Antiviral prodrugs and pharmaceutical compositions thereof

Abstract

To provide antiviral prodrugs and pharmaceutical compositions thereof.SOLUTION: Diesters of 4'-ethynyl-2-fluoro-2'-deoxyadenosine and aqueous parenteral suspensions thereof provide extended in vivo viral suppression of human immunodeficiency virus (HIV).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 898,679, filed September 11, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to antiviral compounds and compositions useful in treating acquired immune deficiency syndrome (AIDS). [Background technology]

[0003] Formula I:

[0004] [ka] 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) (MK-8591), a nucleoside analogue effective as an inhibitor of nucleoside reverse transcriptase (Current Opinion in HIV and AIDS 2018, 13, 294-299), is a potential antiretroviral agent for the treatment of HIV-1 infection and pre-exposure prophylaxis. EFdA is metabolized intracellularly to its active triphosphate anabolite (EFdA-TP), which inhibits HIV reverse transcriptase.

[0005] However, EFdA has a relatively high water solubility and a relatively short time course of plasma concentrations. As a result, when administered to patients to treat human immunodeficiency virus (HIV) infection or for pre-exposure prophylaxis, EFdA can suppress the virus for only a limited period of time. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Current Opinion in HIV and AIDS 2018, 13, 294-299 Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need for formulations that are capable of suppressing viruses for extended periods after administration. The compounds and pharmaceutical preparations of the present invention meet that need.

[0008] The antiviral compounds of the present invention are diesters of EFdA and have limited aqueous solubility. While EFdA has an aqueous solubility of 0.877 mg / mL at physiological pH, the diesters of EFdA of the present invention have an aqueous solubility of less than 0.03 mg / mL, preferably less than 0.002 mg / mL, at physiological pH. These diesters of EFdA are crystalline, useful for providing extended periods of HIV suppression, and can be parenterally administered as a suspension in a pharmaceutically acceptable carrier. A preferred prophylactic dose for human subjects is in the range of about 80 mg to about 800 mg of EFdA diester, parenterally administered at approximately 6-month intervals in a dosage volume of about 0.5 to about 4 milliliters per dose. A preferred therapeutic dose for human subjects is in the range of about 80 mg to about 800 mg of EFdA diester, parenterally administered at approximately 3-month intervals in a dosage volume of about 0.5 to about 4 milliliters per dose.

