tackle those widely diffuse and/or
inoperable lung diseases, notably because of the peculiarity of the lung tissue that is
not easily treated by injection and because of the inadequacy of surgical procedures
in reaching difficultly accessible areas of the diseased tissue. In particular,
bronchioloalveolar cancer (BAC), also known as lepidic lung cancer is a form of
neoplasia affecting the surface of bronchus and alveoli that might benefit from a
topical therapy delivered by aerosol (Anami Y., et al., J. Thorac. Oncol., 2009, 4, 8,
951).
As mentioned above, it has now been found that oxidized avidin can efficiently
reach and link to the lung tissue through aerosol administration. This result was
not predictable based on the data of WO2009016031 and further investigation
showed that the intra-tissue injection was a requirement for the oxidized avidin
linkage to the tissues. In fact, deposition of oxidized avidin on intact skin or eye was
not sufficient to observe a linkage (Figure 1). Linkage of the oxidized avidin was
confirmed to occur only on tissue proteins exposed by surgery also in the pig bladder
(Figure 2). Moreover, nebulised oxidized avidin was found bound to the lung but not
to the trachea thus indicating a specific and unpredictable linkage event in this
organ (Table 1).
The term ‘comprising’ as used in this specification and claims means ‘consisting at
least in part of’. When interpreting statements in this specification and claims
which includes the ‘comprising’, other features besides the features prefaced by this
term in each statement can also be present. Related terms such as ‘comprise’ and
‘comprised’ are to be interpreted in similar manner.
The terms “AvidinOX” or “AvidinOX®” or “OXavidin ” refer to the chemically
HABA
oxidized avidin according to example 1 of WO2009016031. The expression “oxidized
avidin” refers to a chemically oxidized avidin as claimed in WO2009016031 (i.e., an
oxidized avidin in which at least one mannose residue per avidin molecule is
replaced by a residue of the following formula
wherein said oxidized avidin contains about 8 to 15 aldehyde moieties and has a
thermal stability equal or greater than 78°C. Alternatively, the expression “oxidized
avidin” also refers to a compound that was obtained by oxidation of avidin in the
presence of the ligand HABA in order to prevent from oxidation tryptophan residues
that are involved in the biotin-binding site.
The expression “anticancer agent” means an agent capable of fighting tumours. A
non-exhaustive list of anticancer agents consists of chemotherapeutic drugs,
monoclonal antibodies, radiolabelled compounds, effector cells, toxins, cytokines,
viral and plasmid vectors, RNA inhibitors and anticancer cells.
The terms “aerosol”, “aerosolised”, “inhalable” and “nebulised” all refer to the same
concept of breaking into small aerosol droplets, medical solutions/suspensions to
enable them to be directly and efficiently inhaled from the mouth.
The expressions “for conditioning the lung”, “lung conditioning agent” and
“preconditioned lungs” refer to the concept of enabling lung cells to interact with
biotinylated therapeutic agents through oxidized avidin interaction that would have
been inhaled in beforehand.
In particular, the expression “for substantially wholly conditioning the lung” means
that once administered, the oxidized avidin can be detected in at least 95% of the
lung.
Described is a pharmaceutical inhalable composition comprising:
a) the oxidized avidin according to claim 1 or 2,
b) a sterile buffer solution at acidic pH, wherein said buffer is preferably
sodium acetate, and optionally
c) a non-ionic agent selected from the group comprising mannitol, glycerol,
glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol,
polyethylene-glycol, ethanol and isopropanol;
wherein said pharmaceutical composition is delivered through inhalation after
nebulization.
In a preferred embodiment “acidic pH” means that the pH is comprised between 5.0
and 6.9.
In a more preferred embodiment “acidic pH” means that the pH is comprised
between 5.0 and 6.0.
In an even more preferred embodiment “acidic pH” means that the pH is comprised
between 5.0 and 5.5.
In a further embodiment, the pharmaceutical inhalable composition is freeze-dried.
