CA2326760A1 - Use of l-glu-l-trp in the treatment of hiv infection - Google Patents

Abstract

This invention provides methods for the treatment of HIV infection by administering effective amounts of L-Xaa-L-Trp, wherein Xaa is Glu, Ile or Leu, and derivatives and analogs thereof.

Description

USE OF L-GLU-L-TRP IN THE TREATMENT OF HIV INFECTION
REFERENCE TO RELATED APPLICATIONS
The disclosures of the following applications are incorporated herein by reference in their entirety: L.R. Green and N. V. Sinackevich, "Analogs of L-Glu-L-Trp Having Pharmacological Activity," filed April 3, 1998 (Atty docket no. 015542-002600);
and L.R. Green and N.V. Sinackevich, "Ex vivo Uses of Dipeptides," filed April 3, 1998 (Atty docket no. 015542-002700).
BACKGROUND OF THE INVENTION
This invention is directed to prophylactic and therapeutic treatments of retroviral infections and, in particular, HIV infection.
HIV-1 and HIV-2 are retroviruses that infect human T-lymphocytes bearing the cell surface marker CD4. By destroying helper T-cells, they cripple the immune system and lead to severe immune deficiency. Immune deficiency leaves the infected person vulnerable to opportunistic infECtion by invading pathogens and to reduced immune surveillance of malignant cells. Severe immunodeficiency that results from advanced HIV infection is called AIDS, or Acquired Imrnunodeficiency Syndrome.
HIV is spread by exposure to bodily fluids, such as blood and semen, that contain the virus. High risk activities include sexual intercourse with an infected person, use of contaminated hypodermic needles; and transfusion of contaminated blood. The Centers For Disease Control estimate that about four-hundred thousand to about six-hundred thousand persons in the United States are infected with the virus. The threat is even more serious in developing countries, such as those of sub-Saharan Africa, India and Thailand. In those countries, the number of persons infected with HIV is growing at an alarming rate.
Currently, prophylactic treatment of HIV infection focuses on preventing exposure to the virus. This includes educating the public as to how the virus is spread, promoting abstinence from practices that carry a high risk of exposure to the virus and using devices, such as condoms, to establish a physical barrier against the virus. Also, practitioners currently are developing and testing vaccines to arm the immune system against the virus. Such vaccines typically employ antigens derived from HIV
coat proteins, such as gp120. However, no such vaccines are presently in public use.
No completely effective therapeutic treatment of HIV infection has been $ developed. However, significant progress has been made in treating HIV
infection and its sequelae. One class of useful drugs is the inhibitors of reverse transcriptase, such as zyduvine (AZT, 3'-azido-2',3'-dideoxythymidine) and zalcitibine (ddC, 2',3'-dideoxycytidine). These drugs are nucleotide analogs. A second class is HIV
protease inhibitors, such as indinavir sulphate or saquinavir mesylate. These drugs inhibit the activity of HIV protease that cleaves viral polyprotein precursors to generate functional HIV proteins. A third class of drugs is the immunomodulators, such as thymopentin.
These drugs work by enhancing immune system function, thereby making the person's immune system more effective in fighting the infection.
Currently there is great hope in the use of a combination of two reverse-transcriptase inhibitors and a protease inhibitor as a so-called "triple threat" approach against HIV infection. While relatively effective in decreasing viral loads, such therapies suffer from a number of drawbacks, including complicated administration schedules, side effects and expense. Because of dosing schedules and the requirement to take various drugs either on an empty or full stomach, the three-drug approach can involve taking pills at several, specially timed intervals. This requires a discipline that is difficult for many people. The drugs also have side effects, such as nausea, myalgia, liver damage and nephrolithiasis. Also, therapies can cost tens of thousands of dollars a year. Thus, they are out of reach of most people in developing countries and uninsured people in developed countries.
Thymopentin is a pentatpeptide having activity as an immunomodulator.
P. Costigliola et al. ("Thymopentin (TP-5) Therapy During Lymphadenopathy Syndrome," (1987) J. Exp. Pathol., 3:705-712) reported positive results in the treatment of subjects infected with HIV and exhibiting LAS/ARC with thymopentin.
However, the use of immunomodulators in the treatment of HIV infection has not yet gained widespread medical acceptance.
L-Glu-L-Trp, also known as thymogen, is a dipeptide known to normalize immune system function. The drug was found to be the active principle in an extract of the thymus gland called thymosin. (Morozov et al., United States patent x,070,076.) The dipeptide has been shown to be effective in the treatment of immunodeficient, immunodepressed or hyperactive immune states. (Khavinson et al., WO 92/17191;
Khavinson et al., WO 95/03057; and Morozov et al., United States patent 5,538,951.) Pro-drugs of L-Glu-L-Trp, such as cyclized versions of the dipeptide or linear polymers of the dipeptide, are processed by the body into the active compound.
(Khavinson et al. , WO 93/08815.) Two related compounds, L-Ile-L-Trp and L-Leu-L-Trp, also have been shown to have immunomodulating properties similar to L-Glu-L-Trp. (Khavinson et al., WO 94/20063.) In addition to these properties, L-Glu-L-Trp has anti-angiogenic activity. (Green et al., WO 97/12625.) Other related compounds reported to stimulate the immune response are the tripeptide Pyr-Leu-Trp and phoshoramidon (sugar-Leu-Trp). (Polita et al., United States patent 5,143,903.) However, there is no report of these compounds being tested against HIV infection.
These tryptophan-containing dipeptides are believed to function, at least in part, by reversibly associating with specific cellular receptors, namely "CD2"
receptors, thereby inducing conformation changes in the receptor which "trigger"
intracellular mechanisms resulting in up-regulation of adenylate cyclase and an increase in AMP.
They simultaneously increase the affinity of the CD2 receptor for its "target"
ligand.
This increase in affinity is believed to heighten the interaction between these cells and their natural ligands, thereby facilitating such interaction and encouraging cellular response to such interaction. (Khavinson et al., WO 94/20063.) SLTMMARY OF THE INVENT ION
While L-Glu-L-Trp is known to have properties as an immunomodulator, it has exceptional and unexpectedly good properties in the treatment of retroviral infections, and, especially, in the treatment of HIV infection. L-Glu-L-Trp inhibits retroviral replication in persons infected with HIV and decreases viral load by a factor of ten. More particularly, this invention provides treatments for persons suffering from AIDS complicated by Kaposi's sarcoma. Such individuals have a mortality rate twice that.of persons suffering from AIDS without Kapasi's sarcoma. Quite surprisingly, in cases of AIDS with Kaposi's sarcoma, L-Glu-L-Trp decreases the number of lesions and decreases the mortality rate significantly. While not msnmg CO De trr~mcu uy ui~~~y, 1~
as believed that this effect is due to a synergy between the immunomodulatory and anti-angiogenic properties of L-Glu-L-Trp. Accordingly, this invention is directed to methods of treating HIV infection by administering an effective amount of L-Glu-L-Trp.
In one aspect this invention provides a method for inhibiting retroviral infection in a subject comprising the step of administering to the subject a pharmacologically effective amount of a trytophan-containing compound selected from:
(1) L-Xaa-L-Trp; (2) a cyclic form of L-Xaa-L-Trp; (3) a linear or cyclic polymer of L-Xaa-L-Trp, the polymer having no more than 20 or no more than 10 amino acids;
and (4) a derivative of any of the foregoing compounds which hydrolyses in aqueous solution into any of the foregoing compounds, wherein Xaa is Glu, Ile or Leu. In certain embodiments, the compound is L-Glu-L-Trp; Xaa is Glu; or the compound is L-Xaa-L-Trp. In another embodiment, the compound is a pro-drug analog of the formula selected from (I) Naa-L-Xaa-L-Trp, wherein Xaa is Glu, Ile or Leu and Naa is any amino acid or a monosaccharide; and (2) a derivative of Naa-L-Xaa-L-Trp which hydrolyses in aqueous solution into Naa-L-Xaa-L-Trp. In another embodiment, the compound is an analog having the structure of formula 1:
Y X
C
O
/ O
H ~ O_ NH
O