[0009] Parenteral formulations containing the antiviral compounds of the present invention can be dry formulations containing the antiviral compound together with pharmaceutically acceptable excipients, or can be stable suspensions of the antiviral compound in an aqueous or oily vehicle. The present invention provides, for example, the following items. (Item 1) Formula (II): [ka] [In the formula, R1 and R 2 are independently -C(=O)R 3 and R 3 is selected from the group consisting of isopropyl, 3-pentyl, cyclopentyl, phenyl, and phenylmethyl. A compound represented by the formula: (Item 2) Item 1. The compound according to item 1, which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate. (Item 3) Item 1. The compound according to item 1, which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-ethylbutanoate. (Item 4) Item 1. The compound according to item 1, which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-(((cyclopentanecarbonyl)oxy)methyl)-2-ethynyl-tetrahydrofuran-3-yl cyclopentanecarboxylate. (Item 5) Item 1. The compound according to item 1, which is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3-yl 2-phenylacetate. (Item 6) 1. An aqueous parenteral suspension comprising: Formula (II): [ka] [In the formula, R 1 and R 2 are independently -C(=O)R 3 and R 3is selected from the group consisting of isopropyl, 3-pentyl (1-ethoxypropyl), cyclopentyl and phenylmethyl. A compound represented by the formula: nonionic surfactants; suspending agent; and water; 10. The aqueous parenteral suspension comprising: (Item 7) 7. The aqueous parenteral suspension according to item 6, wherein the compound is present in an amount within the range of about 3 to 4.5% by weight of the suspension, the nonionic surfactant is present in an amount within the range of about 0.1 to about 0.5% by weight of the suspension, and the suspending agent is present in an amount within the range of about 0.1 to about 0.5% by weight of the suspension. (Item 8) 7. The aqueous parenteral suspension of item 6, wherein the nonionic surfactant is polyoxyethylene (20) sorbitan monooleate. (Item 9) 7. An aqueous parenteral suspension according to claim 6, wherein the suspending agent is selected from the group consisting of methylcellulose, carboxymethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose. (Item 10) 7. The aqueous parenteral suspension of item 6, wherein the compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2((2-phenylacetoxy)methyl)-tetrahydrofuran-3-yl 2-phenylacetate. (Item 11) 7. The aqueous parenteral suspension of item 6, wherein the compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-ethylbutanoate. (Item 12) 10. A method for suppressing human immunodeficiency virus in vivo in a patient, comprising parenterally administering to said patient an effective amount of the compound of claim 1 in an aqueous suspension. (Item 13) 13. The method of claim 12, wherein the administration is a three-month administration regimen. (Item 14) Item 13. The method according to Item 12, wherein the effective amount is a dose within the range of about 80 to about 800 mg. (Item 15) Item 13. The method according to Item 12, wherein the effective amount is a dose within the range of about 200 to about 400 mg. (Item 16) 13. The method of claim 12, wherein the compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3-yl 2-phenylacetate. (Item 17) 13. The method of claim 12, wherein the compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-ethylbutanoate. (Item 18) 1. A method for preventing HIV infection in an HIV-uninfected human subject, comprising parenterally administering to said human subject an effective amount of the compound of claim 1 in an aqueous suspension in a semi-annual dosing regimen. (Item 19) Item 19. The method of item 18, wherein the compound is administered in a unit dose ranging from about 80 to about 800 mg. (Item 20) Item 19. The method of item 18, wherein the compound is administered in a dosage volume ranging from about 0.5 to about 4 mL per administration. (Item 21) 8. An aqueous parenteral suspension according to item 6 or 7, wherein the nonionic surfactant is polyoxyethylene (20) sorbitan monooleate. (Item 22) 22. An aqueous parenteral suspension according to any one of claims 6, 7 or 21, wherein the suspending agent is selected from the group consisting of methylcellulose, carboxymethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose. (Item 23) 23. The aqueous parenteral suspension of any one of items 6, 7, 21 or 22, wherein the compound is crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2((2-phenylacetoxy)methyl)-tetrahydrofuran-3-yl 2-phenylacetate or crystalline (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((2-ethylbutanoyl)oxy)methyl-2-ethynyltetrahydrofuran-3-yl 2-ethylbutanoate. (Item 24) 10. A method for suppressing human immunodeficiency virus (HIV) in vivo in a patient, the method comprising parenterally administering to the patient an effective amount of a compound according to any one of items 1 to 5 in an aqueous suspension. (Item 25) 25. The method of item 24, wherein the administration is a three-month administration regimen. (Item 26) A method for preventing HIV infection in a human subject not infected with HIV, comprising administering to said human subject an effective amount of a compound according to any one of items 1 to 5 in an aqueous suspension for six months. The method further comprises parenterally administering the compound in a dosing regimen. (Item 27) 27. The method according to any one of items 24 to 26, wherein the effective amount is a dose within the range of about 80 to about 800 mg. (Item 28) 28. The method of any one of items 24 to 27, wherein the compound is administered in a dosage volume ranging from about 0.5 to about 4 mL per administration. (Item 29) 29. The method of any one of items 24 to 28, wherein administration of the compound results in a slow or controlled or sustained release of EFdA from the compound in vivo. (Item 30) 30. The method according to any one of items 24 to 29, wherein the route of administration of the compound is selected from the group consisting of oral, parenteral, subcutaneous injection, intravenous, intramuscular, intrasternal injection, infusion, and release from an implant. (Item 31) 31. The method of any one of items 24 to 30, wherein the compound is formulated as an aqueous suspension, solution, or encapsulated in particles for sustained release. (Item 32) 32. The method according to any one of items 24 to 31, wherein the viral infection is caused by HIV. (Item 33) 32. The method according to any one of items 24 to 31, wherein the viral infection is caused by HBV. (Item 34) 34. The method of any one of items 24 to 33, further comprising administering an additional anti-HIV drug and / or anti-HBV drug. (Item 35) 35. The method of claim 34, wherein the additional agent is selected from the group consisting of cabotegravir, dolutegravir, doravirine, elvitegravir, lersiverine, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, and lamivudine. (Item 36) 12. Use of a compound according to any one of items 1 to 11 for the treatment and prevention of HIV or HBV viral infections. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows the X-ray powder diffractogram (XPRD) of EFdA. [Figure 2] 1 shows data provided by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) of EFdA. [Figure 3] 1 shows DSC and TGA data for Compound 2. [Figure 4] 1 shows XPRD data for compound 3. [Figure 5]1 shows DSC data for compound 3. [Figure 6] 1 shows TGA data for compound 3. [Figure 7] 1 shows XPRD data for compound 5. [Figure 8] 1 shows DSC data for compound 5. [Figure 9] 1 shows TGA data for compound 5. [Figure 10] The data provided in Table 2 is presented in graphical form. [Figure 11] The data provided in Table 3 is presented in graphical form. [Figure 12] The data provided in Table 4 is presented in graphical form. [Figure 13] 1 shows data in graph form for Example 10. [Figure 14] 1 shows XPRD data for compound 2. [Figure 15] 1 shows XPRD data for compound 6. DETAILED DESCRIPTION OF THE INVENTION

