Gonadotropin-releasing hormones for use as adjuvant immunotherapeutics

Abstract

The present invention relates to the use of a gonadotropin-releasing hormone (including GnRH I, a GnRH I analogue, GnRH II, or a GnRH II analogue) as adjuvant immunotherapeutic.

Claims

1. A method to prevent progression of human immunodeficiency virus (HIV) for three years or more in a subject in need thereof, the method comprising: administering to said subject a gonadotropin releasing hormone (GnRH) agonist during a treatment period of one to four weeks, wherein the GnRH agonist is an adjuvant immunotherapeutic; and wherein the administering during the treatment period prevents progression of the HIV in the subject during an immune response maintaining period of three years or more.

2. The method according to claim 1, wherein during the immune response maintaining period, the subject exhibits no symptoms associated with HIV and no progression of the HIV to acquired immunodeficiency syndrome (AIDS).

3. The method according to claim 1, wherein the GnRH agonist is selected from the group consisting of GnRH I, a GnRH I analogue, GnRH II, a GnRH II analogue, and pharmaceutically acceptable salts thereof.

4. The method according to claim 1, wherein the GnRH agonist is selected from the group consisting of deslorelin, avorelin, leuprorelin, triptorelin, buserelin, fertirelin, lutrelin, goserelin, historelin, nafarelin, and pharmaceutically acceptable salts thereof.

5. The method according to claim 1, wherein the GnRH agonist is GnRH II, a GnRH II analogue, or a pharmaceutically acceptable salt thereof.

6. The method according to claim 1, wherein the method further comprises administering one or more naturally-occurring, semi-synthetic, or synthetic sex hormones.

7. The method according to claim 6, wherein the sex hormone is an androgen.

8. The method according to claim 6, wherein the sex hormone is selected from the group consisting of testosterone, dihydrotestosterone, androsterone, dehydroepiandrosterone, dehydroepiandrosterone, androstenedione, methyltestosterone, stanozolol, and pharmaceutically acceptable salts thereof.

9. The method according to claim 6, wherein the sex hormone is an estrogen.

10. The method according to claim 6, wherein the sex hormone is selected from the group consisting of estrogen, estradiol, estriol, estrone, ethynylestradiol, mestranol, dinestrol, diethylstilbestrol, and conjugated estrogens, and pharmaceutically acceptable salts thereof.

11. The method according to claim 6, wherein the method further comprises administering a gestagen.

12. The method according to claim 1, wherein the treatment period is four weeks.

13. The method according to claim 1, wherein the GnRH agonist is administered daily during the treatment period.

14. The method according to claim 4, wherein the GnRH agonist is selected from the group consisting of buserelin, triptorelin, goserelin, and pharmaceutically acceptable salts thereof.

15. The method according to claim 14, wherein the GnRH agonist is buserelin, or a pharmaceutically acceptable salt thereof.

16. The method according to claim 1, wherein the GnRH agonist is not administered to said subject during the immune response maintaining period.

17. The method according to claim 12, wherein the GnRH agonist is not administered to said subject during the immune response maintaining period.

18. The method according to claim 13, wherein the GnRH agonist is not administered to said subject during the immune response maintaining period.

Description

LEGENDS TO FIGURES

(1) FIG. 1: Expression of MHC class I after stimulation of T cells with increasing concentrations of GnRH II. PBMCs from a healthy donor was stimulated with GnRH II and IL-2 for 72 hours. Data points represent mean fluorescent intensity of MCH class I expression on CD4.sup.+ T cells (blue triangles) or CD8.sup.+ T cells (black squares) measured with flow cytometry.

(2) FIG. 2: GnRH receptor expression in human T cells analysed with quantitative realtime PCR. The bars represent ratios of GnRHR I or GnRHR II mRNA normalized to RNA polymerase II expression in sorted naive T cells (white bars) or memory T cells (gray bars). MCF-7 breast cancer cell line (black bar) was used as a positive control.

(3) FIG. 3. Study outline and visit schedule of phase IIa clinical study.

(4) FIG. 4: FSH levels of the 26 patients as measured during the visits of the phase IIa clinical study.