wherein X and Y are independently selected from H, lower alkyls, esters, amides, halides, carbohydrates or oligodideoxyribose groups, or, together can be a ketone group.
The bond between X or Y and the carbon can be non-hydrolyzable. In that case, X or Y
have a mass of less than about 500 D, preferably less than about 100 D. The bond between X or Y and the carbon can be hydrolizable. In that case, any derivatization will result in a analog compound that, when exposed to water or to an enzyme that breaks a hvdolizable bond, will convert or transform to a hydrate or ketone. Such forms are active.

In another embodiment, the treatment is a prophylactic treatment for inhibiting establishment of infection wherein the subject is not infected with HIV. In another embodiment, the compound is administered intranasally or intravenously . In another embodiment, the effective amount is about 1 ~.g/kg to about 10 mg/kg, about 10 ~,g/kg to about 1 mg/kg or about 100 ~.glkg. In one embodiment, the effective amount is about 10 ~.g/kg to about 1 mg/kg and the compound is administered 3 times per week to 7 times per week. In another embodiment, the subject has a CD4-positive T cell count below about 400 cells per ml. In another embodiment, the subject suffers from Kaposi's sarcoma or an opportunistic infection or an opportunistic malignancy. In another embodiment, the method involves co-administering a reverse transcriptase inhibitor or an HIV protease inhibitor. In another embodiment, the pharmaceutically acceptable carrier is normal saline.
In another aspect this invention provides a kit comprising: (1) a pharmacologically effective amount of a tryptophan-containing compound of this invention; and (2) a label instructing the use of the compound in the treatment of retroviral infection.
This invention also provides the use of a tryptophan-containing compound of this invention in the manufacture of a pharmaceutical composition for the treatment of retroviral infection.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graph showing percent inhibition of splenomegaly by L-Glu-L-Trp and AZT in various studies.
Fig. 2A-2E are graphs showing amounts of HIV RNA, number of raised 2j lesions and CD4 count of AIDSIKaposi sarcoma patients treated with L-Glu-L-Trp. Co-treatment with other HIV therapies also is indicated.
DETAILED DESCRIPTION OF THE INVENTION
I. TRYPTOPHA.N-CONTAINING COMPOUNDS
Tryptophan-containing compounds are useful in the methods of this invention for inhibiting retroviral infection. These tryptophan-containing compounds have the formulae:
(1) L-Xaa-L-Trp, wherein Xaa is GIu, IIe or Leu;