[0011] Prolonged in vivo viral suppression is achieved by administering to a subject of Formula (II):

[0012] [ka] [In the formula, R 1 and R 2 are independently -C(=O)R 3 and R 3 is a member of the group consisting of isopropyl, 3-pentyl, cyclopentyl and phenylmethyl. This is achieved by the diester represented by the formula: The diester is prepared by reacting EFdA with the desired acid or anhydride and recovering the diester as a crystalline compound. The following examples illustrate the preparation of preferred diesters.

[0013] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate

[0014] [ka]

[0015] To a mixture of EFdA (compound 1) (3 g, 6.8 mmol, 1 equiv.) and 4-dimethylaminopyridine (DMAP) (499 mg, 2.73 mmol, 0.4 equiv.) in anhydrous dimethylformamide (DMF) (100 mL) was added isobutyric acid (8.4 g, 27.3 mmol, 6 equiv.) dropwise at ambient temperature. The reaction was stirred at room temperature for 5 h. The reaction was monitored by LC-MS, as alkylation of the NH group could be observed with prolonged reaction times. The reaction mixture was then filtered to remove the urea by-product. Acetonitrile was used to rinse the reaction mixture. The reaction mixture was then washed twice with water and once with brine, and the solvent was then dried, filtered, and evaporated under reduced pressure. The resulting crude material was purified by silica gel column chromatography using 60–70% ethyl acetate (EtOAc) in hexane to give compound 2 as a glassy solid. The resulting glassy solid was dispersed in a minimum amount of isopropanol, followed by rotary evaporation to give pure compound 2 as a white solid (2.5 g, 85% yield). LC-MS (ESI+): m / z 434.49 [M+H] + .

[0016] Approximately 250 mg / g of compound 2 was dissolved in aqueous 0.25% CMC-Na / 0.5% TWEEN ( RThe suspension was suspended in PBS-80 (26-gauge needle) and stabilized at 40°C / 75% relative humidity for 2 weeks. Figure 14 provides XPRD data for compound 6 from the incubated suspension before (bottom panel), after 1 week (middle panel), and after 2 weeks (top panel), demonstrating that the compound retains good crystallinity in suspension.

