Prodrug compositions

Abstract

The present invention provides a composition comprising nanoparticles of prodrugs of certain pharmaceutically active agents, wherein the nanoparticles of prodrugs are dispersed within a carrier material. The present invention further provides processes for the making of the same.

Claims

1. A solid or semi-solid composition comprising nanoparticles of a prodrug compound of emtricitabine dispersed within one or more carrier materials, wherein the log P value of the prodrug compound is at least about 1 and greater than the log P value of emtricitabine, wherein the prodrug compound is selected from the group consisting of: ##STR00036## ##STR00037##

2. The composition of claim 1, wherein the one or more carrier materials comprise a hydrophilic polymer and/or surfactant.

3. The composition of claim 2, wherein the hydrophilic polymer is selected from the group consisting of ethylene oxide-propylene oxide block copolymers, polyvinyl alcohol (‘PVA’), polyvinyl alcohol-polyethylene glycol graft copolymer; polyethylene glycol k1 (having average M.sub.w of 1,000), hydroxylpropyl methyl cellulose (HPMC), polyvinylpyrrolidone k30 (‘PVP k30’) and any combination thereof.

4. The composition of claim 2, wherein the surfactant is selected from the group consisting of vitamin-E-polyethylene glycol-succinate, sodium deoxycholate (NDC), polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, dioctyl sulfosuccinate sodium salt (AOT), polyethylene glycol (15)-hydroxystearate and any combination thereof.

5. The composition of claim 1, wherein the one or more carrier materials are provided in any one of the following combinations: poloxamer 188 AND vitamin-E-polyethylene glycol-succinate; poloxamer 188 AND sodium deoxycholate; poloxamer 188 AND polyoxyethylene (20) sorbitan monolaurate; poloxamer 188 AND polyoxyethylene (20) sorbitan monooleate; poloxamer 188 AND dioctyl sulfosuccinate sodium salt; poloxamer 188 AND polyethylene glycol (15)-hydroxystearate; poloxamer 407 AND vitamin-E-polyethylene glycol-succinate; poloxamer 407 AND sodium deoxycholate; poloxamer 407 AND polyoxyethylene (20) sorbitan monolaurate; poloxamer 407 AND polyoxyethylene (20) sorbitan monooleate; poloxamer 407 AND dioctyl sulfosuccinate sodium salt; poloxamer 407 AND polyethylene glycol (15)-hydroxystearate; polyvinyl alcohol AND vitamin-E-polyethylene glycol-succinate; polyvinyl alcohol AND sodium deoxycholate; polyvinyl alcohol AND polyoxyethylene (20) sorbitan monolaurate; polyvinyl alcohol AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol AND dioctyl sulfosuccinate sodium salt; polyvinyl alcohol-polyethylene glycol graft copolymer AND vitamin-E-polyethylene glycol-succinate; polyvinyl alcohol-polyethylene glycol graft copolymer AND sodium deoxycholate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monolaurate; polyvinyl alcohol-polyethylene glycol graft copolymer AND dioctyl sulfosuccinate sodium salt; polyethylene glycol k1 AND sodium deoxycholate; polyethylene glycol k1 AND polyoxyethylene (20) sorbitan monolaurate; polyethylene glycol k1 AND polyoxyethylene (20) sorbitan monooleate; polyethylene glycol k1 AND dioctyl sulfosuccinate sodium salt; polyethylene glycol k1 AND polyethylene glycol (15)-hydroxystearate; hydroxylpropyl methyl cellulose AND vitamin-E-polyethylene glycol-succinate; hydroxylpropyl methyl cellulose AND sodium deoxycholate; hydroxylpropyl methyl cellulose AND polyoxyethylene (20) sorbitan monolaurate; hydroxylpropyl methyl cellulose AND polyoxyethylene (20) sorbitan monooleate; hydroxylpropyl methyl cellulose AND dioctyl sulfosuccinate sodium salt; hydroxylpropyl methyl cellulose AND polyethylene glycol (15)-hydroxystearate; polyvinylpyrrolidone k30 AND vitamin-E-polyethylene glycol-succinate; polyvinylpyrrolidone k30 AND sodium deoxycholate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monolaurate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monooleate; polyvinylpyrrolidone k30 AND dioctyl sulfosuccinate sodium salt; or polyvinylpyrrolidone k30 AND polyethylene glycol (15)-hydroxystearate.

6. The composition of claim 1, wherein the composition is a solid.

7. A process for preparing a composition according to claim 1, the process comprising: (i) preparing an oil-in-water emulsion comprising: an oil phase comprising a prodrug compound as defined in claim 1; and an aqueous phase comprising one or more selected carrier materials, wherein the one or more carrier materials comprise a hydrophilic polymer and/or surfactant; and (ii) removing the oil and water from the oil-in-water emulsion to form the composition.