(2) a cyclic form of L-Xaa-L-Trp;
(3) a linear or cyclic polymer of L-Xaa-L-Trp, the polymer having no more than 20 or no more than 10 amino acids; and (4) derivatives of any of the foregoing compounds which hydrolyze in aqueous solution into any of the foregoing compounds.
Regarding cyclized forms of the compound, it is well known in the art of chemistry that peptides frequently exist in solution in equilibrium between linear and cyclized states, equilibrium favoring the linear state. Therefore, in the blood, cyclic L-Xaa-L-Trp would tend to equilibrate into the linear form. Regarding linear or cyclic polymers of L-Xaa-L-Trp, once introduced into the body, these compounds undergo proteolytic degradation, thereby releasing the most active form of the compound, L-Xaa-L-Trp.
Derivatives of L-Glu-L-Trp also are useful in the treatments of this invention. In one embodiment, a derivative is a pharmaceutically acceptable salt of the above compounds. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.
In another embodiment, a derivative is an analog in which the reactive terminal amine or carboxyl groups are derivatized with amides, imides, esters, anhydrides, ethers, methyl or ethyl-alkyl esters, alkyl, aryl or mixed alkyl/aryl moieties in which the formula weight of the entire compound is less than about 5000 Daltons or less than about 1000 Daltons. Such derivatives are expected to equilibrate into the active form by, for example, hydrolysis in the body.
In another embodiment, the derivative is an analog in which L-amino acids are substituted for D-amino acids, such as L-Glu-D-Trp or D-Glu-L-Trp, and analogs of tryptophan such as 5-hydroxy-tryptamine, 5-hydroxy-indol-acetic acid and pyrole analogs in which the nitrogen in the indole ring of Trp is replaced with carbon (formula 1).
In another embodiment, a derivative is a pro-drug that is metabolized into the active form, e. g. , L-Xaa-L-Trp. One important class of analogs are trimeric compounds of the formula Naa-L-Xaa-L-Trp, wherein Naa is any L-amino acid or a mono-saccharide. Naa-L-Xaa-L-Trp also is degraded upon administration to the body by the cleavage of the amino-terminal moiety into the active L-Xaa-L-Trp. Another class of analogs are those in which the compound is derivatized by the covalent attachment of a moiety at the amino or carboxy terminus. Representative examples include HEW, EWEW; GEW, EWKHG, EWKKHG, EW-NH-NH-GHK-NH,, Ac-L-Glu-L-Trp-OH, Suc-EW, Cpr-EW, But-EW, RKEWY, RKEW, KEWY, KEW, PEW.
II. PHARMACEUTICAL COMPOSITIONS AND MODES OF DELIVERY
The compounds of this invention preferably are delivered as pharmaceutical compositions. "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject. The pharmaceutical compositions of this invention comprise a pharmacologically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and excipients, such as a phosphate buffered saline solution, 5 ~ aqueous solution of dextrose, and emulsions, such as an oil/water yr waterloil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995).
Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent.
The compounds of the invention can be formulated for administration in a variety of ways. Typical routes of administration include both enteral and parenteral.
These include, without limitation, subcutaneous, intramuscular, intravenous, intraperitoneal, intramedullary, inuapericardiac, intrabursal, oral, sublingual, ocular, nasal, topical, transdermal, transmucosal, or anal. The mode of administration can be, e.g., via swallowing, inhalation, injection or topical application to a surface (e.g., eyes, mucus membrane, skin) .
Particular formulations typically are appropriate for specific modes of administration. Various contemplated formulations include, for example, aqueous solutions, solid formulations, aerosol, gas, vapor or dry powder formulations and transdermal formulations.
A. Aqueous Solutions for Enteral, Parenteral Or Transmucosal Administration Examples of aqueous solutions include, for example, water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions and the like. A preferred carrier for delivery of the tryptophan-containing compounds of this invention is normal (0.09 %) saline solution.
The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives can also include additional active ingredients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate.
These compositions can be sterilized by conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
Aqueous solutions are appropriate for injection (e.g. intravenous injection).
Aqueous solutions also are useful for enteral administration as tonics and administration to mucous or other membranes as, e.g., nose or eye drops. The composition can contain the compound in an amount of about 1 ~g/ml to about 10 mg/ml, more preferably about 10 ~cg/ml to about 1 mg/ml, e.g., about i00 ~cglml.
B. Solid and Other Non-Aqueous Compositions For Enteral Or Transdermal Delivery Solid compositions are appropriate for enteral administration. They can be formulated in the form of, e.g., pills, tablets, powders or capsules. For solid compositions, conventional solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed.
The carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, maltose, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
A unit dosage form, such as a tablet, can have about 1 ~.g to about 100 mg of the compound.
C. Topical Administration For Transdermat Or Transmucosal Delivery Systemic administration can also be by transmucosal or transdermal means. Transmucosal delivery is particularly attractive for treatment of HIV
infection because it can be self delivered easily.
For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrations are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays, for example, or using suppositories. Transdermal delivery systems can include, e.g., patches.
For topical administration, the agents are formulated into ointments, creams, salves, powders and gels. In one embodiment, the transdermal delivery agent can be DMSO. The compound can be administered in a toothpaste.
D. Delivery By Inhalation For inhalation, the compound is preferably administered in the form of an aerosol or mist. For aerosol administration, the compound preferably is supplied in finely divided form along with a surfactant and propellant.
The surfactant preferably is soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropyiene derivatives of these esters. Mixed esters, such as mixed or natural glycerides, can be employed. The surfactant can constitute 0.1 %-20% by weight of the composition, preferably 0.25 % -5 % .

