AU763289B2 - Use of GM-CSF to inhibit development of drug resistance in HIV+ patients - Google Patents

Description

WO 00/27196 PCT/US99/24857 Title USE OF GM-CSF TO INHIBIT DEVELOPMENT OF DRUG RESISTANCE IN HIV' PATIENTS Background of the Invention Patients infected with Human Immunodeficiency Virus. (HIV) experience a variable but progressive decline in immune function, resulting in clinically apparent opportunistic infections and other diseases. (Crowe et al., J. Acquir. Immune Defic.

Syndr. 4:770-76, 1991; Moss etal., AIDS 3:55-61, 1989). The onset of severe immunodeficiency in HIV-infected individuals is generally accompanied by a marked increase in viral load and a dramatic decline in circulating CD4' T-lymphocytes. Indeed, one of the clinical criteria for the diagnosis and reporting of adult immunodeficiency syndrome, or AIDS (as established by the Centers for Disease Control and Prevention) is a decrease in the number of CD4' T-lymphocytes to <200 cells/mL. Normal CD4' Tlymphocyte cell counts in healthy HIV' individuals range between 800 and 1600 cells/mL.

A number of drugs have been used to treat HIV' patients. Antiretroviral agents that include protease inhibitors, nucleoside analogs that inhibit reverse transcriptase, and non-nucleoside reverse transcriptase inhibitors, have been administered individually or in combination. The early results achieved with combination therapy, commonly involving administration of three anti-retroviral agents, constituted a significant therapeutic advance, and offered hope for prolonging survival of HIV' patients. Such drug cocktails typically include more than one class of antiretroviral agent, such as a protease inhibitor and at least one (often two) nucleoside reverse transcriptase inhibitors.

Unfortunately, antiretroviral drugs have not achieved complete reconstitution of the immune function. Moreover, inhibition of viral replication by these agents can be temporary, due to the evolution of resistant strains of virus that can grow in the presence of the antiretroviral agents (Cameroni etal., Third Human Retroviral Conference, January 1996, Abstract #LB6a; Lerner et al., Ann. Intern. Med., 129:573-778, 1998; Cohen, O. and A. Fauci, New England J. of Med., 339:341-343, July 30, 1998; and Richman, Scientific American, p. 88, July 1998). Non-compliance with a prescribed treatment regimen is believed to be one factor contributing to the emergence of resistant strains (Lerner et al., supra).

Mutations in the viral genome that alter the amino acid sequence of the viral reverse transcriptase enzyme may confer resistance to drugs that act by inhibiting reverse transcriptase. Likewise, mutations that result in certain changes in the amino acid sequence of HIV-encoded protease may confer resistance to drugs that act by inhibiting that protease. Mutations in the reverse transcriptase gene of multi-drug resistant HIV strains, isolated from patients receiving combination therapy with nucleoside analogs, are discussed in Iversen et al. Virol., 70:1086-1090, February 1996). Molla et al. (Nature Medicine, 2:760-766, July 1996) describe multiple mutations in the HIV protease gene, that confer resistance to ritonavir. Further discussion of mutations associated with drug resistance Multi-drug resistant HIV strains have evolved in patients receiving combination therapy with antiretroviral agents. Further, cross-resistance has developed, wherein an HIV strain resistant to one drug is found to be resistant to other drugs in the same class.

S\ To illustrate, an HIV strain that evolves in a patient treated with one nucleoside reverse •a 15 transcriptase inhibitor may exhibit resistance, not only to the particular drug administered to the patient, but also to other nucleoside reverse transcriptase inhibitors. The evolution of multi-drug resistant and cross-resistant strains has constrained treatment options.

Reports of transmission of drug resistant HIV strains from one individual to another raise further concerns.

20 Significant advances have been made in the treatment of HIV-infected patients.

However, a number of challenges remain, not the least of which is the evolution of drugresistant strains of the virus.

Oa 00 a 4« *a P:\WPDOCS\CRN\P-it\Spad773837O pq2a -deddoc.22O5/3 -2a- Summary of the Invention The present invention provides a method for inhibiting the development of drug-resistant human immunodeficiency virus (HIV) in an HIVinfected patient, by administering granulocyte-macrophage colony stimulating factor (GM-CSF) in conjunction with one or more anti-HIV drugs, to the patient. In particular, embodiments, the anti-HIV drugs are antiretroviral agents selected from protease inhibitors, nucleoside reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors.