8. A process for preparing a composition according to claim 1, the process comprising: (i) preparing a single phase solution comprising a prodrug compound as defined in claim 1, and one or more selected carrier materials, wherein the one or more carrier materials comprise a hydrophilic polymer and/or surfactant, in one or more solvents; and (ii) removing the one or more solvents to form the solid composition.

9. A pharmaceutical or veterinary composition in injectable form comprising a composition according to claim 1, and optionally one or more additional (pharmaceutically acceptable) excipients.

10. An intramuscularly-injectable formulation of nanoparticles of a prodrug compound as defined in claim 1.

11. A subcutaneously-injectable formulation of nanoparticles of a prodrug compound as defined in claim 1.

12. An aqueous dispersion, comprising a plurality of nanoparticles of a prodrug compound as defined in claim 1 dispersed in an aqueous medium, each nanoparticle of the prodrug compound being a core around at least some of which an outer layer composed of one or more carrier materials is provided, wherein the prodrug is present in a concentration of at least 10 mg/mL.

13. An oily dispersion, comprising a plurality of nanoparticles of a prodrug compound as defined in claim 1 and one or more carrier materials dispersed in an oily medium, wherein the prodrug is present in a concentration of at least 10 mg/mL.

14. A method of treating an HIV infection, the method comprising administering a therapeutically effect amount of a composition according to claim 1, or an injectable pharmaceutical or veterinary composition thereof.

15. A prodrug compound selected from any one of the following formulae: ##STR00038## ##STR00039## ##STR00040## ##STR00041##

16. A method of treating an HIV infection, the method comprising administering a therapeutically effect amount of a prodrug compound according to claim 15.

17. A method of treating an HIV infection, the method comprising administering a therapeutically effect amount of a formulation of nanoparticles of the prodrug according to claim 1; wherein the formulation of nanoparticles is an intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, or an oily dispersion, wherein the aqueous dispersion comprises a plurality of nanoparticles of the prodrug compound dispersed in an aqueous medium, each nanoparticle of the prodrug compound being a core around at least some of which an outer layer composed of one or more carrier materials is provided, wherein the prodrug is present in a concentration of at least 10 mg/mL; wherein the oily dispersion comprises a plurality of nanoparticles of the prodrug compound and one or more carrier materials dispersed in an oily medium, wherein the prodrug is present in a concentration of at least 10 mg/mL.

18. The composition of claim 1, wherein the composition is a semi-solid.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 includes table 1 presenting the IUPAC name, structure, yield, NMR data and/or melting point data for compounds of the present invention.

(2) FIG. 2 illustrates hydrolysis measurement measured in pooled mixed gender human liver S9 for compounds of the present invention.

(3) FIG. 3 illustrates kinetic analysis data measuring hydrolysis in pooled mixed gender human liver S9 for compounds of the present invention.

(4) FIG. 4 illustrates half-life measurements in pooled mixed gender human plasma and female skeletal muscle S9 for compounds of the present invention.

(5) FIG. 5 illustrates kinetic analysis data for the conversion of prodrug compound 17 to 13.

(6) FIG. 6 illustrates kinetic analysis data for the conversion of prodrug compound 5 to 13.

(7) FIG. 7 illustrates half-life measurements in human liver S9 for prodrug compounds 20 and 21.

(8) FIG. 8 presents log P measurements for compounds of the present invention.

(9) FIG. 9 illustrates HPLC data for the analysis of cytochrome P450 activity on prodrug compounds of the present invention.

(10) FIGS. 10 and 11 illustrate HPLC data for the UGT enzyme metabolism of prodrugs of the present invention.

(11) FIG. 12 illustrates hydrolysis data measured in mouse or human plasma/liver of compounds of the present invention.

(12) FIG. 13 illustrates half-life measurements for prodrug compound 23.

(13) FIG. 14 illustrates half-life measurements for prodrug compound 25.

(14) FIG. 15 illustrates kinetic analysis data for prodrug compound 25.

(15) FIG. 16 illustrates the hydrolysis analysis of prodrug compound 26 in human plasma.

(16) FIG. 17 illustrates the relationship between log P value and the number of hits identified during initial screening studies as described herein.

(17) FIG. 18 illustrates hydrodynamic diameter distribution traces for prodrug nanoparticles hits of the present invention.

(18) FIG. 19 illustrates a meta-analysis of hits identified across the full range of the prodrug compositions of the present invention.

(19) FIGS. 20 and 21 illustrate hydrodynamic diameter distribution traces for prodrug nanoparticles of the present invention at different wt % loadings.

METHODS AND EXAMPLES

(20) The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. If not mentioned otherwise, all evaporations are performed under reduced pressure, between 50 mmHg and 100 mmHg. The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis, melting point (m.p.) and spectroscopic characteristics, e.g. MS, IR and NMR. Abbreviations used are those conventional in the art.