In a preferred embodiment the oxidized avidin is useful as a lung conditioning
agent through inhalation.
In a more preferred embodiment said oxidized avidin is useful for substantially
wholly conditions the lung through inhalation.
In another embodiment, delivery of oxidized avidin is followed by administration of
a therapeutic agent, wherein said therapeutic agent is biotinylated.
In a preferred embodiment, the pharmaceutical inhalable composition of the
invention is useful for the treatment of the following diseases: tuberculosis, Chronic
Obstructive Pulmonary Disease (COPD), lung cancer in general and with particular
interest on bronchioloalveolar (BAC) cancer, asthma, lung alveolitis, lung
inflammatory diseases, cystic fibrosis and alphaantitrypsin deficiency.
In a further preferred embodiment, the pharmaceutical inhalable composition of the
invention is useful for the treatment of primary lung cancer in multi-focal or
metastatic form.
In a still further preferred embodiment, the biotinylated therapeutic agent is a
biotinylated adduct of an anticancer agent.
In a still further preferred embodiment, the biotinylated anticancer agent is a
biotinylated adduct of well-known anti-cancer drugs selected from the group
comprising anti-EGFR, anti-CEA, anti-MUC1, anti-EpCAM, anti-cMET, anti-CTL4
monoclonal antibodies, TNF, TRAIL, Tweak, gamma interferon, G-CSF, GM-CSF,
IL-2, IL-12 or chemotherapeutics.
In an even more preferred embodiment, said anticancer agent is radioactive
derivative selected from the group comprising of radiolabelled biotin-DOTA
(ST2210). The latter was described in the patent application WO2002066075.
In a still more preferred embodiment, the radioisotope used to label biotin-DOTA is
52 52m 55 64 67 67 68 99m
selected from the group comprising Fe, Mn, Co, Cu, Cu, Ga, Ga, Tc,
111 123 125 131 32 47 90 109 111 149 186 188 211 212 212
In, I, I, I, P, Sc, Y, Pd, Ag, Pm, Re, Re, At, Pb, Bi
and Lu.
In another more preferred embodiment, said anticancer agent is a biotinylated
adduct of well-known anti-cancer drugs selected from the group consisting of viral
or plasmid vectors, RNA inhibitors or anti-cancer effector cells.
In another preferred embodiment the biotinylated agent is an agent useful for the
treatment of lung inflammatory diseases and is a biotinylated adduct of well-known
anti-inflammatory drugs anti-inflammatory agent selected from the group
comprising anti-TNF, anti-Tweak, anti-IL-17, anti-IL-23, anti-IL-6, anti-IL-1
monoclonal antibodies, IL-10 or chemokines.
In another preferred embodiment for lung genetic diseases, a therapeutic agent is a
biotinylated adduct of known deficient protein like CFTCR in cystic fibrosis alpha
antitrypsin.
A further preferred embodiment consists in a kit comprising the pharmaceutical
inhalable composition mentioned herein-above, wherein the latter is either freeze-
dried or dissolved in a sterile buffer solution.
In a further more preferred embodiment said kit comprises the pharmaceutical
inhalable composition mentioned herein-above and a nebulizer.
The amount of oxidized avidin which is required to achieve a therapeutic effect will,
of course, vary with the subject under treatment, and the particular disorder or
disease to be treated. It will further depend on the efficiency of the nebulizer used
and the deposition of the aerosol droplets in the lung. Suitable concentrations of
oxidized avidin within the solution for nebulisation can be in the range of 0.005% to
0.5% (w/v) (i.e. 0.05 mg/ml to 5mg/ml).
Non-ionic agent can be used for adjusting the osmolality of the solution for
nebulisation. Examples of non-ionic agents for adjusting the osmolality, which can
be useful in the present invention are selected from the group comprising mannitol,
glycerol, glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol,
polyethylene-glycol, ethanol and isopropanol.