The balance of the composition is ordinarily propellant. Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure.
Among suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes.

5 Mixtures of the above can also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
A nebulizer or aerosolizer device for administering compounds of this 10 invention typically delivers a dose of about concentration of between about 1 ~.g/m' and about 10 mg/m'.
E, Other Formulations In preparing pharmaceutical compositions of the present invention, it can be desirable to modify the complexes of the present invention to alter their pharmacokinetics and biodistribution. For a general discussion of pharmacokinetics, See, Remington's Pharmaceutical Sciences, supra, Chapters 37-39. A number of methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art. Examples of such methods include protection of the complexes in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers .
The tryptophan-containing compounds of this invention also can be incorporated into foodstuffs. This includes solid foods, such as cereals or chewing gums, as well as liquid foodstuffs, such as mixing the compound with hot water as a tea, or incorporating it in any other beverage. The compound may be incorporated in a soap for transdermal delivery during washing.
III. PROPHYLACTIC OR THERAPEUTIC TREATMENTS FOR INHIBITING
RETROYIRAL INFECTION
This invention provides methods for the prophylactic or therapeutic treatment of retroviral infection, including HIV infection.
"Infection" refers to the multiplication of a parasitic organism, e.g., a virus, in a cell or in the body. A subject is "infected" with a virus if the subject has detectable amount of the virus or antibodies that specifically bind to the virus in their body. A "subject" of treatment is a mammal, including a human. Non-human animals subject to diagnosis or treatment include, for example, domesticated animals such as cats.
"Treatment" refers to prophylactic treatment or therapeutic treatment. A
"prophylactic"
treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
Retroviruses are known to cause a number of diseases. In humans, infection with HIV-1 or HIV-2 can lead to AIDS. Infection with the retrovirus can lead to T cell leukemia. The methods of this invention are useful in inhibiting viral replication of any these retroviruses in any of these diseases. Methods of inhibiting retroviral infection in a subject involve administering to the subject a pharmacologically effective amount of a tryptophan-containing compound of this invention.
Single or multiple administrations of the compositions can be carned out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of a compound of this invention sufficient to treat the patient effectively.
The total effective amount of a compound of the present invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time. One skilled in the art would know that the concentration of a compound of the present invention required to obtain an effective dose in a subject depends on many factors including the age and general health of the subject, the route of administration, the number of treatments to be administered and the judgment of the prescribing physician. In view of these factors, the skilled artisan would adjust the dose so as to provide an effective dose for a particular use.
Because the tryptophan-containing compounds of this invention inhibit infection by retroviruses, they are useful in prophylactic treatments to prevent establishment of infection in a subject who shows no signs of infection, but who may be at risk of exposure. Administration can begin when the person has been exposed or is suspected of having been exposed to the virus. Persons who are continually at risk of WO 99/51256 PC'fNS99/07297 exposure also can be treated with the compound. This includes, for example, health care workers who are exposed to bodily fluids, or intravenous drug users.
In the treatment of HIV infection, an effective amount of a compound of this invention is about 1 ~cg/kg to about 10 mg/kg of the subject body mass. A
more g preferable range is about 10 ~ug/kg to about 1 mg/kg. In clinical trials, about 50 ~ug/kg was found to be an effective amount.
For the therapeutic treatment of a person in whom infection is established, doses can be given at least three times per week and usually not more than once a day (i.e., seven times per week). The course of treatment can continue for months.
The result of such treatment is a decrease in viral load. This decrease can be at least a factor of two and can be at least a factor of 10.
A person who is infected with HN may show no other pathological sign.
However HIV infection usually results in a decrease in CD4-positive cells.
Administration of the compound is useful in such persons as a prophylactic against development of full-blown AIDS.
As the infection progresses, a person's CD4-positive cell count can drop below about 400 cells per mm3 of blood. Such persons are considered to have AIDS .
AIDS patients exhibit immunodeficiency. They are particularly subject to opportunistic infections and to opportunistic malignancies, such as Kaposi's sarcoma. In such cases, the compounds of this invention are useful in their immunostimulatory ability to boost immune response to the infection or malignancy.
Kaposi's sarcoma is an opportunistic malignancy characterized by a profusion of lesions invaded by blood vessels. L-Glu-L-Trp has been shown to be effective in the treatment of Kaposi's sarcoma. Without wishing to be limited by theory, 2~ the anti-angiogenic property of L-Glu-L-Trp may contribute to the effectiveness of the compound in this treatment.
HIV infection currently is being treated with inhibitors of reverse transcriptase and HIV protease inhibitors. In one embodiment, the methods of this invention involve administering the tryptophan-containing compounds of this invention with either or both of these drugs in a treatment regimen.
The following examples are offered by way of illustration, not by way of limitation.

