WO2012095388A2

WO2012095388A2 - Compounds for treatment of feline leukemia in felidae

Info

Publication number: WO2012095388A2
Application number: PCT/EP2012/050243
Authority: WO
Inventors: Hans Lutz; Regina HOFMANN; Valentino CATTORI
Original assignee: Universität Zürich
Priority date: 2011-01-10
Filing date: 2012-01-09
Publication date: 2012-07-19
Also published as: WO2012095388A3

Abstract

The invention provides for inhibitors of HIV integrase, particularly raltegravir, for the treatment of FeLV in felidae, particularly in house cats.

Description

Compounds for treatment of feline leukemia in felidae.

The present invention relates to the use of pharmaceutical compounds for treatment of Feline Leukemia Virus (FeLV) infection in felidae, particularly in cats.

The oncogenic gammaretrovirus feline leukemia virus (FeLV) has been the leading cause of death among domestic cats until the introduction of efficient diagnostics and vaccines in the late 1980's. So far, no efficient treatment for viremic animals is available.

Under crowded conditions, up to thirty to forty percent of cats infected with the gammaretrovirus Feline leukemia virus (FeLV) shed the virus continuously and have a course of the disease known as progressive FeLV infection. The prognosis for progressively infected cats is poor, and 70-90% of those cats die within 18 months to 3 years after developing disease symptoms that range from immune suppression, to anemia and lymphoma/leukemia.

After the introduction of reliable, sensitive diagnostics and efficient vaccines in the late 1980's, the prevalence of the virus dropped significantly, but is still relatively high in some countries, especially among sick cats. In multi-cat households with no specific preventive measures in place it may exceed 20%. Furthermore, FeLV is a threat to the survival of other felid species including the most endangered felid, the Iberian lynx.

The efficacy of previously tested antiretroviral drugs is low in FeLV infection, and many have severe side effects in cats (Hartmann et al., Veterinary Immunology and Immunopathology 35, 167-175 (1992)). There are only a few controlled studies that have demonstrated some effect. Feline interferon omega inhibits FeLV replication in vitro, and treatment of viremic cats with this cytokine has been shown to significantly improve clinical scores and extend survival times (de Mari et al., J Vet Intern Med 18, 477-482 (2004)). However, no viral parameters were measured throughout this study to support the hypothesis that interferon-omega actually exerted an antiviral effect.

The nucleoside analogue 3'-azido-2',3'-dideoxythymidine (AZT) effectively inhibits FeLV replication in vitro and in vivo in experimental infections, but its toxicity may cause unwanted side effects (e.g., non-regenerative anemia) and is therefore not recommended as a first line of therapy for progressively infected domestic cats (Lutz et al., J Fel Med Surg 11, 565-574, 2009). Other antiretroviral therapies (ART) used for the treatment of e.g. lentiviral infections in humans are not equally efficient for gammaretroviruses.

Thus, the provision of highly efficient antiretroviral therapy for treatment of FeLV infection is needed to alleviate the consequences of the infection in domestic cats and other felids. The objective of the present invention is to provide safe and efficacious means for the treatment of feline leukemia virus infection in felids.

The present invention was made during the course of experiments aimed at determining, for a panel of drugs, the efficacy of inhibiting FeLV-A/Glasgow-1 replication in vitro. This strain of FeLV is a well-known standard in FeLV research. It was surprisingly found that HIV integrase inhibitor Raltegravir, which is widely used for the treatment of HIV infection in humans, is highly active against the feline leukemia virus. EC₅₀ values for FeLV-A inhibition in feline cell lines for this drug are in the range of those observed for HIV and xenotropic murine leukemia virus-related gammaretrovirus. More surprisingly, clinical studies demonstrated that raltegravir is tolerated well and leads to a significant decrease in p27 viral protein load in experimentally infected cats, likely to result in clearance of the virus by the cat's immune system.

According to a first aspect of the invention Raltegravir (I; /V-(2-(4Z-(4-fluorobenzylcarbamoyl)- 5-hydroxy-1-methyl-6-oxo-1 ,6-dihydropyrimidin-2-yl)propan-2-yl)-5-methyl-1 ,3,4-oxadiazole-2- carboxamide; CAS No 871038-72-1 ; Isentress, Merck & Co.) is provided for treating infection with feline leukemia virus.

Raltegravir is partly eliminated as its glucuronide, a pathway that is not very efficient in cats. This raises the potential for higher efficiency of Raltegravir at low doses.