An aspect of the present invention is a method of inhibiting the development of a drug-resistant strain of human immunodeficiency virus (HIV) in a human, comprising: administering at least one anti-HIV drug to an HIV+ human who does not have AIDS; and administering granulocyte-macrophage colony stimulating factor (GM- CSF) to said human, wherein the GM-CSF and anti-HIV drugs are administered for at least six months according to a regimen wherein the patient receives an anti-retroviral drug for no more than five consecutive days without also receiving at least one dose of GM-CSF; the GM-CSF is administered in an 20 amount sufficient to inhibit the development of an HIV strain to said drug in said human, and wherein said anti-HIV drugs are selected from the group consisting of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors.

A further aspect of the invention is a method of prolonging the therapeutic effectiveness of a regimen for treating an HIV-infected human, comprising adminstering GM-CSF in conjunction with one or more anti-retroviral drugs, wherein the GM-CSF is administered in an amount and frequency sufficient to inhibit the development of HIV resistant to at least one of said anti-retroviral drugs, in said human, and wherein said anti-retroviral drugs are selected from the group consisting of non-nucleoside reverse transcriptase inhibitors and protease inhibitors.

P:AWPDOCS\CRNPunital\Sp,7738370 page 2 a mmodcd.d-22IO5/03 A further aspect of the invention is a method of inhibiting mutations in the reverse transcriptase gene of human immunodeficiency virus (HIV), wherein said mutations confer resistance to an antiretroviral drug, said method comprising administering GM-CSF in conjunction with at least two antiretroviral drugs that inhibit reverse transcriptase, to an HIV patient who does not have AIDS, wherein the GM-CSF is administered in an amount and frequency sufficient to inhibit the development of HIV resistant to at least one of said drugs, in said human.

Detailed Description of the Invention The present invention provides a method for inhibiting the development of drug-resistant HIV strains in a HIV infected human, by administering a granulocyte- WO 00/27196 PCT/US99/24857 macrophage colony stimulating factor (GM-CSF) to the human, in conjunction with one or more anti-HIV drugs. GM-CSF is administered in an amount and frequency, and for a time period, sufficient to inhibit evolution of drug-resistant HIV in the human patient.

The method provided herein thus serves to maintain or prolong the therapeutic effectiveness of anti-HIV drugs, such as antiretroviral agents.

In the study described in example one below, a lower frequency of mutations associated with AZT resistance was found in HIV strains from patients treated with AZT and GM-CSF, compared to controls who received AZT and a placebo. GM-CSF hindered the evolution of drug resistance genotypes in HIV strains harbored by the patients.

Since mutations conferring drug resistance may occur soon after treatment with antiretroviral agents begins, GM-CSF preferably is added to a patient's first treatment regimen that includes antiretroviral agent(s). In one embodiment of the invention, patients treated in accordance with the method provided herein are HIV', but have not progressed to AIDS.

GM-CSF employed in the practice of the invention may be any pharmaceutically safe and effective human GM-CSF, or any derivative thereof having the biological activity of human GM-CSF. In a presently preferred embodiment, the GM-CSF used in the practice of the subject methods is recombinant human GM-CSF (rhu GM-CSF), such as LEUKINE® (Immunex Corporation, Seattle, Washington). LEUKINE® (sargramostim) is a biosynthetic, yeast-derived, recombinant human GM-CSF, consisting of a single 127 amino acid glycoprotein that differs from endogenous human GM-CSF by having a leucine instead of an arginine residue at position 23. Other natural and synthetic GM- CSFs, and derivatives thereof having the biological activity of natural human GM-CSF, may be employed.

The term "yeast-derived GM-CSF" as used herein means that the recombinant GM-CSF is produced in yeast host cells. Suitable yeast host cells include, but are not limited to, Saccharomyces cerevisiae cells. Although GM-CSF may be produced in prokaryotic host cells, such as E. coli, expression in eukaryotic host cells, yeast or mammalian cells, is preferred.