(5) FIG. 5: LH levels of the 26 patients as measured during the visits of the phase IIa clinical study.

(6) FIG. 6: Testosterone levels of the 26 patients as measured during the visits of the phase IIa clinical study.

(7) FIG. 7. Immunological progression assessed via CD4.sup.+ T cell counts during the period from visit 7 of the phase IIa study and up to 48 months after the visit.

(8) FIG. 8. Virological progression assessed as increase in viral load during the time period from visit 7 of the phase IIa study and up to 48 months after the visit.

EXPERIMENTAL

(9) GnRH I vs GnRH II Assay

(10) Compounds are tested on cells made to express either GnRH I receptors or GnRH II receptors by transfection. The cells are exposed to labelled GnRH compound, washed and then assessed by measuring the label on the cells. The label is either measured directly (radioactive isotope label or fluorescent label) or indirectly (biotin labelled peptide).

(11) Signalling induced by the GnRH compounds is measured in the cell lines expressing either GnRH I receptors or GnRH II receptors. GnRH compounds are investigated for their respective affinity to GnRH I receptors and GnRH II receptors using competition assays. Calcium flux is measured using cells labelled with Fluo-4-Direct either using a flow cytometer or by live cell imaging microscopy, in order to evaluate their potency establishing ED50 values. Signalling is also studied by western blotting using antibodies to p-ERK or p-JNK.

(12) To assess the effects of cellular activation on the production of LH and FSH and compare it with stimulation of immune related functions, the effects of the compounds are studied on pituitary cells and immune cells expressing either GnRH I receptors or GnRH II receptors.

EXAMPLES

Example 1: Open-Label Phase II Clinical Study

(13) An open-label, uncontrolled, single-center phase IIa clinical study was conducted at the Clinical Research Unit at the University of Pretoria, South Africa. The study outline is depicted in FIG. 3. The subjects were HIV 1-infected antiretroviral therapy (ART) naïve male patients (n=26). The inclusion criteria were asymptomatic verified HIV-1 infection as documented by HIV antibody test, male gender, age of 18-50 years, CD4.sup.+ T cell count of >350 cells/μl whole blood, and HLA-A*0205, HLA-A*3002, or HLA-B*1503 haplotype positivity. The patients received 1.2 mg/day (4×300 μg, each time administered as 150 μg into each nostril) of buserelin acetate nasal spray solution (Supretact®, Sanofi-Aventis) for 28 days. To compensate for any endocrine side effects, the patients further received a single intramuscular injection of 150 mg testosterone cypionate (Depo®-Testosterone, Pfizer) at day 7 of the treatment period. The primary study objective was to evaluate the safety and tolerability of intranasal administration of buserelin acetate for 28 days, and the secondary study objectives were to evaluate the effects on T cell populations, HIV viral load and serum concentrations of FSH, LH and testosterone.

(14) In terms of safety, no serious adverse events (SAEs) occurred during the study, and no patients experienced intolerable adverse events causing treatment withdrawal. All adverse events except one was graded as mild, and the great majority of the events were considered as unrelated to treatment. A summary of the adverse events is provided in Table 2.

(15) TABLE-US-00002 TABLE 2 Summary of adverse events (AE) in phase IIa study Type of event Safety (n = 26) Any AE 26 (100.0%) Any SAE 0 (0.0%) Any treatment-related AE 17 (65.4%) Any treatment-related SAE 0 (0.0%) Any AE leading to discontinuation 1 (3.8%) Any AE affecting impotence 6 (23.1%) Any AE affecting libido 8 (30.8%) Death 0.0% For categorical variable, n (%) is presented

(16) In terms of efficacy, the HIV viral load decreased between baseline and visit 6 (end of treatment) and visit 7 (follow-up), respectively, with the viral load reduction observed at visit 7 being more pronounced than the reduction observed at visit 6. Also, HLA class I molecules on CD4.sup.+ T cells increased between baseline and visit 6, and the number of HIV-specific binding sites on CD4.sup.+ T cells (CD8 tetramer MFI) at visit 7 was significantly greater compared to baseline. Moreover, a detailed analysis of 16 immunological variables indicated that the treatment resulted in activation of CD4.sup.+ and CD8.sup.+ T cells.