Another advantage is the potential of the inventive treatment enabling a curative application for FeLV. It has long been known that some cats, after a transient period of viremia of up to 9 months, can efficiently suppress viral replication and fully recover from viremia, indicating the possibility that if viral loads can be significantly reduced for long enough, the immune system of the cat may be able to overcome the infection and antiretroviral drugs withdrawn. Treatment of wild felids heavily affected by FeLV such as the Iberian lynx will be really useful only if, thereafter, the animal has fully recovered from infection and can be released into the wild.

In conclusion, the present invention demonstrates that Raltegravir-mediated FeLV inhibition kinetic is compatible with treatment of infected felids.

According to an alternative of this first aspect of the invention, a compound for treating infection with feline leukemia virus or other gammaretroviruses is provided, wherein said compound is an inhibitor of human immunodeficiency virus (HIV) integrase selected from the group comprised of:

Raltegravir;

Elvitegravir (6-[(3-chloro-2-fluoro-phenyl)methyl]-1 -[1-(hydroxymethyl)-2-methyl- propyl]-7-methoxy-4-oxo-quinoline-3-carboxylic acid, GS-9137, Gilead Sciences):

6-[(3-chloro-4-fluoro-phenyl)methyl]-4-hydroxy-2-isopropyl-N,N-dimethyl-3,5-dioxo-7,8- dihydro-2,6-naphthyridine-1-carboxamide (WO 2006107478):

4-[[4-[(4-fluorophenyl)methoxy]-3-hydroxy-phenyl]methyl]-1 -(3-nitrophenyl)-5-oxo-4H- pyrazole-3-carboxylic acid (Hadi et al. Bioorg. Med. Chem. Lett. , 20(22), 6854-6857, 2010):

N-[(4-fluorophenyl)methyl]-3-hydroxy-9,9-dimethyl-4-oxo-6 ,7-dihydropyrimido[2, 1- c][1 ,4]oxazine-2-carboxamide (WO 2007064502):

S/GSK-1349572 (Shionogi-GlaxoSmithKline Pharmaceuticals, Metifiot et al., Viruses, 2(7), 1347-1366, 2010):

7-[(4-fluorophenyl)methyl]-4-hydroxy-3-[hydroxy-(2-hydroxyethylamino)methyl]-1- methyl-1 ,5-naphthyridin-2-one, (GSK-364735, GlaxoSmithKline, Serrao et ai,

Retrovirology, 6 (25), 2009):

2-(1 , 1 -dioxothiazinan-2-yl)-N-[(4-fluorophenyl)methyl]-5-hydroxy-3-methyl-4-oxo-1 ,2- dihydropyrimidine-6-carboxamide, (BMS-707035; Serrao et al., ibid.):

4-[1 -[(4-fluorophenyl)methyl]pyrrol-2-yl]-2,4-dioxo-butanoic acid, (L-731 ,988; ibid.):

5-(1 , 1 -dioxothiazinan-2-yl)-N-[(4-fluorophenyl)methyl]-8-hydroxy-1 ,6-naphthyridine-7- carboxamide (L-870,810, Shimura, K., et al., J. Virol., 82(2): p. 764-774, 2008):

MK-2048 (Serrao et al., ibid.):

2-[5-[(4-fluorophenyl)methyl]thiazol-2-yl]-3-hydroxy-7-(4-isopropylpiperazin-1 -yl)-9-(3- methyl-2-oxo-imidazolidin-1-yl)pyrido[1 ,2-a]pyrimidin-4-one (Le et al., Bioorganic & Medical Letters, 20(17), 5013-5018, 2009):

1-[(4-fluorophenyl)methyl]-N-hydroxy-N-methyl-pyrrolo[2,3-c]pyridine-5-carboxamide)

(Azaindole N-methyl hydroxamic acid; Pfizer, Tanis et al., Bioorg. Med. Chem. Lett. 20(24), 7429-34) and derivatives thereof:

N-[(3-chloro-4-methyl-phenyl)methyl]-2-(1-ethoxy-4-fluoro-pyrrolidin-2-yl)-5-hydroxy-1- methyl-6-oxo-pyrimidine-4-carboxamide (2-pyrrolidinyl-N-methyl pyrimidone derivate 13, Ferrara et al., Biorg. Med. Chem. Lett , 20(17), 5031-5034):

([(2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-chroman-3-yl]3,4,5- trihydroxybenzoate (Catechin gallat):

Gallocatechin gallat ([(2R,3S)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate):

Epicatechin gallat ([(2R,3R)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-chroman-3-yl] 3,4,5-trihydroxybenzoate):

Epigallocatechin gallat ([(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3- yl] 3,4,5-trihydroxybenzoate):

LEDGF 361-370 (an oligopeptide equal to the amino acid sequence 361-370 of

LEDGF/p75, which is the lens epithelium-derived growth factor, Hayouka et al., Bioorg. Med. Chem. 18(23), 8388-8395, 2010).