U.S. Patent 5,391,485, hereby incorporated by reference in its entirety, provides sequence information for cloned human GM-CSF cDNA and a recombinant human GM- CSF polypeptide encoded thereby. U.S. Patent 5,391,485 also discloses certain analogs of GM-CSF (including the above-described analog in which the arginine residue at WO 00/27196 PCT/US99/24857 position 23 of the native sequence is replaced with a leucine residue), and methods for producing such analogs. The arginine residues at positions 23 and 24 of the native sequence constitute a site susceptible to cleavage by KEX2 protease. The KEX2 site can be inactivated by deleting either or both of the arginine residues at positions 23 and 24, or by replacing either or both arginines with a non-basic residue. Inactivating the KEX2 protease site is advantageous when the GM-CSF is to be expressed in yeast cells, such as S. cerevisiae cells.

LEUKINE® is currently available for use in promoting myeloid cell recovery following bone marrow transplant post-myeloablative therapy for the treatment of malignancies. LEUKINE® has been shown to exhibit the same hematopoietic effects as those induced by endogenous GM-CSF, namely, the stimulation of progenitor cells committed along the granulocyte-macrophage pathway to form neutrophils, monocytes, macrophages, and eosinophils (Technical Product Monograph: LEUKINE® Liquid, Immunex Corp., Seattle, WA, 1997, which is herein incorporated by reference).

LEUKINE®, like endogenous GM-CSF, also promotes the differentiation of progenitor cells giving rise to erythrocytes and megakaryocytes (Ibid.) In addition to stimulating hematopoiesis, LEUKINE® enhances many of the functional activities of mature neutrophils, monocytes and macrophages, such as chemotaxis, growth factor secretion, anti-tumor activity, antibacterial activity, and antifungal activity (Ibid.).

In accordance with the present invention, GM-CSF is administered to an HIV' patient in conjunction with at least one additional drug used to treat HIV infection an "anti-HIV" drug). In particular embodiments, the drug(s) is/are antiretroviral agents. The term "antiretroviral agent", as used herein, includes any pharmacological, biological or cellular agent that has demonstrated the ability to inhibit HIV replication.

A number of antiretroviral agents currently in use act by binding to particular HIV enzymes, thereby hindering the activity of those enzymes. Examples of antiretroviral agents, include, but are not limited to, nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, protease inhibitors. Specific examples of nucleoside reverse transcriptase inhibitors include zidovudine (AZT), didanosine (ddl), lamivudine (3TC), stavudine (d4T), and dalcitabine (ddC). Specific examples of nonnucleoside reverse transcriptase inhibitors include nevirapine and delavirdine. Specific examples of protease inhibitors include indinavir, nelfinavir, ritonavir, and saquinavir.

Further examples of anti-HIV drugs that may be co-administered with GM-CSF in accordance with the present invention are HIV integrase inhibitors, and agents that block WO 00/27196 PCT/US99/24857 viral entry through chemokine receptors. Examples of chemokine receptor blocking agents are small peptides known as CXCR4 and CCR4 blocking peptides. Additional antiretroviral agents not yet approved by the Food and Drug Administration may also be effective.

Methods of the present invention include, but are not limited to, a method for treating an HIV' patient, comprising administering to the patient GM-CSF and at least one antiretroviral drug selected from nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors. Particular therapeutic regimens include GM-CSF and two or three antiretroviral drugs. In particular embodiments, GM-CSF, at least one nucleoside reverse transcriptase inhibitor, and at least one protease inhibitor, are administered. A further embodiment involves administering GM-CSF, two nucleoside reverse transcriptase inhibitors, and a protease inhibitor.

A preferred example of such a drug combination involves administering GM-CSF, the protease inhibitor indinavir, and the nucleoside reverse transcriptase inhibitors zidovudine (AZT) and lamivudine (3TC). Further examples of drug combinations that may be administered in conjunction with GM-CSF include, but are not limited to, cocktails comprising AZT; AZT and ddl; AZT and ddC; AZT and 3TC; or AZT and saquinavir. Another example of an antiretroviral drug cocktail includes zidovudine (AZT), didanosine (ddl), and lamivudine (3TC).

GM-CSF may be administered in conjunction with other drugs against which resistance may develop in HIV patients. Resistance to a particular drug may be recognized as a potential problem if such resistance has developed in other HIV' patients, for example. As discussed above, resistance to agents that are reverse transcriptase inhibitors or protease inhibitors has been associated with certain mutations in the viral reverse transcriptase gene or the viral protease gene, respectively. GM-CSF may be coadministered with other types of drugs, when HIV resistance to the drug is attributable to mutation(s) in a viral gene, particularly a viral gene encoding an enzyme.