Besides the non-ionic agent herein above mentioned, the formulation may contain
one ore more additional suitable excipients. Suitable excipients, which can be
mentioned include agents for modifying the pH of the solution and optionally
preservatives.
The formulation of the invention may be distributed in suitable containers such as
multidose vials or, preferably, unit dose vials for single dosage administration. The
solution for nebulisation can be obtained as follows: oxidized avidin of a first vial
can be dissolved by addition of sterile sodium acetate solution at pH 5.5 of a second
vial.
The formulations of the invention are intended for administration by nebulisation
using suitable apparatus instrument that is capable of generating very fine liquid
droplets for inhalation into the lungs. Suitable instruments are for example jet
nebulisers or ultrasonic nebulisers.
DESCRIPTION OF THE DRAWINGS
Figure 1:
It shows the tissue residence of the complex oxidized avidin/ In-ST2210 in the eye
(drop deposition), tongue (i.m. injection), limb (i.m. injection), skin (drop deposition),
scraped skin (drop deposition).
Figure 2:
It shows the linkage of intravenously injected 68-Ga-ST2210 to a surgical lesion
performed in the bladder of a pig to simulate the removal of a human superficial
bladder carcinoma (arrow). The image was obtained 4 hours after 68-Ga-ST2210
injection, by Positron Emission Tomography (PET).
Figure 3:
It shows the chemical integrity of oxidized avidin after having been nebulised for 1
hour in a 100 mM sodium acetate solution at pH 5.5 and without excipient.
Figure 4:
It shows, at T = 2 hours and T = 24 hours, the distribution of intravenously injected
In-ST2210 in various organs of mice which have received 24 hour before oxidized
avidin or vehicle through inhalation.
Figure 5:
Immunochemistry with mouse anti-avidin antibody of lung sections, 24 hours after
oxidized avidin aerosol exposure.
Figure 6:
PET imaging of Cu-ST2210 intravenously injected mice which were exposed to
oxidized avidin aerosol 24 hours before.
Figure 7:
EGFR+ A431 cells incubated with biotinylated cetuximab at the dose of 5 µg, 0.05
µg, 0.05 ng and 0.05 pg/ml for 1 hour in PBS without (a) or with (b) pre-treatment
with oxidized avidin.
Figure 8:
It shows the inhibition of proliferation of A431 cell line with biotinylated cetuximab
with or without prior treatment with oxidized avidin.
Figure 9:
It show a cytofluorimetry analysis of the induction of apoptosis by biotinylated
cetuximab on two cell lines, one expressing high level of EGFR (i.e., A431) and one
which does not express EGFR (i.e., SKMel28) with or without treatment with
oxidized avidin.
Figure 10:
It shows the inhibition of proliferation induced by biotinylated cetuximab with or
without pre-treatment with AvidinOX on A431, A549 (i.e., lung carcinoma
expressing low levels of EGFR, KRAS mutated) and SKMel28 cells.
EXAMPLES
Example 1
Balb/c mice of about 20 g were treated in the indicated sites with a solution of
avidin or oxidized avidin as obtained following the procedure described at example 1
of WO2009016031 (3.0 mg/ml dissolved in 100 mM sodium acetate, pH 5.5) pre-
complexed with In-ST2210. Twenty four hours after injection/deposition, the mice
were sacrificed by CO asphyxia and the treated sites analysed by means of a
gamma counter. Data are reported in Figure 1, and are expressed as the % of
injected dose/100 mg of tissue (%ID/100 mg). Results show a statistically significant
higher amount of oxidized avidin/ In-ST2210 complex in injected tongue, limb
muscle and on the topically treated scraped skin compared to the Avidin/ In-
ST2210 complex. However, deposition of the oxidized avidin/ In-ST2210 or
Avidin/ In-ST2210 complex on normal skin or in the eye did not lead to similar
results, indicating that the complex does not bind to external tissue surfaces and
consequently that a surgical operation is required to take advantage of the binding
properties of oxidized avidin either as single agent when complexed with a
biotinylated agent.