EXAMPLES
I. L-LEU-L-TRP A_ND L-ILE-L-TRP HAVE AN IMIVILTNOPOTENTIATING
ACTIVITY SIMILAR TO L-GLU-L-TRP
Experiments were conducted demonstrating that L-Leu-L-Trp and L-Ile-L-Trp have activity similar to L-Glu-L-Trp in a mouse acute peritonitis model.
The mouse acute peritonitis model is a standard model for testing immune function. It tests the ability of mice to attenuate a microorganism in the presence or absence of a test compound after a challenge with a pathogen and administration of an antibiotic. The efficacy of the antibiotic depends, in part, on immune system function.
Animals having a strong immune response are more capable of withstanding the challenge than those having a weak immune response.
In the study, animals were given various doses of L-Leu-L-Trp or L-Glu-L-Trp. Then, they were inoculated with a 10 x LD50 dose of a pathogenic microorganism, S. aureus. Within an hour, the animals were administered ampicillin.
The number of surviving animals after various numbers of hours was determined.
The shock of infection challenges both the humoral and cell-mediated immune responses.
Therefore, the test is an excellent indicator of overall immune system function.
Treatment with L-Glu-L-Trp, L-Leu-L-Trp and L-Ile-L-Trp provided similar protection against infection. All animals in the control group died as a result of inoculation. Administration of ampicillin alone resulted in about 30 %
survival . In contrast, administration of between 10 mg/kg and 1000 mg/kg of either L-Leu-L-Trp or L-Glu-L-Trp and ampicillin saved over 80% of the animals.
Based on the fact that L-Leu-L-Trp, L-Ile-L-Trp and L-Glu-L-Trp have similar activity in the acute peritonitis model and the fact that this test measures broad aspects of immune system function, one can expect L-Leu-L-Trp and L-Ile-L-Trp to have similar pharmacological function as L-Glu-L-Trp and to be useful in treatments in which L-Glu-L-Trp is useful.