2-(6-chloro-2-methyl-4-phenyl-3-quinolyl) pentanoic acid (I) (Christ et al, Nat. Chem. Biol., 6(6), 442-8, 2010):

a hexapeptide comprising the amino acid sequence HCKFWW.

a peptide of 37 AA comprising a sequence of the transmembrane protein of FeLV: VEELMKLLEELLKKLEELFKKLEEWFKKWFELSKKFT (Boenzli, E. et al. In vitro inhibition of Feline Leukemia (FeLV) infection by synthetic peptides derived from the transmembrane domain, Antiviral therapy, 16, 905-913, 201 1 ).

The preferred embodiment of this aspect of the invention is raltegravir for treatment of FeLV infection (best mode).

According to a second aspect of the invention, a dosage form for treatment of FeLV infection in felidae is provided, comprising a compound according to the preceding aspect of the invention. In one embodiment, one such dosage form comprises raltegravir.

A compound according to the invention can be applied to a subject in any form suitable to the intended treatment. Such a form may be an oral formulation, nasal inhalant, injection or suppository. An oral formulation is preferred.

According to a preferred embodiment of the invention, said compound may be applied as an additive to feed or water, or as pellets, tablets or oral gel for treatment or prevention of FeLV infections in felidae.

According to yet another aspect of the invention, a treatment regime is provided for FeLV infection in felidae, comprising administration of a compound according to the first aspect of the invention during a time course of 1 , 2, 4, 6, 8 or 12 weeks. A time course of four to eight weeks is preferred. In one embodiment, one such treatment regime comprises administration of raltegravir.

By inactivation of FeLV integrase, viral replication is suppressed, leading to a significant decrease in viral load. Thus, due to the specific kinetics of infection and immune system response in FeLV, a viremic subject will recover after a time-limited treatment with a compound according to the first aspect of the invention and acquire a lifetime immunity.

A dosage form for treatment of FeLV infection in felidae, providing for a sufficient level of a compound according to the first aspect of the invention over a time course of 1 , 2, 4, 6, 8 or 12 or more weeks, preferably of four to eight weeks, is provided.

Also within the scope of the present invention is a method for the treatment of feline leukemia in felidae, comprising the administration of a compound according to the first aspect of the invention to a patient -particularly a cat or another member of the family felidae- in need thereof. Similarly, a method for the manufacture of a medicament for treating infection of feline leukemia virus is provided, comprising a compound according to the first aspect of the invention for administration to a subject in need thereof. Medicaments according to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing.

According to yet another aspect of the invention, an animal feed preparation comprising a compound according to the first aspect of the invention is provided.

The invention is further illustrated by the following figures and examples:

Brief description of the figure:

Fig. 1 shows the effect of raltegravir on FeLV replication in QN 10, FEA and CrFK cells lines.

Fig. 2 shows p27 FelV protein level in experimentally infected cats (as % of positive control) after raltegravir treatment over time.

Example 1 : Inhibition of FeLV replication by Raltegravir in cell culture

To determine the inhibitory concentration of raltegravir in vitro, three feline cells lines (QN10 (a feline fibroblast cell line carrying a defective murine sarcoma virus with the mos oncogene), feline embryonic fibroblasts (FEA) and Crandell Reese feline kidney cells (CrFK)) were cultured in 24-well plates (TPP, Trasadingen, Switzerland), 10Ό00 cells/well, in RPMI medium containing 10% FCS (both Sigma-Aldrich, Buchs, Switzerland), and 1x Penicillin-Streptomycin (Sigma-Aldrich) for 24 hours with polybrene (hexadimethrine bromide, Sigma-Aldrich, 4 μg ml end concentration) and seven different concentrations of raltegravir (SelleckChem, Houston, TX, USA. Concentration range was of 0.196nM - 3μΜ. Raltegravir was diluted in RPMI medium from a stock solution of 50mM in DMSO) in triplicate wells, and infected with 5Ό00 focus-forming units FeLV-A/Glasgow-1 (corresponding to 10x 50% tissue culture infectious doses, TCID50) for two hours before washing the cells 1x with PBS. Cells were consequently incubated for 7 days (QN10: 5 days) in 500 μΙ RPMI medium with daily media change and the same raltegravir concentrations as above. Potential cytotoxicity of raltegravir was monitored using the CellTiter-Blue Cell Viability and Apo-ONE Homogeneous Caspase-3/7 Assay (Promega, Madison, Wl, USA) according to manufacturer's instructions. Concentration of up to 3 μΜ Raltegravir had no appreciable effect on cell viability nor induced apoptosis. 200 μΙ cell culture supernatant were collected, centrifuged in a table-top centrifuge at maximal speed for 2 minutes to pellet cells and debris, and viral TNA extracted using the MagnaPure LC TNA extraction kit (Roche, Basel, Switzerland) according to manufacturer's instructions. FeLV RNA was quantitated by real-time RT-PCR as described (Tandon et al., 2005. J Virol Methods 130, 124-132 (2005)) (Figure 1 ) and inhibition kinetics in QN10 cells determined by sigmoid dose- response curve fitting using GraphPad Prism version 5.0 (GraphPad Inc, La Jolla, CA, USA). Effective 50% inhibitory concentrations (EC₅₀) were of 7.6 nM in QN 10, of 1 .3 nM in FEA and of 2.2 nM in CrFK cells. Importantly, the estimated EC₅₀ - Values are in the range or below of what observed for HIV and XMRV, and well below the minimal plasma concentrations that can be found e.g. in humans (Ter Heine et al., Br J Clin Pharmacol 69, 475-483 (2010)).