(21) Preparation of Prodrug Compounds

(22) In general, compounds 1-26 (as illustrated in FIG. 1) may be prepared according to the general methods described below. The skilled person will appreciate that further compounds of the invention are accessible via these general methods by modification of the starting materials and/or substituent groups included in these general methods.

General Method for Synthesis of 5′-Alkoxycarbonyl Emtricitabine Carbamates (Prodrug Compounds 1-8)

(23) In a flame-dried 25 mL round-bottom flask, cooled under argon, emtricitabine (1.0 eq., 0.5 M) was suspended in DCM. Pyridine (3.0 eq., 1.5 M) was then added to the flask and the resulting mixture was cooled to 0° C. in an ice-water bath. The reaction was initiated by the dropwise addition of the appropriate alkyl chloroformate (2.1 eq., 1.05 M). The reaction mixture was left to stir at 0° C. before then being warmed to room temperature. The reaction was monitored by TLC and considered to be complete after 3 hours. The reaction mixture was condensed under reduced pressure and the resulting crude residue was purified via silica flash chromatography (0-10% MeOH in DCM gradient) to provide prodrug compounds 1-8 as a clear oil.

General Procedure for Synthesis of Emtricitabine Carbamates (Prodrug Compounds 9-16)

(24) In a 20 mL vial, compounds 1-8 (1.0 eq., 0.5 M) were suspended in THF. Lithium hydroxide (10 eq., 5 M) was then added followed by the dropwise addition of water (˜20 drops) to increase the solubility of the reaction mixture. The reaction mixture was then left at room temperature to stir. The reaction was monitored by TLC and was considered to be complete after 18 hours. The reaction mixture was condensed under reduced pressure and the resulting crude residue was purified via silica flash chromatography (0-10% MeOH in DCM gradient) to provide compounds 9-16 as a white solid.

General Procedure for Synthesis of 5′-Acyloxy Emtricitabine Carbamates (Prodrug Compounds 17-22)

(25) In a flame-dried 10 mL round-bottom flask, cooled under argon, pyridine (2.0 eq., 0.4 M) was dissolved in DCM. The flask was then cooled to 0° C. in an ice-water bath. The appropriate acyl chloride (1.2 eq., 0.24 M) was then added dropwise to the flask. The reaction was left to stir at 0° C. under argon for 15 minutes. Compounds 13-16 (1.0 eq., 0.2 M) were added to the acyl chloride-pyridine mixture. The reaction mixture was then left to stir at 0° C. for 3 hours. The reaction was monitored by TLC and once complete, the mixture was condensed under reduced pressure. The resulting crude residue was purified via silica flash chromatography (0-100% EtOAc in Hexanes gradient) to provide compounds 17-22 as a clear oil.

Synthesis of (Z)—N′-(5-fluoro-1-((2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-N,N-dimethylformimidamide (prodrug compound 23)

(26) Emtricitabine (255 mg, 1.03 mmol, 1.0 equiv.) was suspended in 4.1 mL of methanol at room temperature. To the suspension was added N,N-dimethylformamide dimethyl acetal (0.68 ml, 5.15 mmol, 5.0 equiv.) in one portion at room temperature to provide a colourless solution after approximately 5 minutes. After approximately a further 1-2 hours a precipitate formed. The reaction mixture was stirred for 21 hours at room temperature in total. The reaction mixture was then condensed and volatiles removed under reduced pressure using a rotary evaporator. The resulting residue was then dried under high vacuum for 3 hours to afford compound 23 (311 mg; white solid; quantitative yield). Compound 23 was used without further purification.

Synthesis of 2-(4-((1-((2R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-oxathiolan-5-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-methyl-4-oxobutan-2-yl)-3,5-dimethylphenyl acetate (Prodrug Compound 24)

(27) To a solution of 3-(2-acetoxy-4,6-dimethylphenyl)-3-methylbutanoic acid (205 mg, 0.77 mmol) in anhydrous DMF (1.55 ml) was added HATU (294 mg, 0.77 mmol) in one portion at room temperature followed by the addition of diisopropylethylamine (0.30 ml, 1.74 mmol). After stirring at room temperature for 5 minutes, 4-amino-1-((2R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-oxathiolan-5-yl)-5-fluoropyrimidin-2(1H)-one (279 mg, 0.77 mmol) was added in one portion. The reaction mixture was stirred for 3 days at room temperature. The reaction mixture was then diluted with water and saturated aqueous Na.sub.2CO.sub.3 and extracted with EtOAc (3×15 mL). The organic layers were combined, washed with brine, dried with anhydrous MgSO.sub.4 and concentrated in vacuo. The resulting residue was then purified by flash chromatography (0-100% EtOAc in Hexanes gradient) to provide compound 24 (305 mg; 65% yield) as clear colourless glass.