II. L-GLU-L-TRP HAS ACTTVITY IN AN ACCEPTED AIDS MODEL AND
SINBLAR ACTTVITV TO COMPOSITIONS SHOWN TO BE EFFECTIVE
IN THE TREATMENT OF HIV INFECTION
A, Rauscher MuLV Model of Retroviral infection In a marine retrovitus model used to screen treatments of HIV infection, administration of L-Glu-L-Trp inhibited virus proliferation.
The Rauscher MuLV model is established in the scientific community as a model for inhibiting retroviral, and therefore HIV, replication. The FDA
evaluates -results using this model in applications for drugs for the treatment of HIV
infection. The marine leukemia virus, like HIV, is a retrovirus, and it has a pathophysiology that closely pa.Tallels that of HIV . Infection with the marine leukemia virus results in immune incompetence, impairment of the lymphoproliferative system and erythroleukemia.
The coauol group had mean spleen mass of 0.09 gms. The group exposed to the virus inoculum had a mean spleen mass of 2.39 gms. In comparison, animals treated with at least 1 ~.g/kg body mass of L-Glu-L-Trp showed less increase in spleen mass. Most dramatically, mice treated with 10 ~g/kg body mass of L-Glu-L-Trp had mean spleen mass comparable to animals not inoculated with the virus -- 0.08 gms.
These results demonstrate that administration of L-Glu-L-Trp inhibits retrovirus proliferation in the MuLV model. Because the MuLV system is a recognized model for HIV infection, the results of these studies lead to the reasonable conclusion that L-Glu-L-Trp has a therapeutic effect in the treatment of HIV infection.
B, Comparison to thymopentin in a cyciophosphamide-induced immunodeficiency model Another immunomodulating thymic-derived peptide, thymopentin, was known as early as 1987 to increase numbers of lymphocytes and, in particular, to increase numbers of CD4-positive lymphocytes, in HIV-positive subjects exhibiting lymphadenopathy syndrome (LAS) or AIDS-related complex (ARC). Thymopentin is the major active component of a composition called thymostimulin.
L-Glu-L-Trp and thymostimulin were compared for their ability to stimulate T-helper lymphocyte activity in a cyclophosphamide-induced immunodeficiency model. T-helper cells, of course, are CD4-positive. In the assay, L-Glu-L-Trp proved to be active at concentrations about 1000-fold lower than thymostimulin.
L-Glu-L-Trp and thymostimulin were compared for their ability to induce recovery of T-helper function in a cyclophosphamide-induced immunodeficiency animal model. Briefly, mice were immunized with sheep red blood cells on day 1. On day 2, cyclophosphamide was administered to the mice. From days 1-5 L-Glu-L-Trp or thymostimulin was administered to the mice intraperitoneally. T-helper cell activity was then tested with the Jerne plaque forming assay. The results show that L-Glu-L-Trp administered at doses between 0.1 to 10 ~.g/kg body mass had roughly the same activity 10 as the administration of 1 mg/kg body mass of thymostimulin. These results confirm that reasonable evidence from the present results to conclude that L-Glu-L-Trp is an immunomodulating drug.
In conclusion, thymopentin is known to stimulate production of T-helper cells in immunodeficient HIV subjects. The evidence above shows that both L-Glu-L-15 Trp and thymopentin are immunomodulators and stimulate production of T-helper cell activity in an immunodeficiency animal model.
III. L-GLU-L-TRP IS EQUALLY OR MORE EFFECTIVE AT INHIBITING
SPLENOMEGALY IN THE RAUSCHER MULV MODEL, THAN IS AZT
Figure 1 is a graph entitled "L-Glu-L-Trp Influence Inhibiting Splenomegaly in the Murine Leukemia (Rauscher) Mouse Model. " The graph presents the results of three studies on the ability of L-Glu-L-Trp or AZT to inhibit splenomegaly as a function of dose delivered. Increased percentages of inhibition of splenomegaly indicate increased inhibition of retroviral replication. Note that the dosages for L-Glu-L-Trp are plotted in terms of ~cg/kg, while the dosages for AZT are plotted in terms of mg/kg. That is to say, L-Glu-L-Trp is compared with AZT delivered at dosages one thousand times higher. The data presented in Fig. 1 are derived from studies conducted under contract at three institutions: Southern Research Institute ("SRI"), Viromed, and Bio Research Laboratories (~BRL"). The studies were conducted using routine protocols for the MuLV model. As indicated in the graph, the studies differed according to dose schedule. The study conducted at SRI involved a direct comparison of the ability of AZT and L-Glu-L-Trp to inhibit splenomegaly. The studies at BRL and Viromed examined only L-Glu-L-Trp and are presented for purposes of corroboration.

The data presented in Fig. 1 show that AZT tests positive in the Rauscher MuLV model for the ability to inhibit splenomegaly. More specifically, AZT, represented by red triangles, inhibits splenomegaly maximally at about 40 % ( 100 mg/kg in this study). Thus, AZT, a compound that tests positively in the model, is currently being used in the treatment of HIV infection to inhibit retroviral replication.
The data in Fig. 1 also show that L-Glu-L-Trp is equally or more effective than AZT in inhibiting splenomegaly in the Rauscher MuLV model. More specifically, in a direct comparison between AZT (red triangles) and L-GIu-L-Trp (green circles, SRi GIu-Trp mcg/kg), L-Glu-L-Trp showed a greater maximal inhibition of splenomegaly than AZT, about 50% compared with about 40%. Furthermore, L-Glu-L-Trp showed maximal inhibition at about 2000-fold lower concentrations than AZT, that is, about 50 ~,g/kg compared with about 100 mg/kg. These results provide further supporting evidence of the effectiveness of L-Glu-L-Trp in the treatment of HIV
infection.
The data in Fig. 1 also show the reproducibility of activity for L-GIu-L-Trp between the several studies. More specifically, in each of the three studies, L-GIu-L-Trp had maximal inhibitory activity in the range of about 10 ~ug/kg to about ~,g/kg. It should be noted that, because the studies involved different administration regimens and involved methods in which the ability of animal groups to become infected with the virus varied, there is some scatter in the results.
In conclusion, this evidence shows that (1) AZT tests positive for inhibition of splenomegaly (an indicator of retroviral reproduction inhibition) in the Rauscher MuLV model; (2) L-Glu-L-Trp is equally or more effective than AZT at inhibiting splenomegaly in the Rauscher MuLV model and (3) the ability of L-Glu-L-Trp to inhibit splenomegaly in the Rauscher MuLV model is corroborated by three different laboratories .
IV. L-GLU-L-TRP INHIBITS HIV VIRAL REPLICATION IN HUMANS AND
DECREASES NUMBERS AND SEVERITY OF CANCEROUS LESIONS IN
PATIENTS WITH KAPOSI'S SARCOMA, A FREQUENT
COMPLICATION OF AIDS
L-Glu-L-Trp was tested in a phase I/II investigation of the AIDS related malignancy known as Kaposi's sarcoma. The protocol involved patients who have acquired a type of malignancy called Kaposi's sarcoma, which commonly occurs with AIDS patients who are at an advanced stage of their disease. Every patient in the study must have AIDS in order to be enrolled in the study protocol. Most of the patients already were taking protease inhibitors. Study subjects are assigned to a specific dose regimen of L-Glu-L-Trp to be given intranasally in a single daily dose, either every other day, or on a schedule where they take the product for 5 consecutive days, followed by a rest off of the peptide for 5 days, after which the cycle is repeated.
During the course of their therapy, the subjects are closely monitored to determine viral load and to determine if the tumor changes (for the better or worse).
(Other laboratory studies are conducted at periodic intervals.) The test for viral load for HIV was conducted by drawing blood from patients and storing it for analysis in a "batch" processing procedure. The viral copies are determined at a contract laboratory experienced in such procedures (Southern Research Institute) at a time when a sufficient number of specimens are collected. The procedure done in this manner is known to be more reliable and accurate, and has the additional advantage of making the test procedure economical.
The effects of treatment with L-Glu-L-Trp on five study subjects are shown here and in Figs. 2A-2E. The figures for each subject reveal the information as follows: (# raised cancer lesions, absolute CD4 count, medications and time of change in medications, viral copies HIV-RNA, and months from start time T=O on the study peptide L-Glu-L-Trp);
~ The horizontal axis falon~ the bottom of the graph) shows the time months and a point to the left "0" when they were first started on the peptide L-Glu-L-Trp. The time interval to the left of the starting time, represents the time prior to starting on L-Glu-L-Trp for which no change, or adverse changes were being followed, and at the ton of each ~rayh is a _horizontal dark line indicating the medication (protease inhibitor) then were taking, and the time when a change in medication occurred. In the example Pt b7/J-S, the patient was taking no inhibitor.
~ L,~t vertical axis: indicates # Raised lesions lsauare ls) .... darkest solid line on graph), and the absolute CD4 count ldiamond (~ ) ..... dash line).

