Patent Application
20050159389
Feline immunodeficiency virus (FIV) is a lentivirus which causes immunodeficiency syndrome in domestic cats (Pedersen et al., 1987; Siebelink et al., 1990). FIV closely resembles human immunodeficiency virus (HIV) in genomic, biochemical, and morphologic characteristics as well as clinical and hematological manifestations (Johnson et al., 1994; Pedersen et al., 1987; Yamamoto, Sparger et al., 1988). As a result, FIV infection of domestic cats is considered to be an excellent small animal model for testing prophylactic and therapeutic strategies against AIDS viruses (Gardner, 1991; Johnson et al., 1994). A number of antiretroviral drugs for HIV, including the prototype nucleoside analogue azidothymidine (AZT), has been tested using the FIV model (Hart et al., 1995; Hartmann et al., 1992; Hayes et al., 1993; Hayes et al., 1995; Meers et al., 1993; North et al., 1989; Smyth et al., 1994).
The therapeutic use of AZT has been unremarkable in cats and was unable to delay the spread of FIV infection in vivo (Hart et al., 1995; Hartmann et al., 1992). Prophylactic AZT treatment of experimental cats caused either a delay or decrease in both infected blood lymphocyte numbers and plasma virus load (Hayes et al, 1993; Hayes et al., 1995; Meers et al., 1993; Smyth et al., 1994). In addition, a delay in FIV antibody production was observed in some animals (Smyth et al., 1994). However, prophylactic therapy with AZT did not protect cats from FIV infection (Meers et al., 1993; Hayes et al., 1993; Hayes et al., 1995; Smyth et al., 1994). As reported for HIV therapy, withdrawal of the drug resulted in a resurgence of the virus in these cats. When compared to the untreated group, significantly higher CD4 and CD8 cell counts were observed shortly after the withdrawal of the drug (Hayes et al., 1993; Hayes et al., 1995). However, CD4/CD8 ratios were not significantly different from the untreated cats. In contrast, FIV-infected cats therapeutically treated with AZT had no change in FIV antigen or anti-FIV antibody titers but had transient improvement in CD4/CD8 ratios and clinical signs (Hart et al., 1995; Hartmann et al., 1992). These findings suggest that monotherapy with AZT has limited benefit as a therapy for FIV infection. Similar observations have been made with AZT monotherapy of HIV-infected individuals (Harrigan, 1995; Staszewski, 1995).
In recent trials, combination therapies with AZT and other antiretroviral drugs, such as phosphonomethoxyethyl) adenine and dideoxycytidine 5′-triphosphate, had minimal to no effect in preventing or controlling FIV infection in cats (Hartmann et al., 1992; Magnani et al., 1994; Philpott et al., 1992). The in vivo use of viral protease inhibitors or new nucleoside analogue combinations, such as, for example, lamivudine (3TC) and AZT has yet to be reported in FIV-infected cats. Commercially available HIV protease inhibitors (e.g., Sequinavir (SQV), Indinavir (IDV), Ritonavir, Nelfinavir) do not inhibit FIV replication in PBMC in vitro. Unlike other nucleoside analogues, 3TC rapidly induces mutations which can phenotypically reverse the mutations caused by AZT, enabling the antiviral activity of AZT to persist in the host (Boucher et al., 1993; Larder, 1995; Tisdale et al., 1993). This unique feature of 3TC makes it a prime candidate for use in combination with AZT. In HIV-positive individuals, the combination AZT/3TC therapy had synergistic or additive effects at decreasing plasma virus load and increasing CD4 cell counts and function (Katlama et al., 1994; Lange, 1995; Paul et al., 1995; Staszewski, 1995). The addition of an HIV protease inhibitor to this combination further decreased the viral load and improved the CD4 cell count (Deeks et al., 1997; Torres et al., 1997).
The subject invention concerns methods for therapeutic and prophylactic treatment of feline animals against infection by FIV. Methods of the present invention utilize a combination of antiretroviral compounds. In one embodiment, an effective amount of a composition comprising AZT and another nucleoside such as 3TC. In another embodiment, cats are given an effective dose(s) of a composition comprising AZT, a nucleoside analog such as 3TC and a retroviral protease inhibitor. In an exemplified embodiment, the protease inhibitor is HBY-793 (Hoescht-Bayer).