In accordance with the present invention, GM-CSF is administered to an HIVinfected patient in amounts and for a time sufficient to inhibit the development of resistance to an anti-HIV drug with which the patient is being treated. The optimal dose, frequency of administration, and duration of treatment with GM-CSF may vary from patient to patient.

WO 00/27196 PCT/US99/24857 In particular embodiments of the method provided herein, GM-CSF is administered in doses greater than or equal to about 100 micrograms (mcg), preferably greater than or equal to about 150 mcg, and more preferably greater than or equal to about 250 mcg. In another embodiment, the dosage of GM-CSF is at least 125 mcg/m 2 examples of suitable dosages include 125 mcg/m 2 or 250 mcg/m 2 In particular embodiments of the method provided herein, GM-CSF is administered at a frequency of least once per week, at least two times per week, or at least three times per week. Preferably, GM-CSF is administered two or three times per week.

In other alternatives, GM-CSF is administered once per day or more. In particular embodiments, GM-CSF is administered for a period of time greater than about four weeks, preferably greater than about eight weeks, more preferably for at least six months, and even more preferably for at least one year.

Since the degree of glycosylation of biosynthetic GM-CSFs appears to influence half-life, distribution, and elimination, the most effective dose of GM-CSF may vary depending on the source of the GM-CSF (Lieschke and Burgess, N. Engl. J. Med. 327:28- 1992; Dorr, Clin. Ther. 15:19-29, 1993; Horgaard et al., Eur. J. Hematol. 50:32- 36, 1993). The degree of glycosylation may vary according to the type of host cells in which recombinant GM-CSF is expressed, for example. The optimal dose of GM-CSF may be adjusted, if a GM-CSF other than LEUKINE® is employed.

The dosage of anti-retroviral agent(s) is chosen according to standard practices and conventional criteria. GM-CSF may be added to a standard treatment regimen that involves administration of antiretroviral drugs.

It should be understood that the optimal dose, frequency of administration, and duration of treatment may vary from patient to patient, depending on the individual patient's condition and response to the treatment, and is best determined by monitoring the patient's response during the course of the treatment. Other factors affecting dosage include, for example, the route of administration, drug formulation, and the other anti- HIV drugs with which the patient is being treated. It should further be understood that administration of higher doses may permit less frequent administration, and lower doses may require more frequent administration in order to achieve a clinically significant response.

GM-CSF may be administered by any suitable means. Suitable modes or routes of administration include, but are not limited to, parenteral, oral, by inhalation (via nose or mouth), or infusion. The term "parenteral" includes injection, by subcutaneous, WO 00/27196 PCT/US99/24857 intravenous, intradermal, or intramuscular routes. Subcutaneous injection is preferred. In particular embodiments, GM-CSF is administered nasally, using an atomizer, or orally, wherein the patient swallows a liquid preparation of GM-CSF.

Any suitable formulation of GM-CSF may be employed. Such formulations may include a physiologically acceptable diluent, excipient, or carrier, for example.

Formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16' Ed., 1980, Mack Publishing Company, Easton, PA.

Examples of GM-CSF formulations include sustained release or slow release formulations, such as those described in WO 97/13502, hereby incorporated by reference.

In another alternative, GM-CSF is administered in the form of a bolus.

A preferred formulation of GM-CSF is described in example 1 below. One alternative is LEUKINE®Liquid (Immunex Corporation, Seattle, WA), which is a sterile, preserved injectable solution containing 500 mcg Sargramostim; 40 mg mannitol, USP; mg sucrose, NF; and 1.2 mg tromethamine, USP; in Bacteriostatic Water for Injection, which includes 0.9% benzyl alcohol.

Treatment in accordance with the present invention advantageously commences shortly after HIV infection, and preferably is initiated in a patient whose health has not significantly deteriorated. AIDS patients may be treated in accordance with the present invention; however, treatment preferably is initiated in an HIV' individual whose condition has not progressed to AIDS. The therapeutic method provided herein preferably is employed in the induction phase of treatment, rather than being delayed until the intensification or salvage phases. Even more preferably, treatment commences during the acute infection stage, immediately after the patient is infected with the virus in the first 3 to 6 weeks post-infection).