Example 2
Anesthetised female pig of about 40 kg was subjected to surgery to generate two 2
cm superficial lesions on the bladder wall. Then, 30 ml of oxidized avidin solution
(3.0 mg/ml dissolved in 100 mM sodium acetate, pH 5.5) were instilled via a
catheter and let interact for 1 hour. Bladder was then washed with saline and 0.5
µg 68-Ga-ST2210 was i.v. administered. After 4 hours the pig was subjected to PET.
Results as shown in Figure 2, demonstrate that only the region that had been
subjected to surgery (i.e., 2 mm lesion) enabled the binding of oxidized avidin,
meanwhile intact bladder tissue proved to be completely inert to the aldehyde
moieties of oxidized avidin. Those surprising data are however, in agreement with
those of example 1 wherein non-surgically damaged tissue were inert to oxidized
avidin.
Example 3
A 100 mM sodium acetate solution at pH 5.5 containing oxidized avidin at the
concentration of 3.0 mg/ml was nebulised by means of a the Nose-Only inExpose
System (Scireq-EMKA technologies) for 1 hour at room temperature. The particle
size of the protein solution midst was 5 µm. The nebulised solution was recovered in
a falcon tube and analyzed by HPLC. Data in Figure 3 show the same elution
profile for the oxidized avidin solution pre and post nebulisation indicating perfect
stability of the protein. This result was not obvious as many proteins and nucleic
acid need extensive formulation studies to select a condition that preserves
integrity and potency of such drugs during nebulisation (Geller D.E., et al., J.
Aerosol Med. Pulm. Drug Deliv., 2010, 23 Suppl 1, S55; Markovic S.N., et al., Am. J.
Clin. Oncol., 2008, 31, 6, 573; Choi W.S., et al., Proc. Natl. Acad. Sci., 98, 20, 11103).
It was very interesting to note that notwithstanding the fact that aldehyde
derivatives are highly susceptible to hydration in the presence of water, no such
phenomena was observed during oxidized avidin nebulisation process. Indeed, the
number of aldehyde moieties per molecule, as determined by Purpald’s method
(Quesenberry M.S., et al., Anal. Biochem., 1996, 234, 1, 50), was found to be
substantially the same (considering the variability of the assay) before and after
nebulisation at both pH 5.0 and pH 5.5, with and without mannitol excipient as
demonstrated by data in Table 1.
Table 1
Number of CHO moieties per
molecule
Oxidized avidin batch
Before
After nebulisation
nebulisation
Mannitol formulation pH 5.0 19.7 19.2
Mannitol formulation pH 5.5 17.1 19.4
Acetate buffer formulation pH 5.5 16.4 16.7
Example 4
Biological activity and biostribution of nebulised oxidized avidin were evaluated in
rat by measuring the uptake of 5 µg of Indium radiolabelled biotinDOTA (i.e.,
In-ST2210) intravenously injected, 24 hours after oxidized avidin, avidin (3.0
mg/ml solution, 0.8ml/min, 1 hour nebulisation) or vehicle inhalation exposure, in
the lung and non target organs.
Twenty Sprague Dawley rats were divided in three different groups. Each group
received the following treatments:
o the first group (made of 4 rats) was exposed to nebulised vehicle only (i.e., 100
mM sodium acetate solution at pH 5.5) and 24 hours later the rats received a 5
mg i.v. dose of In-ST2210 in 0.5 ml of saline.
o the second group (made of 8 rats) was exposed to nebulised avidin (about 10
mg/kg) in a 100 mM sodium acetate solution at pH 5.5 and 24 hours later the
rats received a 5 mg i.v.dose of In-ST2210 in 0.5 ml of saline.
o the third group (made of 8 rats) was exposed to nebulised oxidized avidin (about
mg/kg) in a 100 mM sodium acetate solution at pH 5.5 and 24 hours later the
rats received a 5 mg i.v. dose of In-ST2210 in 0.5 ml of saline.