Synthesis of 2-(4-((5-fluoro-1-((2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-methyl-4-oxobutan-2-yl)-3,5-dimethylphenyl acetate (Prodrug Compound 25)

(28) To a solution of 2-(4-((1-((2R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-1,3-oxathiolan-5-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)amino)-2-methyl-4-oxobutan-2-yl)-3,5-dimethylphenyl acetate (compound 24) (218 mg, 0.36 mmol) in THF (1.8 mL) was added trimethylamine trihydrofluoride (292 μL, 1.79 mmol) in one portion at room temperature. After approximately 5 hours at room temperature, TLC indicated that the starting material was fully consumed. To the reaction mixture was then added water (15 mL) and EtOAc (15 mL). The resulting mixture was then extracted with EtOAc (3×10 mL), the organic layers were combined, washed with brine, dried with anhydrous MgSO.sub.4 and concentrated in vacuo. The resulting residue was then purified by reverse phase (C18) flash chromatography (0 to 100% MeCN in H2O gradient) to provide compound 25 (153 mg; 86% yield) as a clear colourless glass.

Synthesis of (2S)-isopropyl (((((R)-1-(6-(3-(2-acetoxy-4,6-dimethylphenyl)-3-methylbutanamido)-9H-purin-9-yl)propan-2-yl)oxy)methyl)(phenoxy)phosphoryl)-L-alaninate (Prodrug Compound 26)

(29) To a solution of 3-(2-acetoxy-4,6-dimethylphenyl)-3-methylbutanoic acid (284 mg, 1.08 mmol) in 0.4 M NMM/DMF (2.5 mL) was added HATU (391 mg, 1.03 mmol) in one portion at room temperature. After stirring at room temperature for 5 minutes, to the mixture was added (2S)-isopropyl2-((((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)(phenoxy)phosphoryl)amino)propanoate (466 mg, 0.99 mmol) in one portion and the resulting reaction mixture was stirred for 3 days at room temperature. Saturated aqueous Na.sub.2CO.sub.3 was then added the mixture then extracted with EtOAc (3×15 mL). The organic layers were combined, washed with brine, dried with anhydrous MgSO.sub.4 and concentrated in vacuo. The resulting residue was then purified by flash chromatography (0 to 10% MeOH/CHCl.sub.3 gradient) to provide compound 26 (87 mg; 12% yield) as clear colourless glass.

(30) Kinetic Analysis

(31) General Procedure for Preliminary Hydrolysis Measurements of Emtricitabine Carbamate Prodrug Compounds 10-16 and Capecitabine

(32) To evaluate the hydrolysis rates of compounds 10-16, HPLC coupled with UV detection was used. This was performed using the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes. Pooled mixed gender human liver S9 (20.0 mg/mL, Xenotech) was pre-incubated at 37° C. for 5 minutes. Compounds 10-16 (1 mM) were then added and the mixture incubated at 37° C. Aliquots were taken at each time point and quenched in an equal volume of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted 10 fold into Tris buffer (30 mM, pH 7.4) and injected onto the HPLC for analysis.

(33) Hydrolysis measurements of compounds 10-16 at 1 mM in 20.0 mg/mL pooled mixed gender human liver S9 are shown in FIG. 2 and demonstrate that emtricitabine carbamates with longer alkyl chains are more rapidly cleaved.

(34) General Procedure for Kinetic Analysis of Emtricitabine Carbamate Prodrug Compounds 9-16 and Capecitabine Via UV

(35) Reaction mixtures containing Tris buffer (30 mM, pH 7.4) and mixed pooled gender liver S9 (10.0 mg/mL) were pre-incubated at 37° C. for 5 min. Reactions are initiated by the addition of emtricitabine prodrug compounds 9-16 and capecitabine (concentrations vary from 200 uM to 10 mM for capecitabine and compounds 13-16, concentrations up to 20 mM for compound 12 and concentrations up to 60 mM for compounds 9-11). After incubation at 37° C., aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted into Tris buffer (30 mM, pH 7.4) to yield concentrations appropriate for analysis and read spectrophotometrically monitoring a decrease in substrate at 305 nm.

(36) The results of the kinetic analysis of emtricitabine carbamate compounds 9-16 is illustrated in FIG. 3. FIG. 3 demonstrates that carbamate to emtricitabine hydrolysis, measured in pooled mixed gender human liver S9, results in emtricitabine carbamates with longer alkyl chains having a higher intrinsic clearance. This is indicative of a more rapid rate of hydrolysis.

(37) General Procedure for Half-Life Measurements of Prodrug Compounds 9-16 in Plasma and Muscle S9

(38) To evaluate the hydrolysis rates of emtricitabine compounds 9-16, HPLC coupled with UV detection was used with the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes. Male human plasma (Bioreclamation) or female human skeletal muscle S9 (7.96 mg/mL, Bioreclamation) was pre-incubated at 37° C. for 5 minutes. Compounds 9-16 (1 mM) were added and the reaction was incubated at 37° C. Aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted 10 fold into Tris buffer (30 mM, pH 7.4) and injected onto the HPLC for analysis.