(17) As expected, the serum levels of FSH, LH and endogenous testosterone decreased during the treatment period, but had recovered at the time of the follow-up visit (FIGS. 4-6).

Example 2: Follow-Up Study and Interim Analysis

(18) To investigate the long-term effects of GnRH treatment in patients suffering from HIV/AIDS, in particular with the intend of developing means to minimize the risk of disease progression, a follow-up study and interim analysis was conducted 36-48 months after the open-label phase II clinical study. The aim of the study was to assess progression of the HIV infection during the time period from completion of the phase IIa clinical study (Example 1), based on an assessment of the past and current clinical, immunological and virological status.

(19) The follow-up included 13 out of the original 26 patients, of which nine patients were subject to full clinical examination. For the interim analysis, disease progression was based on immunological criteria and defined as a medical condition in which a patient had experienced any AIDS-defining disease such as tuberculosis infection during the time period from the last day of treatment in the clinical trial until the day of follow up, or whose CD4.sup.+ T cell count measured in whole blood had decreased by more than 50 cells/μl to a value below 500 cells/μl. The interim analysis revealed that six out of the nine patients subjected to full evaluation demonstrated no immunological disease progression. In these patients, the long-term effects of treatment included sustained immunological activation of CD4.sup.+/CD8.sup.+ cells (resembling normal physiological levels), no increase in viral load, and no clinical progression of HIV.

(20) A summary of the findings of the 13 patients included in the follow up study is provided below: History of start of ART treatment after initial GnRH treatment: seven of the 13 patients (54%) had started ART treatment by the time of reevaluation (36-48 months after the phase IIa study—Example 1). History of HIV-associated diseases (AIDS defining diseases including TB): two of the 13 patients (15%) had a history of pulmonary TB without dissemination since completion of the phase IIa study. The TB cases were treated successfully. No other events or severe diseases were reported by the reviewed patients or were seen at the follow-up examination after 36-48 months. Immunological status 36-48 months after initial GnRH treatment: To assess the immunological status, the last CD4.sup.+ T cell count results of the initial clinical study at visit 6 (=end of treatment in Example 1) and visit 7 (=end of follow-up in Example 1), respectively, any traceable (routine) CD4 result that had been done since the completion of the initial study (Example 1) as well as the CD4 test which was done as part of the follow up study (Example 2), were evaluated. In order to assess immunological changes since completion of the initial study (Example 1), the last results of the initial study and the last ART naive CD4 result were evaluated. 11 patients (85%) qualified for such an analysis, the two patients that had to be exempted from this analysis had started ART but no CD4 analysis after completion of the phase IIa study (Example 1) and prior to the ART start was done or could be traced. “Immunological progression” was defined as any CD4 result below 500 cells/mm.sup.3 and a drop of 50 cells/mm.sup.3 or more from baseline (visit 6 or visit 7, respectively, Example 1) at the follow up examination (Example 2). Using visit 6 as reference, five out of 11 patients (45%) did not “progress immunologically” during an observation period of at least 36 months. Taking visit 7 as reference, six out of 11 patients (55%) did not “progress immunologically” during the same observation period (FIG. 7). Virological status: To assess the virological status, the last viral load results of the initial clinical study (visit 6=end of treatment, visit 7=end of follow-up, Example 1), any traceable (routine) viral load result that has been done since the completion of initial phase IIa study (Example 1) as well as the viral load test which was done as part of the follow up clinical study (Example 2) were evaluated. In order to assess virological changes, since completion of the phase IIa study (Example 1), the last viral load results of the initial study (Example 1) and the last ART naive viral load result were evaluated. six patients (46%) qualified for such an analysis and the seven patients that could not be included into the analysis had all started ART and no viral load tests or results after completion of the initial clinical study (Example 1) or prior to the start of ART treatment were done or could be traced. “Virological progression” was defined as an increase in viral load of 0.33 log or more from baseline (visit 6 or visit 7, respectively, Example 1) and the last ART naive viral load. Of the six patients that qualified for this analysis, two patients (33%) did not progress virologically, irrespective which visit was chosen as baseline, over a period of at least 37 months (FIG. 8). Combined immunological and virological progression: Combining the virological results of the six patients for whom virological changes were determined with their immunological results revealed that three (50%) progressed immunologically and virologically, one (17%) did progress immunologically but not virologically, one (17%) did progress virologically but not immunologically, and one (17%) did neither progress virologically nor immunologically. These findings are summarized in Table 3.