In one embodiment, treatment in accordance with the method provided herein is initiated in a patient who has not developed neutropenia. In another embodiment, a patient has a CD4' T-lymphocyte count (also referred to as a T cell count) greater than 200 cells/mm 3 preferably greater than 300 cells/mm 3 most preferably greater than 500 cells/mm 3 when treatment with GM-CSF and the other drug(s), in accordance with the present invention, commences. A "CD4' T-lymphocyte count" means the number of circulating CD4' T-lymphocytes in the patient's blood, expressed as CD4' T-lymphocyte cells per unit volume. Generally, CD4' lymphocyte counts are expressed as cells/mm 3 of whole blood.

WO 00/27196 PCTIUS99/24857 The method provided herein involves administering GM-CSF in conjunction with one or more anti-HIV drugs. As used herein, such reference to administration of GM- CSF in conjunction with other drug(s) is not limited to simultaneous administration of GM-CSF and other drug(s). Rather, GM-CSF and the other drug(s) are included in the same treatment regimen, as discussed herein.

Advantageously, a patient receives an anti-retroviral drug for no more than 5 days, preferably for no more than 3 days, without also receiving at least one dose of GM-CSF.

A patient preferably receives an initial dose of GM-CSF within one day of starting treatment with an antiretroviral agent. In one embodiment of the method provided herein, treatment with GM-CSF, and with at least one antiretroviral agent, commence on the same day.

Patients preferably have not already developed resistance to any antiretroviral drug, when treatment in accordance with the present invention commences. In a preferred embodiment, no antiretroviral agent has been administered to the patient, prior to administration of GM-CSF in conjunction with at least one antiretroviral agent.

Cross-resistance, wherein a patient treated with one drug develops resistance not only to the administered drug, but also to other drugs in the same class, has been reported.

To illustrate, a patient treated with one protease inhibitor may become resistant to several protease inhibitors. Thus, in one embodiment, a drug chosen for administration in conjunction with GM-CSF, in accordance with the present invention, is from a class of drugs not previously administered to the patient.

The presence of a drug-resistant HIV strain in a patient may be detected by any suitable technique. In one approach, the presence of mutations that confer drug resistance are detected by genotype analysis, conducted on HIV isolated from a patient. Genotypes of drug resistant retroviral strains are discussed in Schinazi et al., "Mutations in retroviral genes associated with drug resistance", International Antiviral News, 4:95-107, 1996; and Mellors et al., "Mutations in HIV-1 reverse transcriptase and protease associated with drug resistance", International Antiviral News 3:8-13, 1995; Molla et al., Nature Medicine, 2(7):760-766, July 1996; and Iversen et al., J. Virol. 70:1086-1090, February 1996, each of which is incorporated by reference herein.

One genotype analysis technique that may be employed to detect HIV strains harboring mutations that confer drug resistance is described in Stuyver et al., "Line Probe Assay (LiPA) for the detection of drug-selected variants in the HIV reverse transcriptase gene", International Antiviral News, 5(3):38-40, 1007, and Stuyver et al., Antimicrobial WO 00/27196 PCT/US99/24857 Agents and Chemotherapy, 41(2):284-291, Feb. 1997, which are hereby incorporated by reference. The assay technique described in the Stuyver et al. references, supra, employs specific probes to detect nucleotide polymorphisms at resistance-related codons.

To illustrate, the technique of Stuyver et al. may be employed to detect resistancerelated mutations in the reverse transcriptase gene of an HIV strain. Briefly, viral RNA is isolated, and cDNA is synthesized on the RNA template. DNA fragments corresponding to the relevant region of the HIV reverse transcriptase gene are isolated and amplified by PCR. The resulting fragments are biotinylated and hybridized with immobilized oligonucleotide probes, chosen to detect polymorphisms associated with resistance to drugs that inhibit reverse transcriptase. Detection of PCR fragment/probe hybrids involves biotin-streptavidin coupling, using a colorimetric system.

Commercial kits are available for genotype analysis, and may be employed for detecting mutations associated with drug resistance in HIV strains. The use of one such kit, available from Abbott Laboratories, Abbott Park, Illinois, is illustrated in Example 1.

The kit-based assay is based on the principles of the line probe assay technique described in Stuyver et al., supra.