[0017] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-(((2-ethylbutanoyl)oxy)methyl)-2-ethynyltetrahydrofuran-3-yl 2-ethylbutanoate

[0018] [ka]

[0019] A mixture of EFdA (1 g, 3.4 mmol, 1 equiv.), 2-ethylbutanoic anhydride (4.4 g, 20.4 mmol, 6 equiv.), and triethanolamine (TEA) (3.8 mL, 27.2 mmol, 8 equiv.) in anhydrous acetonitrile (MeCN) (43 mL) was cooled to 0 °C, and 4-dimethylaminopyridine (DMAP) (83 mg, 0.68 mmol, 0.2 equiv.) was added to it at 0 °C. The resulting mixture was stirred at 0 °C for 0.5 h and then at room temperature for 5 h. The reaction was monitored by LC-MS, as alkylation of the NH group could be observed with prolonged reaction time. The reaction mixture was quenched with methanol, and the solvent was evaporated under reduced pressure. The crude material was purified by silica gel column chromatography using 60–70% ethyl acetate (EtOAc) in hexane to give compound 3 as a glassy solid. The resulting glassy solid was dispersed in a minimum amount of isopropanol, followed by rotary evaporation to give pure compound 3 as a white solid (1.33 g, 80% yield). LC-MS (ESI+): m / z 490.56[M+H] + .

[0020] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-(((cyclopentanecarbonyl)oxy)methyl)-2-ethynyltetrahydrofuran-3-yl cyclopentanecarboxylate

[0021] [ka]

[0022] Compound 4 was prepared using a method similar to that for compound 2, except that cyclopentanoic acid was used instead of isobutyric acid. LC-MS (ESI+): m / z 486.44 [M+H] + .

[0023] Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-((2-phenylacetoxy)methyl)tetrahydrofuran-3-yl 2-phenylacetate

[0024] [ka]

[0025] A mixture of EFdA (499 mg, 1.7 mmol, 1 equiv.), 2-phenylacetic anhydride (2.6 g, 10.2 mmol, 6 equiv.), and triethanolamine (TEA) (1.9 mL, 13.6 mmol, 8 equiv.) in anhydrous acetonitrile (MeCN) (22 mL) was cooled to 0 °C, and 4-dimethylaminopyridine (DMAP) (42 mg, 0.34 mmol, 0.2 equiv.) was added to it at 0 °C. The reaction was stirred at 0 °C for 0.5 h and then at room temperature for 3 h. The reaction was monitored by LC-MS, as alkylation of the NH group could be observed with prolonged reaction time. The reaction mixture was quenched with methanol, and the solvent was evaporated under reduced pressure. The crude material was purified by silica gel column chromatography using 60–70% ethyl acetate (EtOAc) in hexane to give compound 5 as a glassy solid. The resulting glassy solid was dispersed in a minimum amount of isopropanol, followed by rotary evaporation to give pure compound 5 as a white solid (694 mg, 77% yield). LC-MS (ESI+): m / z 530.52 [M+H] + .

[0026] Table 1 shows the characterization data of the compounds obtained by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

[0027] (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-((benzyloxy)methyl)-2-ethynyl tetrahydrofuran-3-yl benzoate, compound 6

[0028] Compound 6 was prepared using a method similar to that used for compound 2, using 4.5 equivalents of the corresponding acid. LC-MS (ESI+): m / z 502.41 [M+H] + Approximately 275 mg / g of compound 6 was suspended (26-gauge needle) in aqueous 0.25% CMC-Na / 0.5% TWEEN-80 and stabilized at 40°C / 75% relative humidity for 2 weeks. Figure 15 provides XPRD data for compound 6 from the incubated suspension before (bottom panel), after 1 week (middle panel), and after 2 weeks (top panel) of incubation, demonstrating that the compound retains good crystallinity in suspension.

[0029] [Table 1] JPEG2025137788000010.jpg214170 JPEG2025137788000011.jpg49170 [Example]

[0030] The foregoing discussion and examples are illustrative and should not be taken as limiting. Still other variations within the spirit and scope of the invention are possible and will be readily apparent to those skilled in the art.