(21) TABLE-US-00003 TABLE 3 Combined immunological and virological progresssion Virological Virological Progression Non-progression Immunological Progression 3 1 Immunological Non-pro- 1 1 gression

CONCLUSION

(22) In conclusion, over a period of 36 to 48 months after four weeks of treatment with a GnRH analogue, about half of the patients who was reevaluated after treatment with the GnRH analogue in the initial clinical study (Example 1) did not progress immunologically and about one third did not progress virologically. Combined, there was one patient (17%) who did neither progress immunologically nor virologically.

(23) Specific embodiments of the invention are given in the list of items:

(24) Item 1

(25) A GnRH analogue for use in minimizing the risk of progression of viral disease.

(26) Item 2. The GnRH analogue for use in minimizing the risk of progression of viral disease according to item 1, wherein the viral disease is selected from Adenovirus Infection, Alphavirus Infection, Arbovirus Encephalitis, Borna Disease, Bunyavirus Infection, Calicivirus Infection, Chickenpox, Condyloma Acuminata, Coronavirus Infection, Coxsackievirus Infection, Cytomegalovirus Infection, Dengue fever, Contageous Ecthyma, Epstein-Barr Virus Infection, Erythema Infectiosum, Hantavirus Infection, Viral Hemorrhagic Fever, Viral Hepatitis, Herpes Simplex, Herpes Zoster, Infectious Mononucleosis, Influenza, Lassa Fever, Measles, Molluscum Contagiosum, Mumps, Paramyxovirus Infection, Phlebotomus Fever, Polyomavirus Infection, Rabies, Respiratory Syncytial Virus Infection, Rift Valley Fever, Rubella, Slow Virus Diseases, Smallpox, Subacute Sclerosing Panencephalitis, Tumor Virus Infections, West Nile Fever, Yellow Fever, and HIV/AIDS.

(27) Item 3. The GnRH analogue for use according to items 1 or 2, wherein the viral disease is HIV/AIDS.

(28) Item 4. The GnRH analogue for use according to any of the preceding items, wherein the GnRH analogue is selected from GnRH I, a GnRH I analogue, GnRH II, a GnRH II analogue, or any pharmaceutically acceptable salts thereof.

(29) Item 5. The GnRH analogue for use according to item 4, wherein the GnRH analogue is a GnRH I analogue.

(30) Item 6. The GnRH analogue for use according to any of the preceding items, wherein the GnRH analogue is a GnRH I analogue selected from deslorelin, avorelin, leuprorelin, triptorelin, buserelin, fertirelin, lutrelin, goserelin, historelin, and nafarelin.

(31) Item 7. The GnRH analogue for use according to any of the preceding items, wherein the GnRH analogue is a GnRH I analogue selected from triptorelin, goserelin, and buserelin.

(32) Item 8. The GnRH analogue for use according to any of the preceding items, wherein the GnRH analogue is buserelin.

(33) Item 9. The GnRH analogue for use according to any one of items 1-4, wherein the GnRH analogue is GnRH II.

(34) Item 10. The GnRH analogue for use according to any one of items 1-4, wherein the GnRH analogue is a GnRH II analogue.

(35) Item 11. A GnRH analogue for use according to any of the preceding items in combination with one or more natural, semi-synthetic or synthetic sex hormones, or any pharmaceutically acceptable salts thereof.

(36) Item 12. A GnRH analogue for use according to item 11, wherein the sex hormone is an androgen or an agent exhibiting a corresponding physiological effect.