The subject invention concerns methods for therapeutic and prophylactic treatment of cats against infection by FIV. Methods of the present invention utilize a combination of antiretroviral compounds. In one embodiment, cats can be administered an effective amount of a composition comprising AZT and another nucleoside analog. Preferably, the nucleoside analog is 3TC.
In another embodiment of the methods of the present invention, cats are given an effective dose(s) of a composition comprising AZT, another nucleoside analog and a retroviral protease inhibitor. Preferably, the nucleoside analog is 3TC. In an exemplified embodiment, the protease inhibitor is HBY-793. The structure of HYB-793 is shown in
FIV-infected cats treated according to the methods of the present invention can also be given bone marrow transplantation after total body irradiation in conjunction with the antiretroviral drug combination therapy. The bone marrow transplanted can be either allogeneic or autologous.
The antiretroviral compositions of the subject invention can be administered using standard procedures known in the art. For example, the compositions can be administered as oral or nasal formulations. The compositions can also be administered by parenteral injection, i.e., intravenous, intramuscular, or subcutaneous injection. The amounts and dosage regimens for administration can readily be determined by the ordinarily skilled clinician.
Cats that are not infected with FIV can be treated according to the methods of the present invention to provide effective prophylactic treatment against FIV infection. FIV-infected cats can be treated according to the subject methods to provide effective therapy for controlling, inhibiting or eliminating FIV infection in that cat.
Results from studies described herein show that the addition of a nucleoside analog like 3TC to prophylactic AZT therapy will completely protect cats against FIV infection. This observation is supported by the in vitro findings demonstrating that an AZT/3TC combination was more effective at inhibiting FIV replication in PBMC cultures than single-drug treatments using AZT or 3TC alone. The AZT/3TC combination is effective when used prophylactically or immediately upon FIV exposure. In addition, the combination of antiretroviral drugs AZT/3TC/FIV-PI can be used as an anti-FIV therapy to treat chronically infected animals.
The present invention also concerns kits comprising in one or more containers AZT, another nucleoside analog and an inhibitor of a retroviral protease. Preferably, the nucleoside analog is 3TC. In a preferred embodiment, the retroviral protease inhibitor is HBY-793.
The following abbreviations of FIV strains are used herein:
All references cited herein are incorporated by reference.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
In the first set of in vitro studies, feline T-cell lines chronically infected with FVPet (FL-4 cells) or FIVBang (FIVBang/FeT-J cells) at 2×105 cells/ml were treated for 3 weeks with a single drug or various combinations of AZT, 3TC, an FIV protease inhibitor (FIV-PI; Hoescht-Bayer HBY-793), and HIV protease inhibitors (HIV-PI) (
Both single and combination treatments with AZT and 3TC had minimal to no effect at inhibiting RT activity in FIVBang/FeT-J cells (20-50% inhibition) and FL-4 cells (0-10% inhibition). In contrast, FIV-PI treatment inhibited FIV replication by 70-80% in both cell lines (
As a means to simulate in vivo conditions, primary peripheral blood mononuclear cells (PBMC) from specific pathogen free (SPF) cats were next used as the indicator cells. Primary PBMC isolated by ficoll hypaque method were stimulated with concanavalin A for 3 days and cultured for an additional 2 weeks before their use in drug studies (Staszewski, 1995). Antiretroviral drug(s) were added to the PBMC cultures (1×106 cells/ml) immediately before FIVBang (subtype B) or FIVUK-8 (subtype A) inoculation of 100 50% tissue culture infectious dose (TCID50). Both single and combination treatments with AZT and 3TC inhibited the FIV replication in PBMC at doses which were not toxic to the cells (
Based on the findings from in vitro studies, the prophylactic use of AZT/3TC combination was next tested in experimental cats. Four of the eight SPF cats (16-20 weeks of age) received oral administration of AZT and 3TC (75 mg/kg each) twice a day (BID), while remaining cats received placebo. This treatment dose was based on the in vivo research, in which six SPF cats (2 cats per treatment group) treated (BID) with either AZT or 3TC at 100 mg/kg or AZT/3TC combination at 50 mg/kg each had no hematological or clinical abnormalities after two weeks of treatment. In this study, all cats except for one treated cat (#RU1) were inoculated with 100 50% cat infectious dose (C1D50) of FIVUK-8 at 3 days after the first drug or placebo treatment. FIVUK-8 was used in this study because this strain gave more consistent CD4/CD8 ratio inversion in a larger number of infected cats than did infection with FIVBang or FIVPet. All cats received either the drug or placebo treatments throughout the first 11 weeks after FIV inoculation, unless stated otherwise. The cats were monitored daily for clinical signs and twice a month for hematological changes, FIV load in PBMC and plasma, anti-FIV antibody titers, and CD4/CD8 ratio and absolute counts (Diehi et al., 1995; Green et al., 1993; Okada et al., 1994; Tellier et al., 1997; Yamamoto et al., 1991).