[0031] Synthesis of stable crystalline forms Example 1 Compound 2 (approximately 200 mg) was completely dissolved in a minimum amount of acetone with stirring. The solvent was then slowly evaporated at ambient temperature. As a result, the recrystallized sample was obtained as a white powder. Other solvents such as ethyl acetate, methanol, and tetrahydrofuran can also be used.

[0032] General Examples of Formulations of Compounds of the Invention All formulation protocols produced stable aqueous suspensions with 26-gauge syringeability.

[0033] EFdA preparation EFdA was crushed and sieved through a #80 sieve. A preformed solution of 0.25% carboxymethylcellulose sodium (CMC sodium) and 0.1% polyoxyethylene (20) sorbitan monooleate (TWEEN-80) was added to approximately 300 mg of EFdA (Compound 1) to obtain a suspension of approximately 1 g (300 mg of Compound 1 + approximately 700 mg of polymer solution) of the final formulation (approximately 300 mg / g). The suspension was then bath sonicated in an ice bath for 10 minutes. The density of the formulation was 1.064 g / mL, and the concentration of EFdA was 319.2 mg / mL.

[0034] Example 2 The recrystallized Compound 2 was ground and sieved through a US Standard Sieve Series No. 80 sieve (nominal mesh size 0.180 mm) for wire cloth screens. 250 mg of Compound 2 was placed in a suitable container and pre-formed 0.25% sodium carboxymethylcellulose (CMC) and 0.1% TWEEN-80 solution was added to obtain a final formulation (approximately 250 mg / g) of 1 g (250 mg of Compound 2 + approximately 750 mg of polymer solution). The suspension was then probe sonicated in an ice bath for 5 minutes (sonication time: 5 minutes; pulse amplitude: 20; pulse on time: 30 seconds; pulse off time: 20 seconds).

[0035] Example 3 Compound 3 was ground and sieved through a US Standard Sieve Series No. 80 sieve for Wire Cloth Screens (nominal mesh size 0.180 mm). 250 mg of Compound 3 was placed in a suitable container and a preformed solution of 0.25% CMC sodium and 0.5% TWEEN-80 was added to obtain a final formulation (approximately 250 mg / g) of 1 g (250 mg of Compound 3 + approximately 750 mg of polymer solution). The suspension was then probe sonicated in an ice bath for 5 minutes (sonication time: 5 min; pulse amplitude: 20; pulse on time: 30 s; pulse off time: 20 s). The density of the formulation was 1.004 g / mL. The suspension contained 250.88 mg / mL of Compound 3 (approximately 150 mg / mL of EFdA).

[0036] Example 4 Compound 5 was ground and sieved through a US Standard Sieve Series No. 80 sieve for Wire Cloth Screens (nominal mesh size 0.180 mm). 200 mg of Compound 5 was placed in a suitable container and a preformed solution of 0.25% CMC sodium and 0.5% TWEEN-80 was added to obtain a final formulation (approximately 200 mg / g) of 1 g (200 mg of Compound 5 + approximately 800 mg of polymer solution). The suspension was then probe sonicated in an ice bath for 5 minutes (sonication time: 5 min; pulse amplitude: 20; pulse on time: 30 s; pulse off time: 20 s). The density of the formulation was 1.047 g / mL. The suspension contained 209.4 mg / mL of Compound 5 (approximately 115.97 mg / mL of EFdA).

[0037] Pharmacokinetic (PK) studies animal: Animals (male SD rats, approximately 200–250 g, and male rhesus monkeys, approximately 2–3 kg) were obtained from approved sources (SLAC Laboratory Animal Co. Ltd., Shanghai, China, and / or Topgene Biotechnology, Wuhan city, Hubei Province, China).

[0038] Acclimatization / Quarantine: Upon arrival, the animals were assessed for general health by veterinary staff or other authorized personnel. Animals were allowed to acclimate for at least 3 days before being subjected to the study.