~ Ri;~ht vertical axis: indicates HIV RNA (conies per ml) (sauare (~))....
Lighter solid line.
The slides are labeled as follows and represent 5 human subjects where evidence is strong showing L-Glu-L-Trp's activity in reducing viral copies of HIV: Pt 61/TAR, Pt 62/F-G, Pt 64/JFS, Pt 67/J-S, Pt 68/IRD.
1. Fig. 2A: Pt 61/TAR
The patient was on Crixivan~ (Merck & Co.) (indinavir sulphate) for a period of 4 months before starting on L-Glu-L-Trp. During that period, his viral load had remained essentially unchanged. His cancer situation had worsened with a substantial increase in the number of raised lesions. This subject had a very low CD4 count, approximately 8 to 40 over the entire period recorded.
After starting on the peptide at T=O, the viral load droped from 194,097 to 31,000 copies per ml. Protease inhibitors were changed to Norvir +
Saquinavir (possibly because of patient intolerance of Crixivan), and the picture is not interpretable since one cannot say how much effect is due to the new combination and how much is due to L-Glu-L-Trp. The patient responded to treatment with L-Glu-L-Trp reducing cancer lesions and sustaining remission out to 7 months tracking.
2. Fig. 2B: Pt 63/F-G
The patient had been taking the protease inhibitor Saquinavir for 2 months prior to starting on the protocol with L-Glu-L-Trp. Cancer lesions had remained unchanged, and viral load was increasing.
The viral load reversed and dropped from 4,116 to 784 copies per ml on L-Glu-L-Trp. The cancer lesions responded and the patient is in complete remission, sustained to 8 months tracking.

3. Fig. 2C: Pt 64/JFS
The patient was on Crixivan for a period of 1 month before starting on L-Glu-L-Trp. The cancer and HIV viral load were both rapidly increasing.
After starting on the protocol, the cancer lesions reduced and he went into complete remission sustained to 7 months tracking. The viral load for HIV
dropped from 19,326 to 1,138 copies per ml. Note that while on Crixivan, the viral load had increased from 641 to 19,326.
4. Fig. 2D: Pt 67/J-S
The patient was taking no protease inhibitors prior to the study, and had no change in lesions prior to the start of the study.
After starting therapy with L-Glu-L-Trp the viral load dropped from 12,655 to 701, and then rose to 2,445 copies per ml while on the study out to 2 months.
More data is anticipated within next batch of blood studies to be processed.
The tumor responded and sustained response to 5 months tracking.
5. Fig. 2E: Pt 68/IRD
The patient had been on Crixivan for 12 months prior to the start of L-Glu-L-Trp. During this period there was a constant and elevated viral load near 1 million copies per ml.
After starting on L-Glu-L-Trp the viral load dropped from 947,913 to 166,121 copies per ml HIV-RNA. The cancer showed response sustained to 5 months tracking.
V . SIIwIMARY
In summary, L-Leu-L-Trp and L-Ile-L-Trp have similar activity to L-Glu-L-Trp, as demonstrated by the acute peritonitis model, a model of general immune system function. L-Glu-L-Trp demonstrates the ability to inhibit proliferation of retroviruses in an accepted model of HIV infection. The administration of L-Glu-L-Trp as a nose drop inhibits viral replication. Of the five human subjects presented, each responded to peptide administration with a nearly 10-fold drop in viral copies per ml. A