At 4 weeks of treatment, severe anemia was observed in all challenged and unchallenged cats treated with AZT/3TC; therefore, the doses of each drug were lowered to 34 mg/kg each at 4 weeks of treatment and subsequently to 5-10 mg/kg each at 5 weeks of treatment. AZT/3TC treatment was terminated in one cat (#3 GB) at 6 weeks of treatment, and the treatment was resumed 6 days later at 5 mg/kg each. Based on virus isolation and PCR analyses, one cat (#101) from the placebo group was positive for FIV by 3 weeks post infection (pi) and had anti-FIV antibodies by 5 weeks pi (Table 1). However, plasma viral RNA levels of this cat were not detected throughout the study; even though the virus load in the PBMC was similar to the levels detected in the remaining placebo cats. These placebo cats (#NK4, #NK6, #IH5) were positive for FIV titers in the plasma and PBMC and for anti-FIV antibodies by 7 weeks pi. Furthermore, all placebo cats, except for cat #101, had transient or persistent CD4/CD8 inversion starting 11 weeks pi. In contrast, all AZT/3TC-treated cats were negative for FIV and had no CD4/CD8 inversion throughout the study. Both drug and placebo treatments were terminated at 11 weeks pi and all cats were monitored for additional 6-13 weeks. In the previous reports, an increase in FIV load of the PBMC was observed after the withdrawal of AZT treatment in FIV-infected cats (Hayes, et al., 1993; Hayes et al., 1995; Meers et al., 1993). Thus, if low levels of FIV infection undetectable by current assays existed in AZT/3TC-treated cats, then such infection should rebound when the drugs are removed. In this study, all AZT/3TC-treated cats remained negative for FIV in PBMC and anti-FIV antibodies throughout the 6-13 weeks after the withdrawal of the drug treatment. Virus isolation and PCR of bone marrow and lymph node cells performed at the termination of the study further confirmed the FIV-free status of these cats. Thus, complete protection of cats against experimental FIV infection was achieved with prophylactic AZT/3TC therapy.
Based on the in vivo toxicity observed in the prophylactic study, three cats (#101, #NK6, #144) chronically infected with FIVUK-8 for 16 weeks were treated at 20 mg/kg of each drug (BID), while an additional three infected cats (#1H5, #NK4, #158) received placebo. These cats were treated with either drug combination or placebo for 8 weeks and monitored an additional 4 weeks for changes in FIV load and CD4/CD8 values. All parameters monitored were identical to those of the prophylactic study. All treated cats developed either mild or severe anemia by 3.5 weeks of treatment. As a result, both drug doses were lowered to 10 mg/kg. Nevertheless, the anemia in one cat (#144) became so severe by 6 weeks of treatment that the drug treatment was terminated for 1 week and resumed thereafter at a low dose of 5 mg/kg of each drug (BID). Unlike the prophylactic study, no significant differences in either FIV load or CD4/CD8 ratios and absolute counts were observed between the treated and placebo cats (Table 2). This study in combination with the previous studies suggest that doses even as low as 20 mg/kg of each drugs when used over moderate period of time (3.5 weeks or longer) will cause anemia in cats. However, short-term treatment (2 weeks) with high dose combination (75 mg/kg each) is well tolerated by cats.