Example 2: in vivo administration of Raltegravir for treatment of FeLV viremia

Experimental design:

Animals and experimental infection: Specified pathogen-free cats acquired from Liberty Research, Inc. (Waverly, NY) at the age of 12 weeks were kept under barrier conditions in large rooms furnished with running boards, climbing trees, ladders, hammocks and elevated sleeping places, under optimal ethological conditions. They were infected intraperitoneally with 1 Ό00Ό00 focus-forming units of FeLV-A/Glasgow strain. Seven viremic animals were followed and observed twice daily for absence of clinical signs and were carefully clinically examinated once per week by a veterinarian. At the occasion of the clinical examination, the weight of the cats was determined.

Blood collection:

Blood specimens were obtained by jugular venipuncture using evacuated tubes (Becton Dickinson, Plymouth, UK) containing K3 EDTA weekly after experimental infection. Blood was used for hematological analysis and plasma for the deterimination of virological parameters including FeLV p27.

Determination of p27:

FeLV p27 antigen was detected by a sandwich ELISA (Lutz et al. J.Immunol. Methods, 56, 209-220, 1983). Results are shown as percentages in comparison to a positive control, cell culture supernatant of FL74 feline lymphoblastoid cell line, which was considered to be 100%. In this study, p27 values above 5% were considered to be positive. The detection limit was defined as the lowest concentration giving an increase in absorbance per unit time (10 min) above the concentration of the mean of the zero standard increased by 3 S.D.. Cats positive for p27 are considered to be antigenaemic.

Treament with raltegravir:

Raltegravir was purchased from a pharmacy and repacked in small gelatine capsules containing 40 mg/capsule. To treat FeLV viremia, each cat was given one capsule in the morning and evening. As the body weight was around 4 kg, this resulted in a daily dose of 20 mg/kg bodyweight. Results:

Outcome of experimental infection:

First signs of infection (positive p27 results) were observed in week 2 p.i. By week 3, most cats were clearly positive for p27 and by week 12, 7 cats had become persistently p27 positive and remained positive.

Effect of treatment:

After treatment was started in week 15, the p27 levels in all 7 cats decreased constantly during the 4 weeks observation period. This systematic decrease in 7 of 7 cats is considered as proof of effectivesness of the raltegravir treatment.

It is interesting to note that cats tolerate raltegravir well. In this and a previous experiment, in which doses of up to 80 mgs per day and cat had been applied, no side effects were observed as determined by clinical examination and laboratory results (hematology and clinical biochemistry).

Discussion:

After treatment, the levels of p27 decreased steadily. The observation that p27 did not drop immediately after onset of treatment can be explained by the fact that cells already infected continue to produce virus as the integrase inhibitor does not affect viral replication but that infection of additional cells is diminished. With increasing time of treatment the number of infected cells will decrease as they will be affected by the immune reaction, which is constantly eliminating a certain percentage of the infected cells. With time, the immune system may be able to keep the numbers of infected cells low and thus, control viremia. Therefore, it may not be necessary to continue treatment for longer periods resulting in cats that remain immune to FeLV viremia.

Claims

1. Raltegravir (/V-(2-(4-(4-fluorobenzylcarbamoyl)-5-hydroxy-1-methyl-6-oxo-1 ,6- dihydropyrimidin-2-yl)propan-2-yl)-5-methyl-1 ,3,4-oxadiazole-2-carboxamide) for treatment of FeLV infection in felidae, particularly in cats (felis catus).