[0039] Animal Care: Animals were housed in groups during the acclimation period and individually during the study period. The animal room environment was controlled (target conditions: temperature 18-26°C, relative humidity 30-70%, 12 hours of artificial light, 12 hours of darkness). Temperature and relative humidity were monitored daily.

[0040] Animal cannulation: none. Animals were fasted for at least 12 hours prior to dosing. All animals were given free access to Certified Rodent and Non-Rodent Diet (Catalog # M01-F, SLAC Laboratory Animal Cl. Ltd., Shanghai, China) 4 hours after dosing. Water was autoclaved and then provided to the animals ad libitum. Water was analyzed periodically and the results were retained. The food and water were free of known contaminants at detectable levels that would be expected to interfere with the purpose, conduct, or results of this study.

[0041] 1. Dosage Formulation SC formulation:Suspensions were prepared on the day of dosing according to the procedures described above in Examples 2-5 and Tables 2-4. Animals were administered suspensions within 4 hours of preparation. Two 20 μL aliquots of each prepared suspension were transferred into 1.5 mL polypropylene microcentrifuge tubes, and dose verification was performed by LC / UV or LC-MS / MS.

[0042] 2. Dose Administration The suspension was administered by subcutaneous injection (SC) according to the institution's standard operating procedures (SOP).

[0043] 3. Sample Collection Approximately 200 μL of blood was collected from the saphenous vein of rats and 0.5 mL of blood was collected from rhesus monkeys at each time point. All blood samples were transferred to microcentrifuge tubes containing 4 μL of K2EDTA (0.5 M) as an anticoagulant and placed on wet ice until processed for plasma.

[0044] 4. Blood / Plasma Processing blood: Blood samples were processed for plasma within 30 minutes of collection by centrifugation at 3000 g for 15 minutes at approximately 4° C. Plasma samples were stored in polypropylene tubes, flash frozen on dry ice, and kept at −70±10° C. until LC / MS / MS analysis.

[0045] 5. Sample Analysis Verification of dosage concentration Aliquots of the prepared suspensions were taken in duplicate at the center of each suspension. The concentration of the active ingredient in each aliquot was determined by LC / UV or LC / MS / MS.

[0046] Bioanalytical methods and sample analysis · Developed a non-GLP LC-MS / MS method for the quantification of the active ingredient (test compound) in the corresponding biological matrix; A calibration curve with eight non-zero calibration standards was applied to the method including the lower limit of quantitation (LLOQ); A set of quality control (QC) samples consisting of low, medium and high concentrations was applied to the method; · Test sample analysis was carried out simultaneously using LC-MS / MS method with one set of calibration standards and two sets of QC samples (if the number of samples was more than 48, two calibration curves with two sets of QC samples were applied); Acceptance Criteria: Linearity: ≥6 calibration standards were back-calculated to within ±20% of the nominal value in plasma; Accuracy: ≥4 of 6 QC samples were back-calculated to within ±20% of the nominal value in plasma; Specificity: The average calculated concentration in a single blank matrix must be 0.5 times the LLOQ; Sensitivity: LLOQ is targeted at 1–3 ng / mL; Carryover: The average calculated carryover concentration in a single blank matrix immediately after the highest standard injection should be the LLOQ. If the carryover cannot meet the criteria, the percent carryover should be estimated according to the in-house analytical SOP.

[0047] 6. Data Analysis Plasma concentration versus time data were analyzed using a non-compartmental approach using the Phoenix WinNonlin 6.3 software program. max , T max , T 1 / 2 , AUC (0-t) , AUC (0-inf) , MRT (0-t) , MRT (0-inf) , %F and plasma concentration versus time profile graphs were obtained.

[0048] Example 5 Several prodrugs, including EFdA (1) as a control, were subjected to a single-dose subcutaneous rat PK study. All animals were injected with an EFdA-equivalent dose of 10 mg / kg and a concentration equivalent to 4 mg / mL as an aqueous suspension formulation in 0.5% CMC-Na and 0.5% TWEEN-80. Similar exposures were observed, but all compounds 2, 3, and 5 exhibited significantly lower C than EFdA. max for more than a week at the lower limit of quantification (LLO) The plasma levels of EFdA exceeded those of the control group (Q).