(39) The results of the half-life analysis in pooled mixed gender human plasma and female skeletal muscle S9 are illustrated in FIG. 4. FIG. 4 indicates long hydrolysis half-lives for compounds 9-16 regardless of alkyl chain length.

(40) General Procedure for Kinetic Analysis of 5′-Acetoxy Emtricitabine Carbamates Prodrug Compound Hydrolysis

(41) A reaction mixture containing Tris buffer (30 mM, pH 7.4) and mixed pooled gender liver S9 (1 mg/mL, 3.0 mg/mL, or 4.0 mg/mL) are pre-incubated at 37° C. for 5 minutes. To the reaction mixtures is added compound 17 which initiates the hydrolysis reaction (concentrations of compound 17 ranging from 200 μM to 10 mM). After incubation at 37° C., aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 3 minutes. The supernatant was diluted into Tris buffer (30 mM, pH 7.4) to yield samples with concentrations appropriate for analysis. The samples were analyzed by HPLC with UV detection using the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes.

(42) Kinetic analysis data for the conversion of compound 17 to compound 13 via 5′-acetoxy hydrolysis is illustrated in FIG. 5. The hydrolysis was performed under conditions in which the pentyl carbamate group of compound 17 is stable in 1.0 mg/mL pooled mixed gender human liver S9. It was observed that the intrinsic clearance for the 5′-acetoxy hydrolysis is greater than that for the carbamate group hydrolysis.

(43) Kinetic analysis data for the conversion of compound 5 to compound 13 via 5′-pentoxycarbonyl hydrolysis is illustrated in FIG. 6 (concentrations of compound 5 ranging from 200 μM to 10 mM). The hydrolysis was performed under conditions in which the pentyl carbamate group is stable in 3.0 mg/mL pooled mixed gender human liver S9. It was observed that the intrinsic clearance for 5′-pentoxycarbonyl hydrolysis is greater than that for carbamate group hydrolysis but reduced when compared to the intrinsic clearance of the 5′-acetoxy hydrolysis. Kinetic analysis data for the conversion of compound 17 to compound 13 via 5′-acetoxy hydrolysis and the conversion of compound 5 to compound 13 via 5′-pentoxycarbonyl hydrolysis is presented in Table 1 below.

(44) TABLE-US-00001 TABLE 1 Intrinsic Clearance Compound K.sub.M (mM) V.sub.max (nmol/h/mg) (μL/h/mg) 17 1.31 4877 3723 5 0.6 948 1580

(45) Kinetic analysis data for compounds 20 and 21 in human liver S9 was generated in accordance with the general procedure described above. The data, as illustrated in FIG. 7 shows comparable half-lives for compounds 20 and 21.

(46) Procedure for Log P Determinations of Emtricitabine Carbamate Prodrug Compounds

(47) Carbamate compounds were dissolved in 1-octanol and mixed vigorously with an equal volume of deionized water. The mixture was then allowed to separate and each layer collected, diluted and measured by HPLC coupled with a UV detection system (5% to 100% acetonitrile/Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1. The log P values for emtricitabine carbamate prodrug compounds, taken as an average of triplicate measurements, are illustrated in FIG. 8. It was observed that there is an increase in log P as the carbamate group alkyl chain length increases.

(48) Procedure for Analysis of Cytochrome P450 Activity on Emtricitabine and Prodrug Compound 13

(49) Reaction mixtures containing phosphate buffer (100 mM, pH 7.4), mixed pooled gender human liver microsomes (2.0 mg/mL), NADP+ (1 mM), glucose-6-phosphate (5 mM), and glucose-6-phosphate dehydrogenase (1.5 Unit/mL) were pre-incubated at 37° C. for 5 minutes. The reactions were then initiated via the addition of emtricitabine or compound 13 (1 mM). After incubation at 37° C., aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 4 minutes. The supernatant was then diluted into phosphate buffer (100 mM, pH 7.4) to yield samples with concentrations suitable for analysis. The samples were analyzed by HPLC with UV detection using the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes.

(50) it was observed that emtricitabine was not metabolized by CYP enzymes in 2.0 mg/mL pooled mixed gender human liver microsomes. However, compound 13 was metabolized only in the presence of NADPH. Further LC-MS analysis suggests that the compound 13 metabolite was hydroxylated, as shown in FIG. 9.

(51) Procedure for Analysis of UGT Activity on Emtricitabine and Prodrug Compound 13

(52) Reaction mixtures containing phosphate buffer (100 mM, pH 7.4), mixed pooled gender human liver S9 (2.0 mg/mL), MgCl.sub.2 (10 mM), UDPGA (8 mM), and D-saccharic acid-1,4-lactone (2 mM) were pre-incubated at 37° C. for 5 minutes. The reactions were then initiated by the addition of emtricitabine or compound 13 (1 mM). After incubation at 37° C., aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 4 minutes. The supernatant was then diluted into phosphate buffer (100 mM, pH 7.4) to yield samples with concentrations suitable for analysis. The samples were analyzed by HPLC with UV detection using the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes.