(37) Item 13. A GnRH analogue for use according to items 11 or 12, wherein the sex hormone is selected from testosterone, dihydrotestosterone, androsterone, dehydroepiandrosterone, dehydroepiandrosterone, androstenedione, methyltestosterone, and stanozolol.

(38) Item 14. A GnRH analogue for use according to any one of items 11-13, wherein the sex hormone is selected from testosterone, methyltestosterone, and stanozolol.

(39) Item 15. A GnRH analogue for use according to any one of items 11-14, wherein the sex hormone is testosterone.

(40) Item 16. A GnRH analogue for use according to item 11, wherein the sex hormone is an estrogen or an agent exhibiting a corresponding physiological effect.

(41) Item 17. A GnRH analogue for use according to items 11 or 16, wherein the sex hormone is selected from estrogen, estradiol, estriol, estrone, ethynylestradiol, mestranol, dinestrol, diethylstilbestrol, and conjugated estrogens.

(42) Item 18. A GnRH analogue for use according to any one of items 11, 16-17, wherein the sex hormone is selected from estrogen, estradiol, ethynylestradiol, mestranol, dinestrol, and diethylstilbestrol.

(43) Item 19. A GnRH analogue for use according to any one of items 11, 16-18 further in combination with a gestagen or an agent exhibiting a corresponding physiological effect.

(44) Item 20. A GnRH analogue for use according to item 19, wherein the gestagen is selected from progesterone, hydroxyprogesterone, medroxyprogesterone, norethisterone, megestrol, medrogestone, and norgestrel.

(45) Item 21. A pharmaceutical composition comprising a GnRH analogue or any pharmaceutically acceptable salts thereof and a least one pharmaceutically acceptable excipient as defined in any of items 1-20 for use in minimizing the risk of progression of viral disease.

(46) Item 22. A pharmaceutical kit, comprising in a single package: i) a first composition comprising a supraphysiological amount of GnRH or a GnRH analogue, ii) optionally, a second composition comprising one or more naturally occurring, semi-synthetic or synthetic sex hormones, and iii) instructions for use, for use in minimizing the risk of progression of viral disease.

(47) Item 23. The pharmaceutical kit according to item 22, further comprising a third composition comprising a gestagen.

(48) Item 24. The pharmaceutical kit according to items 22 or 23, wherein the viral disease is HIV/AIDS.

(49) Item 25. A method comprising administering to a human or animal subject suffering from a viral disease an effective amount of a GnRH analogue or any pharmaceutically acceptable salts thereof for minimizing the risk of progression of viral disease.

(50) Item 26. A method according to item 25, wherein the effective amount of the GnRH analogue is a supraphysiological amount.

(51) Item 27. The method according to items 25-26, wherein the viral disease is selected from Adenovirus Infection, Alphavirus Infection, Arbovirus Encephalitis, Borna Disease, Bunyavirus Infection, Calicivirus Infection, Chickenpox, Condyloma Acuminata, Coronavirus Infection, Coxsackievirus Infection, Cytomegalovirus Infection, Dengue fever, Contageous Ecthyma, Epstein-Barr Virus Infection, Erythema Infectiosum, Hantavirus Infection, Viral Hemorrhagic Fever, Viral Hepatitis, Herpes Simplex, Herpes Zoster, Infectious Mononucleosis, Influenza, Lassa Fever, Measles, Molluscum Contagiosum, Mumps, Paramyxovirus Infection, Phlebotomus Fever, Polyomavirus Infection, Rabies, Respiratory Syncytial Virus Infection, Rift Valley Fever, Rubella, Slow Virus Diseases, Smallpox, Subacute Sclerosing Panencephalitis, Tumor Virus Infections, West Nile Fever, Yellow Fever, and HIV/AIDS.

(52) Item 28. The method according to items 25-27, wherein the viral disease is HIV/AIDS.

(53) Item 29. The method according to items 25-28, the method further comprising administering to a human or animal subject an effective amount, of one or more natural, semi-synthetic or synthetic sex hormones or pharmaceutically acceptable salts thereof, to compensate for any negative endocrine effects caused by the GnRH analogue.