Allogeneic bone marrow transplantation (BMT) in combination with total body irradiation (TBI) and anti-FIV drug therapy was evaluated as an immune reconstitution therapy for FIV-infected cats. The rationale for this therapy is as follows: (1) TBI will decrease FIV load by destroying recipient's hematopoietic cells, including FIV-infected immunocytes. (2) Anti-FIV drug therapy can block the infection of engrafted donor cells in the BMT recipients. (3) BMT with donor BM cells from uninfected cats will reconstitute normal hematopoietic system. The TBI/BMT combination alone was unable to decrease the virus load due to rapid infection of engrafted donor cells. A majority of FIV-infected recipients of allogeneic BMT succumbed to graft-versus-host disease, accelerated FIV-related diseases, or their combination. As a result, studies were performed to identify antiretroviral drugs that can be combined with TBI/BMT. Prophylactic therapy with AZT/3TC combination protected 100% of the cats from FIVUK 8 challenge infection. Moreover, the only FIV-infected cat to survive allogeneic BMT also received concurrent AZT/3TC therapy. This cat had complete hematopoietic engraftment including normal CD8 counts. However, its CD4 counts were only slightly higher than the levels observed before BMT. Furthermore, only slight decrease in plasma virus load was observed during high-dose AZT/3TC therapy. Nonetheless, its anti-FIV antibody titers were 100-fold lower than those before BMT. This cat was still healthy at one year post-BMT and is still responsive to AZT/3TC therapy.
Recent findings with anti-HIV triple-drug combination have revealed that triple-drug cocktails are unable to immune reconstitute the patient with normal numbers and repertoire of T cell populations or to completely decrease/remove the virus load in the lymphoid tissues within feasible duration of time. As such, autologous bone marrow transplantation (BMT) was tested in combination with antiretroviral drugs as an immune reconstitution therapy for FIV-infected cats. Based on preliminary results, no significant decrease in FIV load or improvement in CD4/CD8 ratios or counts were detected in infected cats that received autologous BMT one (1) day after total body irradiation (TBI). These cats survived the autologous BMT and are currently alive over two years after BMT. This is in contrast to the results from allogeneic BMT of FIV-infected cats, whereby all cats except the one on AZT/3TC therapy succumbed to GVHD, accelerated FIV-disease, or their combination. The extension of the time of BMT after TBI will decrease the infected cell reservoir load and, consequently, fewer infected cells will be available to infect engrafted cells. Addition of antiretroviral drug therapy will prevent any remaining infected cell reservoir from contaminating the engrafted cells.
Antiviral compounds of the invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention. In general, the compositions of the subject invention will be formulated such that an effective amount of the antiviral compounds are is combined with a suitable carrier in order to facilitate effective administration of the composition. It should, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies.
The compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable, and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art. Preferably, the compositions of the invention are in the form of a unit dose.
Once improvement in condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
aThe AZT/3TC treatment was started 3 days before FIV inoculation (−0.4 post-infection) at a dose of 75 mg/kg each and decreased to 34 mg/kg at 4 wk post-infection (pi) and then to 10 mg/kg at 5 wk pi. In one cat (#3GB), the AZT/3TC treatment was withdrawn at 6 wk pi and resumed at a low dose of 5 mg/kg at
bSamples before drug or placebo treatment (Pre) and those at various weeks post-infection (wk) were tested for FIV levels, FIV antibodies, and CD4/CD8 rations. FIV levels were determined by virus isolation (VI), PCR for FIV provirus in PBMC, and RT-PCR for plasma viral RNA (vRNA). FIV antibodies were determined
cInverted CD4/CD8 ratios are bolded.
aThe doses of each drug were decreased from 20 mg/kg to 10 mg/kg at 3 weeks treatment. Treatment was withdrawn in one cat (#144) at 6 weeks of treatment and resumed one week later at 5 mg/kg. The changes in doses of each drug, including amount (mg/kg) and time (wk after initial treatment), are shown.
bSamples before drug or placebo treatments (0 wk) and those at various weeks after initial treatment (wk) were tested for FIV levels, FIV antibodies, and CD4/CD8 ratios. FIV loads were determined by the number of PBMC (50 to 5 × 106 PBMC cocultured with 5 × 106 feeder PBMC) needed for positive virus
This application is a continuation of U.S. application Ser. No. 09/763,037, filed Jul. 24, 2001, which is the national stage of international application No. PCT/US99/11940, filed May 28, 1999, which claims the benefit of U.S. provisional application Ser. No. 60/087,281, filed May 29, 1998, now abandoned.
The subject invention was made with government support under a research project supported by NIH Grant AI30904. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60087281 | May 1998 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09763037 | Jul 2001 | US |
| Child | 11029078 | Jan 2005 | US |