(53) HPLC analysis, as shown in FIG. 10, illustrates that emtricitabine is not metabolized by UGT enzymes. HPLC analysis, as shown in FIG. 11, illustrates that compound 13 is also not metabolized by UGT enzymes. The only reaction observed was the conversion of compound 13 into emtricitabine via carbamate hydrolysis.

(54) General Procedure for Analysis of Prodrug Hydrolysis in Mouse, Rat, and Human Liver S9 or Plasma

(55) Reaction mixtures containing either mouse liver S9 (20.8 mg/mL), mouse plasma, rat liver S9 (1 mg/mL) and phosphate buffer (100 mM, pH 7.4), rat plasma, human liver S9 (1.0 mg/mL) and phosphate buffer (100 mM, pH 7.4) or human plasma were pre-incubated at 37° C. for 5 minutes. Reactions were then initiated by addition of compounds 4, 8 or 16 (1 mM in mouse, 0.5 mM in rat and human). After incubation at 37° C., aliquots are taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was then diluted into phosphate buffer (30 mM, pH 7.4) to yield samples with concentrations suitable for analysis. The samples were analyzed by HPLC with UV detection and the resulting hydrolysis data can be seen in FIG. 12.

(56) It was observed that the hydrolysis rate for emtricitabine carbamate compound 16 (1 mM) is comparable in mouse and human liver S9. However, the hydrolysis rate of compound 16 in mouse plasma is dramatically enhanced relative to human plasma (illustrated in FIG. 3).

(57) Table 2 shows half-life data for compounds 4 and 8 (0.5 mM) for both rat plasma/liver and human plasma/liver. In all cases, the half-lives for compound 4 are shorter than that of compound 8. The data indicates that the octyl carbonate hydrolysis of compound 8 occurs less readily than butyl carbonate hydrolysis of compound 4. In all cases, the half-life indicates the depletion of starting material compounds. In rat compartments, the evidence suggests that the carbamate group of the prodrug compounds is cleaved before the carbonate groups. In human compartments, only carbonate cleavage is observed.

(58) TABLE-US-00002 TABLE 2 Human Human Rat Plasma t.sub.1/2 Rat Liver t.sub.1/2 Plasma t.sub.1/2 Liver Compound (min) (min) (min) t.sub.1/2 (min) 4 5.8 4.6 59.2 10.3 8 16.7 33.3 578 151

(59) General Procedure for Half-Life Measurements of Emtricitabine Amidine Prodrug Compound 23

(60) To evaluate the hydrolysis rates of emtricitabine amidine prodrugs, HPLC coupled with UV detection was used with the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes. Normal human serum (Bioreclamation) or phosphate buffer (100 mM, pH 7.4) was pre-incubated at 37° C. for 5 minutes. Emtricitabine amidine compound 23 (1 mM) was then added and the reaction mixture was incubated at 37° C. Aliquots were taken at each time point and quenched in three volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 7 minutes. The supernatant was injected onto a HPLC system for analysis, the resulting data can be seen illustrated in FIG. 13. Half-life data for emtricitabine amidine compound 23 can be seen as presented in Table 3 below.

(61) TABLE-US-00003 TABLE 3 Conditions Half-Life (h) Phosphate Buffer (100 mM, pH 7.4) 95 Human Serum 5.8

(62) General Procedure for Kinetic Analysis of Emtricitabine Trimethyl Lock Prodrugs Via UV

(63) A reaction mixture containing Tris buffer (30 mM, pH 7.4) and mixed pooled gender liver S9 (10.0 mg/mL) was pre-incubated at 37° C. for 5 minutes. The reaction was then initiated by the addition of emtricitabine trimethyl lock compound 25 (concentration of compound 25 ranging from 100 μM to 8 mM). After incubation at 37° C., aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted into Tris buffer (30 mM, pH 7.4) to yield samples having concentrations appropriate for analysis and read spectrophotometrically monitoring a decrease in substrate at 312 nm. The kinetic analysis data has been summarized in Table 4 below.

(64) TABLE-US-00004 TABLE 4 Intrinsic Clearance Compound K.sub.M (mM) V.sub.max (nmol/h/mg) (μL/h/mg) 25 2.4 1659 691

(65) General Procedure for Half-Life Measurements of Emtricitabine Trimethyl Lock Prodrugs

(66) To evaluate the hydrolysis rates of emtricitabine trimethyl lock prodrugs, HPLC coupled with UV detection was used with the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes. Human plasma (Bioreclamation) or human skeletal muscle S9 (7.96 mg/mL, Bioreclamation) was pre-incubated at 37° C. for 5 minutes. Emtricitabine trimethyl lock compound 25 (1 mM) was added and the reaction was incubated at 37° C. Aliquots were taken at each time point and quenched in three volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted in Tris buffer (100 mM, pH 7.4) and injected onto the HPLC for analysis. Half-life data for emtricitabine trimethyl lock compound 25 is presented in Table 5 below.