All rats were sacrificed 2 hours after the i.v. administration of In-ST2210, and
samples of blood, spleen, kidney, liver, stomach, brain, trachea, and tissue samples
of different portions of lung were collected, weighed and analyzed with the use of a
gamma-counter (Perkin Elmer). Data were expressed as the % of injected dose/gram
of tissue (%ID/g).
As shown in Table 2, oxidized avidin inhalation enables to increase in a statistically
significant fashion In-ST2210 concentration in the lung, meanwhile other organs
did not show any statistical differences of concentration of In-ST2210 compared
to avidin or vehicle groups.
Table 2
In-ST2210 %ID/g
Groups Oxidized
Vehicle Avidin
avidin
Blood 0.011±0.005 0.009±0.005 0.012±0.004
Spleen 0.105±0.159 0.027±0.003 0.032±0.002
Kidney 0.426±0.082 0.378±0.048 0.414±0.033
Liver 0.046±0.009 0.041±0.007 0.051±0.005
Stomach 0.031±0.011 0.067±0.085 0.276±0.246
Brain 0.002±0.001 0.002±0.000 0.002±0.000
Trachea 0.021±0.007 0.022±0.008 0.029±0.007
Lung 0.024±0.004 0.023±0.006 0.054±0.009***
Lung sn 0.024±0.004 0.023±0.006 0.053±0.008***
Lung dx (caudal lobe) 0.024±0.004 0.022±0.006 0.055±0.009***
Lung dx (cranial, middle and
0.025±0.004 0.023±0.005 0.055±0.011***
accessory lobe)
*** p < 0.001 one way Anova versus avidin
Example 5
A biodistribution experiment aimed at determining the lung uptake selectivity and
stability of In-ST2210 was conducted. Balb/c mice of about 20 g (5 mice/group)
were exposed to nebulised (i.e., by means of Nose-Only inExpose System -Scireq-
EMKA technologies) oxidized avidin (3 mg/ml solution) or vehicle (3 ml) for 1 hour;
1 hour exposition corresponding to about a 90 mg/kg dose. Twenty four hours after,
all mice received In-ST2210 intravenously (i.e., 1 mg in 0.2 ml of saline), and after
further 2 or 24 hours, the animals were sacrificed by CO asphyxia. The lung and
non target organs were collected weighed and counted in a gamma counter. Data
were expressed as the % of injected dose/gram of tissue.
Results, as exposed in Figure 4, demonstrate a specific and significant uptake of
In-ST2210 only in the lung which had been pre-treated with oxidized avidin.
Example 6
Balb/c mice of about 20 g were exposed to nebulised oxidized avidin according to the
protocol described at example 3, and were sacrificed by asphyxia 24 hours after. The
lungs were removed and fixed in formalin and paraffin embedded. Serial sections
obtained by means of a microtome were processed and incubated with a HRP-
conjugated rabbit anti-avidin antibody (GeneTex, USA) and then with DAB
substrate.
Figure 5 shows the presence of oxidized avidin at broncho/epithelial level down to
terminal bronchioles. This distribution was found to be homogenous in all lung
compartments.
Example 7
Balb/c mice of about 20 g were exposed to nebulised oxidized avidin or vehicle
according to the protocol described at example 5. After 24 hours, they received an
64 64
intravenous 1 mg dose of Cu-ST2210. Cu-ST2210 distribution was examined by
PET imaging 4 hours after. Figure 6 shows the presence of a radioactive signal in
the lungs of the mouse pre-treated with nebulised oxidized avidin but not in the
lungs of a vehicle treated mouse. Kidneys, bladder are visible in both groups of
mice. This observation is consistent with the physiological elimination of ST2210 at
that time point. A radioactive signal in one limb is also visible in both groups of
mice since they had been i.m. pre-treated with oxidized avidin at the time of aerosol
in order to have an internal positive control in the experiment.