The present invention provides a method for inhibiting mutation(s) in the reverse transcriptase gene of human immunodeficiency virus (HIV), wherein said mutations confer resistance to an antiretroviral drug, said method comprising administering GM- CSF in conjunction with an antiretroviral drug that inhibits reverse transcriptase, to an HIV' patient. Likewise, a method for inhibiting mutations in an HIV protease gene comprises administering GM-CSF in conjunction with an antiretroviral drug that acts by inhibiting an HIV protease, to an HIV patient. GM-CSF is administered in an amount and frequency sufficient to inhibit the development of HIV resistant to the drug, in said human.

The term "inhibit development of drug-resistant HIV" as used herein includes reduction or delay in the occurrence of drug resistance. A delay in evolution of drug resistant strains is beneficial to HIV' patients, since the duration of therapeutic effectiveness of a particular treatment regimen is prolonged, and the need to identify and substitute other drugs, to which the patient is not resistant, is postponed.

WO 00/27196 PCT/US99/24857 Example 1 Administration of GM-CSF and Antiretroviral Agent(s) to AIDS Patients 105 HIV-seropositive individuals diagnosed with AIDS were enrolled in a placebo-controlled, double-blind study, conducted as follows.

The subjects, ages 18 to 55, were randomized to receive 125 mcg/m 2 of yeastderived GM-CSF (LEUKINE®) or a placebo. GM-CSF or placebo was administered by subcutaneous injection two times per week for twenty-six weeks, by medical staff at study sites. All subjects received AZT at a dose of 300mg/day. AZT, which is 3'-azido- 2',3'-dideoxythymidine, also known as azidothymidine or zidovudine, is a thymidine analog. Certain of the subjects received additional antiretroviral drugs, as discussed below.

The formulation of recombinant human GM-CSF utilized in this study was LEUKINE® (Sargramostim, Immunex Corporation, Seattle, WA). LEUKINE® was supplied as a lyophilized powder, in vials containing 500 mcg of recombinant human GM-CSF; 40 mg mannitol, USP; 10 mg sucrose, NF; and 1.2 mg tromethamine, USP.

The drug was reconstituted in 1 ml Bacteriostatic Water for Injection, USP.

The placebo control, also supplied as a sterile lyophilized powder, contained only the inactive excipients present in the LEUKINE® preparation mannitol, sucrose, and tromethamine, as listed above); and was reconstituted in Bacteriostatic Water for Injection, USP. Vials containing placebo were labeled in a fashion identical to vials containing LEUKINE®. Vials of placebo and LEUKINE® were stored refrigerated at 2- 8°C (36-46 0

F).

Patients were eligible for inclusion in the study if the following parameters were met: AIDS diagnosis within 3 months of study enrollment, less than 6 months exposure to zidovudine, CD4' cell count at baseline <300 cells/Wl, Kamofsky performance status of 60%, hemoglobin >9 mg/dl, platelet count >75,000 cells/dl, liver function tests within 3x normal limits, and creatinine <2 mg/dl. Patients were ineligible for inclusion in the study if any of the following exclusion criteria were met: prior use of any immunomodulating agents, or the presence of active AIDS defining infections. All enrolled subjects provided written informed consent.

Groups were balanced for demographics, Karnofsky performance score, and the type and duration of antiretroviral therapy. However, randomization resulted in a GM- CSF treatment group with a significantly lower baseline CD4' cell count, compared to the placebo group (median 80 cells/pl v. 136 cells/pl; p= 0.029).

WO 00/27196 PCT/US99/24857 At baseline and during treatment, subjects were evaluated for viral load, lymphocyte subsets, and infections. Prophylaxis for opportunistic infections was permitted. Prophylactic trimethoprim-sulfamethoxazole was administered when CD4* cell counts were <200 cells/pl.

As noted above, subjects eligible for inclusion in the study were required to have had less than 6 months exposure to zidovudine. Some patients had no previous exposure to AZT. In the GM-CSF group 29 patients received AZT for the first time, on the day the first dose of GM-CSF was administered to them. In the placebo group 32 patients were started on AZT and placebo on the same day.

Patients were allowed to receive newer antiretroviral agents as they became available, but AZT maintenance was required throughout the study. Seventy of the subjects received additional antiretroviral agents at some time during the study, as indicated in Table 1. The number and percentage of subjects are presented, for the indicated treatment regimens.