response in tumor reduction is noted as well. Although most of the patients were taking protease inhibitors, it will be seen that even while on the protease inhibitors, the viral loads were not under control, and that it was after the peptide was administered that the viral load (copies per ml} dropped by nearly 10-fold. One of the subjects was taking 5 only the peptide L-Glu-L-Trp (Pt 67/J-S), and was not on protease inhibitors, and dropped the viral load by nearly 10-fold, after starting on the study.
The present invention provides novel materials and methods for the prophylactic and therapeutic treatment of retroviral and, in particular, HN
infection.
10 While specific examples have been provided, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.
15 All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted.
Applicants do not admit by their citation of various references in this document that any particular reference is "prior art" to their invention.

Claims (29)

WHAT IS CLAIMED IS:

1. A method for inhibiting retroviral infection in a subject comprising the step of administering to the subject a pharmacologically effective amount of a compound selected from:
(1) L-Xaa-L-Trp;
(2) a cyclic form of L-Xaa-L-Trp;
(3) a linear or cyclic polymer of L-Xaa-L-Trp, the polymer having no more than 20 or no more than 10 amino acids; and (4) a derivative of any of the foregoing compounds which hydrolyses in aqueous solution into any of the foregoing compounds, wherein Xaa is Glu, Ile or Leu.

2. The method of claim 1 wherein the subject is a feline and the retroviral infection is feline leukemia virus.

3. The method of claim 1 wherein the subject is a human.

4. The method of claim 3 wherein the retroviral infection is HLVP1 infection.

5. The method of claim 3 wherein the retroviral infection is HIV
infection.

6. The method of claim 5 wherein the compound is L-Glu-L-Trp, or a pharmaceutically acceptable salt thereof.

7. The method of claim 5 wherein Xaa is Glu.

8. The method of claim 5 wherein the compound is L-Xaa-L-Trp or a pharmaceutically acceptable salt thereof.

9. The method of claim 6 for inhibiting establishment of infection wherein the subject is not infected with HIV.

10. The method of claim 6 for therapeutic treatment wherein the subject is infected with HIV.

11. The method of claim 6 wherein HIV is HIV-1.

12. The method of claim 6 wherein HIV is HIV-2.

13. The method of claim 6 wherein the compound is administered in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of the compound.

14. The method of claim 6 wherein the compound is administered intranasally or intravenously.

15. The method of claim 6 wherein the effective amount is about 1 µg/kg to about 10 mg/kg.

16. The method of claim 6 wherein the effective amount is about 10 µg/kg to about 1 mg/kg.

17. The method of claim 10 wherein the effective amount is about 10 µg/kg to about 1 mg/kg and the compound is administered 3 times per week to 7 times per week.

18. The method of claim 10 wherein the subject has a CD4-positive T
cell count below about 400 cells per ml.

19. The method of claim 10 wherein the subject suffers from Kaposi's sarcoma.

20. The method of claim 10 wherein the subject suffers from an opportunistic infection or an opportunistic malignancy.

21. The method of claim 10 further comprising co-administering a reverse transcriptase inhibitor or an HIV protease inhibitor.

22. The method of claim 13 wherein the pharmaceutically acceptable carrier is normal saline.

23. A kit comprising:
(1) a pharmacologically effective amount of a compound selected from:
(a) L-Xaa-L-Trp;
(b) a cyclic form of L-Xaa-L-Trp;
(c) a linear or cyclic polymer of L-Xaa-L-Trp, the polymer having no more than 20 or no more than 10 amino acids; and (d) a derivative of any of the foregoing compounds which hydrolyses in aqueous solution into any of the foregoing compounds, wherein Xaa is Glu, Ile or Leu; and (2) a label instructing the use of the compound in the treatment of retroviral infection.

24. The kit of claim 23 wherein the retroviral infection is HIV
infection.

25. The kit of claim 24 wherein the compound is L-Glu-L-Trp or a pharmaceutically acceptable salt thereof.

26. The kit of claim 25 wherein the compound is formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of the compound.

27. The kit of claim 26 wherein the pharmaceutically acceptable carrier is normal saline.

28. A method for inhibiting retroviral infection in a subject comprising the step of administering to the subject a pharmacologically effective amount of a compound selected from:
(1) Naa-L-Xaa-L-Trp, wherein Xaa is Glu, Ile or Leu and Naa is any amino acid or a monosaccharide; and (2) a derivative of Naa-L-Xaa-L-Trp which hydrolyses in aqueous solution into Naa-L-Xaa-L-Trp.

29. A kit comprising:
(1) a pharmacologically effective amount of a compound selected from:
(a) Naa-L-Xaa-L-Trp, wherein Xaa is Glu, Ile or Leu and Naa is any amino acid or a monosaccharide; and (b) a derivative of Naa-L-Xaa-L-Trp which hydrolyses in aqueous solution into Naa-L-Xaa-L-Trp; and (2) a label instructing the use of the compound in the treatment of retroviral infection.