Example 8
Human EGFR , epidermoid carcinoma cells A431 were incubated in PBS for 1 hour
with 1 ml of an anti-EGFR monoclonal biotinylated antibody (i.e., biotinylated
cetuximab) in the dose range of 0.05 pg/ml to 5 µg/ml. In one experiment the cells
were previously incubated with oxidized avidin meanwhile in the first experiment,
cells were only treated with biotinylated cetuximab.
After washings, biotinylated cetuximab binding was detected by cytofluorimetry
after incubation with a mouse anti-human antibody conjugated with phycoerythrin
(PE). As demonstrated in Figure 7, in the presence of oxidized avidin, the binding to
A431 cells of low doses of biotinylated cetuximab such as 0.05 ng and 0.05 pg/ml is
still visible.
Example 9
Data reported in Figure 8 show that the anti proliferative activity of biotinylated
cetuximab (experiment being conducted as reported in example 8) was increased at
least 3 times owing to its immobilization through binding to the membrane bound
oxidized avidin even at doses as low as 0.05 ng and 0.05 pg/ml. Proliferation
inhibition was measured by Cell Titer Glow assay, Promega. Data are expressed as
% of inhibition of cell proliferation in the absence of oxidized avidin (i.e., white bars)
or in its presence (i.e., black bars). Moreover, as also previously reported
(Petronzelli F., et al., Basic Clin. Pharmacol. Toxicol., 2011, 233), oxidized avidin
did not affect cell proliferation as observed in a comparison experiment involving
medium only (data not shown).
Example 10
Induction of apoptosis by biotinylated cetuximab with or without pre-treatment
with AvidinOX was tested on EGFR A431 cells (vulvar squamous cell carcinoma,
expressing high levels of EGFR, KRAS wild-type) and EGFR SKMel28 cells (which
do not express EGFR). The cells were incubated for 15 minutes with biotinylated
cetuximab (b-cetuximab) in the presence or absence of AvidinOX pre-treatment.
After washings, the cells were incubated in complete medium for 18 hours. Annexin
V positive cells were analysed by cytofluorimetry with FITC-Annexin V apoptosis
detection kit I (BD Pharmingen). Data in Figure 9 indicate that the pro-apoptotic
effect of biotinylated cetuximab is increased at least 3 times when AvidinOX is
anchored on the membrane of the EGFR cells but not of the EGFR cells thus
indicating the specificity of pro-apoptotic activity of the AvidinOX-anchored
biotinylated cetuximab.
Example 11
Inhibition of proliferation by biotinylated cetuximab with or without pre-treatment
with AvidinOX was tested on A431, A549 (i.e., lung carcinoma, low EGFR, KRAS
mutated) and SKMel28 cells. For testing the effect of anchored biotinylated
cetuximab on cell proliferation, 5x10 A431, A549 and SKMel28 cells, with and
without pre-incubation with AvidinOX, were seeded in 96 wells microtiter plates
(2x10 cells/well) in DMEM, 10% FCS. After adhesion, 100 µl of biotinylated
cetuximab were added, in triplicates, in the range of 0.05 pg/ml to 5 µg/ml, in
DMEM 1% FCS. The cells were washed after 15 minutes and cultivated for 48
hours in DMEM 1% FCS. Cell viability was detected by CellTiter-Glow® (Promega).
Data in Figure 10 show that biotinylated cetuximab was able to inhibit proliferation
of cells expressing high levels of EGFR (i.e., human vulvar carcinoma A431 cells)
and low levels of EGFR (i.e., KRAS mutated, human lung adenocarcinoma A549
cells) but not proliferation of EGFR cells (SKMel28). Surprisingly, such effects were
significantly improved when biotinylated cetuximab was AvidinOX-anchored on the
cell surface of these EGFR cells.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the
purpose of providing a context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents is not to be
construed as an admission that such documents, or such sources of information, in
any jurisdiction, are prior art, or form part of the common general knowledge in the
art.