[0049] Table 2 shows rat PK data for compounds 1, 2, 3, and 5 after subcutaneous (SC) administration at a dose equivalent to 10 mg / kg of EFdA. The data, in graphical form, are shown in Figure 10.

[0050] [Table 2]

[0051] Example 6 After optimization, SC rat PK studies were again performed on EFdA at a dose equivalent to 100 mg / kg higher at concentrations equivalent to 120 mg / mL for compound 3, 116 mg / mL for compound 5, and 19 mg / mL for EFdA, respectively. Compound 3 showed a significantly higher C max The activity of Compound 3 and Compound 5 was delayed and 100-fold lower. Furthermore, prolonged half-lives and mean residence times were also observed. This indicates that Compound 3 and Compound 5 are suitable for prophylaxis. is doing.

[0052] Tables 3A and 3B show rat PK data for Compounds 1, 3, and 5 after SC administration. Data for Compound 3 are presented in graphical form in FIG.

[0053] [Table 3]

[0054] [Table 4]

[0055] Example 7 After rat PK analysis, the focus shifted to non-rodents, i.e., rhesus monkeys. Compound 5 and EFdA were subjected to a single-dose rhesus monkey PK study by subcutaneous administration at an EFdA-equivalent dose of 50 mg / kg. The aqueous suspension contained 0.25% CMC-Na and 0.1% / 0.5% TWEEN-80, with EFdA concentrations of 116 mg / mL and 319 mg / mL of Compound 5 and EFdA, respectively. For Compound 5, plasma levels of EFdA above the LLOQ were observed for over one month, and the C was 24-fold lower than that of EFdA itself. max was observed.

[0056] Table 4 shows the rhesus monkey PK data for compounds 1 and 5 after SC administration at an EFdA-equivalent dose of 50 mg / kg. The data are presented in graphical form in Figure 12.

[0057] [Table 5]

[0058] The suspension medium for the compound of the present invention can be an aqueous vehicle such as water for injection (WFI) or a vegetable oil vehicle such as sesame oil, olive oil, etc. The suspension medium can further contain pharmaceutically acceptable excipients such as non-ionic surfactants, suspending or flocculating agents, preservatives, buffers, toxicity adjusters, chelating agents, antioxidants, etc.

[0059] For prophylaxis, a preferred prophylactic dose for a human subject is in the range of about 80 mg to about 800 mg of the antiviral compound administered parenterally at approximately six-month (half-year) intervals in a dosage volume of about 0.5 to about 4 milliliters (mL) per dose.

[0060] For treating human patients, an effective amount of the antiviral compounds of the invention is preferably in the range of about 80 mg to about 800 mg per dose in a 3-month dosing regimen with a dosing volume of about 0.5 to about 4 mL, and more preferably, the effective amount is in the range of about 200 to about 400 mg, although this dosing regimen can vary depending on the time interval between doses in a particular dosing regimen.

[0061] As used in this specification and claims, the term "effective amount" refers to the amount of the antiviral compound that, after administration, is sufficient to inhibit HIV reverse transcriptase, sufficient to inhibit HIV replication, sufficient to exert a prophylactic effect, and / or sufficient to exert a therapeutic effect.

[0062] The term "administration" and variations thereof, such as "administering a compound," in reference to the claimed methods of treatment means providing an antiviral compound to a patient and encompasses self-administration as well as administration to a patient by another person.

[0063] Preferably, parenteral suspensions suitable for injection contain the antiviral compound of the present invention in an amount ranging from about 3 to 45% by weight based on the weight of the suspension. Preferred particle sizes are about 50 micrometers (μm) or less, more preferably an average particle size ranging from about 6 μm to about 15 μm.

[0064] Preferred flocculating or suspending agents are linear polymers, especially substituted celluloses such as methylcellulose, carboxymethylcellulose (CMC), hydroxypropylcellulose, hydroxypropylmethylcellulose, and the like.