Table 1 Antiretroviral Drugs received by Subjects Placebo GM-CSF (n=52) (n=53) AZT only 18 17 (32%) AZT 2" drug during study 16 18 (34%) AZT 2nd drug prior to study 18 18 (34%) Receiving any 2n d agent 34 36 (68%) ddl 32 26 (49%) ddC 4 8 3TC 4 5 saquinavir 1 Subject withdrawal was similar between groups (13/52 for placebo and 15/53 for GM-CSF). Adverse events accounted for only 7 subject withdrawals: 2 in the placebo group (1 anemia, 1 thrombocytopenia) and 5 in the GM-CSF group (1 confusion, 4 anemia). The remaining subjects were withdrawn for either disease progression or death (4 in each group), non-compliance (3 in each group), lost to follow-up (2 in each group), and reasons listed as other (1 for GM-CSF, 2 for placebo). One subject, randomized to placebo, received saquinavir during the last 8 weeks of study. Data for that subject at the 6 month timepoint were excluded from the analysis.

WO 00/27196 PCT/US99/24857 Heparinized blood and plasma were collected at month 1, 3, and 6 during the study, for lymphocyte subset analysis by flow cytometry and virological assays. Plasma was cryopreserved at a central laboratory, and batch analyzed to quantify HIV-RNA concentration by nucleic acid sequence based branched chain assay (NASBA, Organon- Teknika).

Samples collected at baseline and 6 months were also evaluated to determine the frequency of codon mutations associated with AZT resistance. HIV genotype variation at codon 41, 69, 70, 74, 184, 214, and 215 was analyzed. This genotype analysis involved amplifying HIV nucleic acid by PCR, using an Amplicor" kit (Hoffman LaRoche, Nutley, New Jersey), and specific probe sequence hybridization using a LiPA HIV-1 RT kit (Murex Immunogenetics, Research Triangle Park, North Carolina; kit available from Abbott Laboratories, Abbott Park, Illinois). The genotype analysis that was conducted using the LiPA HIV-1 RT kit is based on the line probe assay described in Stuyver et al, International Antiviral News, 5(3):38-40, 1997, and Stuyver et al., Antimicrobial Agents and Chemotherapy, 41(2):284-291, February 1997.

This genotype analysis was conducted on samples from 46 subjects that had been blindly and randomly selected from 77 subjects who had completed the treatment phase with a minimum of 95% study drug compliance and had sufficient cryopreserved blood to evaluate all timepoints. All assays were performed per manufacturer's recommendations.

Comparisons between the treatment groups were made using the likelihood ratio chisquare test for categorical data (Fisher's exact test in the case of zero frequencies) and the Wilcoxon rank-sum test for continuous data (Hollander, and D. A. Wolfe, Non parametric Statistical Methods, John Woley Sons, New York, 1973, pp 68-74).

Results of Genotype Analysis At baseline, 6/23 of placebo subjects and 5/20 of GM-CSF subjects (in the genotype analysis group) had evidence of resistance to AZT. After 6 months of therapy, virus sequences from 20/25 of placebo subjects and 9/18 of GM- CSF subjects (p=0.04) demonstrated new resistance mutations, in individuals with amplifiable HIV sequence. Viral sequences were not amplified in one placebo subject and two GM-CSF subjects at six months.

Mutations at codon 41, which is associated with 3TC resistance, were observed in 3 subjects at six months (2 placebo subjects, 1 GM-CSF subject). Genotype mutations associated with resistance to DDI, DDC, and D4T were not detected.

13 The genotype analysis demonstrated a lower frequency of codon mutations associated with AZT resistance among those receiving GM-CSF compared to placebo (80% v. 50%, respectively, p=0.04). Thus, GM-CSF limited the evolution of resistance genotypes.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters 15 formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

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Claims (19)

1. A method of inhibiting the development of a drug-resistant strain of human immunodeficiency virus (HIV) in a human, comprising: administering at least one anti-HIV drug to an HIV* human who does not have AIDS; and administering granulocyte-macrophage colony stimulating factor (GM-CSF) to said human, wherein the GM-CSF and anti-HIV drugs are administered for at least six months according to a regimen wherein the patient receives an anti-retroviral drug for no more than five consecutive days without also receiving at least one dose of GM-CSF, the GM-CSF is administered in an amount sufficient to inhibit the development of an HIV strain resistant to said drug in said human, and said anti-HIV drugs are selected from the group consisting of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors.