WE
Claims (15)
1. Use of an oxidized avidin in the manufacture of a lung conditioning agent for the treatment of lung cancer diseases, lung inflammation diseases selected from the group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha antitrypsin deficiency, wherein the lung conditioning agent is for administration by inhalation.
2. The use according to claim 1, for substantially wholly conditioning the lung.
3. The use according to claim 1 or claim 2, wherein the inhalation of the lung conditioning agent is to be followed by administration of a biotinylated therapeutic agent.
4. Use of an oxidized avidin and a biotinylated therapeutic agent in the manufacture of a medicament for the treatment of lung cancer diseases, lung inflammation diseases selected from the group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha antitrypsin deficiency, wherein the medicament is formulated for administration of that biotinylated therapeutic agent after the oxidized avidin, and wherein the oxidized avidin is formulated for administration by inhalation.
5. The use according to claim 3 or claim 4, wherein the biotinylated therapeutic agent is selected from the group consisting of radioactive agent, monoclonal antibodies, cytokines, chemokines, enzymes, chemotherapeutics, viral or plasmid vectors and cells.
6. The use according to claim 5, wherein the monoclonal antibody is a biotinylated derivative of a monoclonal antibody selected from the group consisting of anti- EGFR, anti-CEA, anti-MUC1, anti-EpCAM, anti-cMET, anti-CTL4, anti-TNF, anti-Tweak, anti-IL-17, anti-IL-23, anti-IL-6, anti-IL-1, anti-TNF, anti-Tweak monoclonal antibodies.
7. The use according to claim 5, wherein the biotinylated cytokine is a biotinylated adduct of a cytokine selected from the group consisting of TNF, Tweak, TRAIL, gamma interferon, G-CSF, GM-CSF, IL-2, IL-12.
8. The use according to claim 5, wherein the biotinylated chemokine is a biotinylated adduct of a chemokine selected from the group consisting of CXC and CC chemokine families.
9. The use according to claim 5, wherein the biotinylated enzyme or is a cystic fibrosis transmembrane conductance regulator protein.
10. The use according to claim 5, wherein the biotinylated radioactive agent is biotin-DOTA labelled with a radioisotope selected from the group comprising 52 52m 55 64 67 67 68 99m 111 123 125 131 32 47 90 Fe, Mn, Co, Cu, Cu, Ga, Ga, Tc, In, I, I, I, P, Sc, Y, 109 111 149 186 188 211 212 212 177 Pd, Ag, Pm, Re, Re, At, Pb, Bi and Lu.
11. A pharmaceutical inhalable formulation for nebulization comprising: a) oxidized avidin in a first vial in a concentration from 0.005% to about 0.5% (w/v), b) a sterile buffer solution comprises sodium acetate at pH comprised between 5.0 and 6.9 in a second vial and optionally c) a non-ionic agent selected from the group comprising mannitol, glycerol, glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol, polyethylene- glycol, ethanol and isopropanol.
12. The pharmaceutical inhalable composition of claim 11 which is freeze-dried.
13. The pharmaceutical inhalation composition of claim 11 or 12 for use in the treatment of lung cancer diseases, lung inflammation diseases selected from the group comprising COPD, asthma, lung alveolitis and cystic fibrosis and alpha antitrypsin deficiency.
14. The use as claimed in any one of claims 1 to 10, substantially as herein described with reference to any example thereof.
15. The composition as claimed in any one of claims 11 to 13, substantially as herein described with reference to any example thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11006338.5 | 2011-08-02 | ||
EP11006338 | 2011-08-02 | ||
PCT/EP2012/064576 WO2013017494A1 (en) | 2011-08-02 | 2012-07-25 | Pharmaceutical composition of oxidised avidin suitable for inhalation |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ620202A NZ620202A (en) | 2016-05-27 |
NZ620202B2 true NZ620202B2 (en) | 2016-08-30 |
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