2. A compound for treating infection with feline leukemia virus or other gammaretroviruses, wherein said compound is an inhibitor of human immunodeficiency virus (HIV) integrase selected from the group comprised of:

Raltegravir (/V-(2-(4-(4-fluorobenzylcarbamoyl)-5-hydroxy-1-methyl-6-oxo-1 ,6- dihydropyrimidin-2-yl)propan-2-yl)-5-methyl-1 ,3,4-oxadiazole-2-carboxamide):

6-[(3-chloro-4-fluoro-phenyl)methyl]-4-hydroxy-2-isopropyl-N,N-dimethyl-3,5-dioxo- 7,8-dihydro-2,6-naphthyridine-1 -carboxamide:

4-[[4-[(4-fluorophenyl)methoxy]-3-hydroxy-phenyl]methyl]-1-(3-nitrophenyl)-5-oxo- 4H-pyrazole-3-carboxylic acid):

N-[(4-fluorophenyl)methyl]-3-hydroxy-9,9-dimethyl-4-oxo-6,7-dihydropyrimido[2,1 - c][1 ,4]oxazine-2-carboxamide):

S/GSK-1349572

7-[(4-fluorophenyl)methyl]-4-hydroxy-3-[hydroxy-(2-hydroxyethylamino)methyl]-1 - methyl-1 ,5-naphthyridin-2-one:

2-(1 ,1 -dioxothiazinan-2-yl)-N-[(4-fluorophenyl)methyl]-5-hydroxy-3-methyl-4- 1 ,2-dihydropyrimidine-6-carboxamide:

- 4-[1-[(4-fluorophenyl)methyl]pyrrol-2-yl]-2,4-dioxo-butanoic acid:

- 5-(1 ,1 -dioxothiazinan-2-yl)-N-[(4-fluorophenyl)methyl]-8-hydroxy-1 ,6- naphthyridine-7-carboxamide:

- MK-2048 (Serrao et al., Retrovirology, 6 (25), 2009);

2-[5-[(4-fluorophenyl)methyl]thiazol-2-yl]-3-hydroxy-7-(4-isopropylpiperazin-1-yl)-9- (3-methyl-2-oxo-imidazolidin-1 -yl)pyrido[1 ,2-a]pyrimidin-4-one:

1 -[(4-fluorophenyl)methyl]-N-hydroxy-N-methyl-pyrrolo[2,3-c]pyridine-5- carboxamide):

N-[(3-chloro-4-methyl-phenyl)methyl]-2-(1-ethoxy-4-fluoro-pyrrolidin-2-yl)-5- hydroxy-1 -methyl-6-oxo-pyrimidine-4-carboxamide:

Catechin gallat ([(2R,3S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-chroman-3-yl] 3,4,5-trihydroxybenzoate):

Gallocatechin gallat ([(2R,3S)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3- yl] 3,4,5-trihydroxybenzoate):

Epigallocatechin gallat ([(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)- chroman-3-yl] 3,4,5-trihydroxybenzoate):

LEDGF 361-370;

2-(6-chloro-2-methyl-4-phenyl-3-quinolyl) pentanoic acid:

a hexapeptide comprising the amino acid sequence HCKFWW.

3. A compound according to claim 2, for infection with feline leukemia virus in felidae, particularly in cats (felis catus).

4. A dosage form for treatment of FeLV infection in felidae, comprising a compound according to claim 2.

5. A dosage form according to claim 3, whereby a compound according to claim 2 is applied as an orally formulation, nasal inhalant, injection, suppository or feed preparation.

6. A treatment regime for FeLV infection in felidae, comprising administration of a compound according to claim 2 during a time course of 1 , 2, 4, 6, 8 or 12 weeks.

7. A treatment regime according to claim 6, wherein the time course of the treatment is of a duration of four to eight weeks.

8. A dosage form for treatment of FeLV infection in felidae according to claim 4, comprising daily oral dosages of a compound according to claim 2 for a treatment regime over a time course of 1 , 2, 4, 6, 8 or 12 weeks,

9. A dosage form according to claim 8, wherein the time course of the treatment is four, six or eight weeks.

10. A method for the treatment of FeLV infection in felidae, comprising the administration of a compound according to claim 2 to a patient in need thereof.

1 1 . A method for the manufacture of a medicament for FeLV infection in felidae, comprising a compound according to claim 2 for administration to a subject in need thereof.

12. An animal feed preparation comprising a compound according to claim 2.