(67) TABLE-US-00005 TABLE 5 Conditions Half-Life (h) Human plasma 0.4 Human muscle S9 (7.96 mg/mL) 1

(68) General Procedure for Kinetic Analysis of Emtricitabine Trimethyl Lock Prodrugs in Mouse Liver S9 Via UV

(69) A reaction mixture containing Tris buffer (30 mM, pH 7.4) and mixed pooled gender mouse liver S9 (1.0 mg/mL) were pre-incubated at 37° C. for 5 minutes. The reaction is then initiated by the addition of emtricitabine trimethyl lock compound 25 (concentration of compound 25 ranging from 600 μM to 6 mM). After incubation at 37° C., aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted into Tris buffer (30 mM, pH 7.4) to yield concentrations appropriate for analysis and read spectrophotometrically monitoring a decrease in substrate at 312 nm. The kinetic analysis data in mouse liver S9, as illustrated in FIG. 15, has been summarized in Table 6 below.

(70) TABLE-US-00006 TABLE 6 Intrinsic Clearance Compound K.sub.M (mM) V.sub.max (nmol/h/mg) (μL/h/mg) 25 1.6 13200 8300

(71) General Procedure for Half-Life Measurements of Tenofovir Alafenamide Trimethyl Lock Prodrug in Human Plasma

(72) To evaluate the hydrolysis rates of tenofovir alafenamide compound 26, HPLC coupled with UV detection was used with the following method (0% to 100% acetonitrile in Et.sub.3NHOAc (50 mM, pH 8) gradient) over 10 minutes at a flow rate of 3 mL min.sup.−1, held at 100% acetonitrile for 3 minutes, ramped down to 5% acetonitrile over 0.2 minutes and then held for 2.8 minutes. Human plasma (Bioreclamation) was pre-incubated at 37° C. for 5 minutes. Compound 26 (1 mM) was added and the reaction was incubated at 37° C. Aliquots were taken at each time point and quenched in two volumes of ice-cold methanol. The quenched aliquots were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted in Tris buffer (100 mM, pH 7.4) and injected onto the HPLC for analysis.

(73) The HPLC analysis data of tenofovir alafenamide compound 26 in human plasma, illustrated in FIG. 16, shows that hydrolysis (through two potential pathways) occurs with a half-life of 1.1 h.

(74) Single-Round Infectivity Assays

(75) Procedure for Single-Round Infectivity Assays

(76) Based on the kinetic analysis data provided above, compounds 16 and 25 were demonstrated to be most efficiently hydrolysed and, therefore, were selected for single-round infectivity assays.

(77) Peripheral blood mononuclear cells were isolated from healthy blood donors using Hypaque-Ficoll gradient centrifugation. CD4+ T cells were selected by magnetic beads (Miltenyi) and activated using anti-CD3 and anti-CD28 antibodies. Activated cells were seeded onto a 96-well plate at a concentration of 1×10.sup.5 cells/well in RPMI1640 supplemented with 10% FBS, cytokine-rich supernatant, and 50% heat-inactivated human serum. Test compounds were added at this step and maintained throughout the culture. Cells were incubated at 37° C. for 21 hours. Pseudotype (GFP-tagged) HIV was then added to the culture via spinoculation and incubated for 3 days. Cells were then washed and stained with Zombie Red viability stain. Infectivity was quantified using flow cytometry.

(78) The IC.sub.50 for emtricitabine obtained closely matches literature reported values. In addition, viability staining indicates that there is little to no toxicity of FTC on CD4+ T cells. The IC.sub.50 obtained for compounds 25 and 16 shows comparable potency to the parent emtricitabine. Viability staining also indicates little to no toxicity of 16 and 25 on CD4+ T cells. The IC.sub.50 data for these compounds is presented in Table 7 below.

(79) TABLE-US-00007 TABLE 7 Compound IC.sub.50 (nM) ± SD Emtricitabine 14.4 ± 4.1 16  39.4 ± 17.6 25 28.6 ± 1.2

(80) Stability Assessment of Single-Round Infectivity Assay Well Media

(81) Well media used in the single-round infectivity assays as described above, comprised of RPMI1640 medium supplemented with 10% FBS, cytokine-rich supernatant, 50% heat-inactivated human serum and HEPES buffer (12 mM). The emtricitabine test compounds were incubated in well media at 37° C. At various time points, aliquots were quenched in two volumes of ice-cold methanol. Quenched mixtures were then centrifuged at 14000 rpm for 5 minutes. The supernatant was diluted 10-fold into Tris buffer (30 mM, pH 7.4) and then analyzed via HPLC coupled with UV detection.