[0065] Preferred surfactants are nonionic surfactants, and a particularly preferred surfactant is polyoxyethylene (20) sorbitan monooleate (TWEEN-80).

[0066] In addition to parenteral dosage forms, the compounds of the present invention can also be administered in oral dosage forms and can be administered as implants.

[0067] Dosage forms containing the compounds of the invention may further include additional anti-HIV and / or anti-HBV agents such as cabotegravir, dolutegravir, doravirine, eviregravir, resiverin, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, lamivudine, and the like.

[0068] The present invention further provides, among various embodiments, a method for preventing viremia or treating a viral infection in a patient for whom reverse transcriptase inhibition is medically indicated, comprising administering to the patient an effective amount or concentration of a compound of Formula (II). More specifically, the compound of Formula (II) can be administered in a formulation that provides sustained, controlled, or prolonged release of EFdA from these prodrugs. More specifically, the compound of Formula (II) can be formulated as an aqueous suspension or solution, and encapsulated in sustained-release particles, including PLGA and other such materials known in the art. More specifically, the viral infection can be caused by HIV or HBV. Routes of administration for these prodrugs include, but are not limited to, oral, parenteral, and release from implants (drug delivery compositions and devices). In methods of treating or preventing a viral infection, the method can further include an additional anti-HIV and / or anti-HBV drug, including but not limited to, cabotegravir, dolutegravir, doravirine, elvitegravir, lersiverine, tenofovir disoproxil fumarate, tenofovir alafenamide fumarate, or lamivudine.

[0069] Example 8 The surfactant concentration was optimized using 0.3% and 0.5% methylcellulose in a formulation containing 400 mg / gm of micronized Compound 5. Formulations were prepared using various concentrations of TWEEN-80 (0.1, 0.2, 0.3%). No significant effect of surfactant concentration was observed on the viscosity, flowability, and redispersion time of the formulations after 10 days of storage. The observed results are reported in Table 5.

[0070] [Table 6]

[0071] Example 9 Bulk batches (40 gm) of suspensions of Compound 5 were prepared with concentrations of 0.3% methylcellulose and 0.2% TWEEN-80. Polymer and surfactant concentrations were optimized at a drug concentration of 40 wt%, but bulk batches with a drug concentration of 35 wt% were also prepared for accelerated and long-term stability studies. The compositions of the formulations filled into the stability chambers are shown in Table 6. The detailed manufacturing procedures and detailed compositions for the preparation of the formulations are shown below.

[0072] [Table 7]

[0073] Methylcellulose was slowly added to the required amount of water for injection (WFI) in a glass bottle while stirring. The resulting mixture was stirred using a magnetic stirrer until the solution was clear and free of lumps. TWEEN-80 (0.08 g) was added to the resulting solution and stirred thoroughly.

[0074] Micronized Compound 5 (14 g; average particle size 11 μm) was slowly added to the prepared polymer-surfactant solution under vigorous stirring at 1400 rpm using a magnetic stirrer. After complete addition of Compound 5, the resulting suspension was stirred (600 rpm) for 20 minutes to ensure uniform particle dispersion.

[0075] The prepared formulations were characterized for various physicochemical properties such as assay, pH, purity %, redispersibility, needle passability, polymorphic form and particle size, etc. The results are shown in Table 7.

[0076] [Table 8]

[0077] The formulations shown in Table 6 are considered acceptable based on the properties shown in Table 7.

[0078] Example 10 Aqueous suspensions of EFdA, Compound 3, and Compound 5 were administered subcutaneously to rhesus monkeys, and peripheral blood mononuclear cells (PBMCs) were periodically collected and evaluated for pharmacokinetic data. The observed results are shown in Tables 8, 9, and 10 for both compounds, and in Figure 13 for Compound 5.

[0079] [Table 9]

[0080] [Table 10]

[0081] [Table 11]

Claims

[Claim 1] The invention described in the specification.

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