2. The method of claim 1, wherein three of said anti-HIV drugs are administered to said human.

3. The method of claim 1 or 2, wherein at least one protease inhibitor, and at least one nucleoside reverse transcriptase inhibitor, are administered to said human. '0 4. The method according to any one of claims 1 to 3, wherein: said nucleoside reverse transcriptase inhibitor is selected from the group consisting of zidovudine (AZT), didanosine (ddl), lamivudine (3TC), 25 stavudine (d4T), and dalcitabine (ddC); P:\WPDOCS\CRN\Puitj\Spm773837.mkdedd cdimd-22/O5/03 said non-nucleoside reverse transcriptase inhibitor is selected from the group consisting of nevirapine and delavirdine; and said protease inhibitor is selected from the group consisting of indinavir, nelfinavir, ritonavir, and saquinavir.

5. The method according to any one of claims 1 to 4, wherein at least one of the anti-HIV drugs is zidovudine.

6. The method according to any one of claims 1 to 5, wherein at least one of said antiretroviral agents is selected from zidovudine, lamivudine, and indinavir.

7. The method according to any one of claims 1 to 6, wherein the GM-CSF is recombinant GM-CSF.

8. The method according to claim 7, wherein the recombinant GM-CSF is produced in yeast host cells.

9. The method according to claim 7 or 8, wherein the 15 recombinant GM-CSF consists of a single 127 amino acid glycoprotein that differs from endogenous human GM-CSF by having a leucine instead of a proline at position 23. The method according to any one of claims 1 to 9, wherein the GM-CSF is administered at a dose of greater than or equal to 100 mcg.

11. The method according to any one of claims 1 to 10, wherein the GM-CSF is administered at a dose of 250 mcg. ?:\WPDOCS\CRNPunita\Spc1i77738370.smid clims.oc22O5/03 -16-

12. The method according to any one of claims 1 to 10, wherein the GM-CSF is administered at a dose of greater than or equal to 125 mcg/m 2

13. The method according to any one of claims 1 to 12, wherein the GM-CSF is administered at least two times per week.

14. The method according to any one of claims 1 to 12, wherein the GM-CSF is administered two or three times per week. The method of any one of claims 1 to 14, wherein the patient has a CD4 T lymphocyte count greater than 200 cells/mm 3 when administration of GM-CSF and said drug commences.

16. The method according to any one of claims 1 to 15, wherein said patient is not treated with said antiretroviral drug prior to administration of GM-CSF to said patient.

17. The method according to any one of claims 1 to 16, wherein *9 said anti-HIV drug is administered to said human for no more than 3 consecutive 15 days, without administration of at least one dose of GM-CSF.

18. A method of prolonging the therapeutic effectiveness of a regimen for treating an HIV-infected human, comprising administering GM-CSF, in conjunction with one or more anti-retroviral drugs, wherein the GM-CSF is administered in an amount and frequency 20 sufficient to inhibit the development of HIV resistant to at least one of said "anti-retroviral drugs, in said human; and wherein said anti-retroviral drugs are selected from the group consisting of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. 9 99 P:\WPDOCS\CRN\P-it\Sp.\7738370.Med cImsdo-22/O5O3 -17-

19. A method of inhibiting mutations in the reverse transcriptase gene of human immunodeficiency virus (HIV), wherein said mutations confer resistance to an antiretroviral drug, said method comprising administering GM-CSF in conjunction with at least two antiretroviral drugs that inhibit reverse transcriptase, to an HIV+ patient who does not have AIDS, wherein the GM-CSF is administered in an amount and frequency sufficient to inhibit the development of HIV resistant to at least one of said drugs, in said human. The method according to any one of claims 1 to 17, wherein one of the anti-HIV drugs is AZT and the duration of GM-CSF administration is sufficient to induce a statistically significant reduction in the number of mutations at a codon associated with AZT resistance in the HIV genome, wherein said mutation is at codon 41, 69, 70, 74, 184, 214, or 215 of the HIV genome.

21. Use of at least one anti-HIV drug and GM-CSF in the manufacture of a kit for use in a method according to any one of claims 1 to

22. A method according to any one of claims 1, 18 and 19, substantially as hereinbefore described. S

23. Use according to claim 21, substantially as hereinbefore described. DATED this 22nd day of May, 2003 SCHERING AKTIENGESELLSCHAFT S By its Patent Attorneys 25 DAVIES COLLISON CAVE 0* 0 e •e