(82) Stability studies indicate that both compounds 16 and 25 were hydrolyzed to the parent emtricitabine in a time-frame matching the time of pre-incubation in single-round infectivity assays. This is likely due to the presence of heat-inactivated serum still containing active esterases. The antiviral potency of 16 and 25 is thus due to parent emtricitabine revealed following carbamate hydrolysis. Well media half-life data is provided in Table 8 below.

(83) TABLE-US-00008 TABLE 8 Compound Well Media Half-Life (h) 16 18 25 0.83

(84) Preparation of Prodrug Nanoparticles

(85) In general, prodrug nanoparticles of the present invention may be prepared according to the general methods described below. The skilled person will appreciate that further nanoparticles of the invention are accessible via these general methods by modification of the prodrug compound, carrier materials, reagents or the reaction conditions included in these general methods.

(86) Initial Screening Approach for Prodrug Nanoparticles

(87) Prodrug nanoparticles based on prodrug compounds 1-26, prepared and investigated as described above, were synthesised. The prodrug nanoparticles were formed using carrier materials (i.e. polymers and surfactants) selected based on their ability to stabilise nanoparticles in aqueous environments as well as their safety profiles according to the FDA's GRAS list. These selected carrier materials are listed in Table 9 below.

(88) TABLE-US-00009 TABLE 9 Polymer Surfactant Pluronics ® F-68 Vitamin-E-polyethylene glycol-succinate (TPGS) Pluronics ® F-127 Sodium deoxycholate (NDC) Polyvinyl alcohol (PVA) Polyoxyethylene (20) sorbitan monolaurate (Tween ™ 20) Kollicoat ™ Polyoxyethylene (20) sorbitan monooleate (Tween ™ 80) Polyethylene Glycol (PEG) Dioctyl sulfosuccinate sodium salt (AOT) k1 (Mw 1,000) Hydroxylpropyl methyl Polyethylene glycol (15)-hydroxystearate cellulose (HPMC), (Solutol ™ HS) Polyvinylpyrrolidone k30 (‘PVP k30’)

(89) Screening of Prodrug Particles Containing Prodrug Compounds at 10 wt % Loadings

(90) For each prodrug compound 1-23 and 25 42 binary combinations (i.e. each polymer was systematically combined with each surfactant) of the polymers and surfactants (listed in Table 9) were used to prepare prodrug nanoparticles, loaded with 10 wt % prodrug compound, for the initial screening. The prodrug compound loadings for each of the prodrug nanoparticles were 10 wt % compared to carrier materials. The 10 wt % loaded prodrug nanoparticles were prepared according to the procedure below.

(91) Procedure for the Preparation of 10 wt % Loaded Prodrug Nanoparticles

(92) Stock solutions of carrier materials were dissolved in water at a concentration of 22.5 mg/ml and left to roll overnight to ensure full dissolution. Prodrugs were dissolved in chloroform at a concentration of 10 mg/mL, using a magnetic stirrer bar and magnetic stirrer plate, at room temperature to ensure full dissolution. The resultant prodrug solutions were not left rolling overnight, due to the likely hydrolysis of the modified carbamate and/or carbonate groups.

(93) Into a 4 mL glass vial was added 266.6 μL of polymer solution, along with 133.4 μL of surfactant solution. To this total of 400 μL aqueous phase, 100 μL of prodrug solution in chloroform was added giving a ratio of 1:4 of organic phase to aqueous phase. This two phase mixture was then sonicated (Covaris S2x ultrasonicator, with a duty cycle of 20%, an intensity of 250, cycles per burst of 500 in frequency sweeping mode). This provided homogenous emulsions. Following sonication, the emulsions were immediately frozen in liquid nitrogen prior to being freeze dried for 48 hours using a Benchtop K freeze dryer (Virtis) at a setting of −100° C. and a pressure of <20 pBar.

(94) At the end of the 48 hours freeze drying process, the samples were sealed and placed in a humidity controlled desiccator prior to analysis. The dried prodrug nanoparticle monoliths had a composition of 1 mg prodrug, 3 mg surfactant, and 6 mg polymer.

(95) Physical characterisation of the prodrug nanoparticles (after dispersion in water) such as hydrodynamic diameter and polydispersity index (PDI) were analysed using Dynamic Light Scattering (DLS) (also referred to as Photon Correlation Spectroscopy (PCS)). Dynamic light scattering measurements were carried out at 25° C. on a Malvern Instruments Ltd. Zetasizer Nano S spectrometer using the following parameters.

(96) Particle Type: Nanoparticles (Refractive Index 1.330, Absorption 0.010)

(97) Dispersant: Water (Viscosity 0.8872 cP, Refractive Index 1.330)

(98) Temperature: 25° C.

(99) Cell Type: Polystyrene disposable cuvette

(100) Measurement Angle: 172° Back Scatter

(101) Number of Measurements: 3

(102) Number of runs per measurement: Automatic

(103) Prodrug nanoparticles “hits” were then identified based on their ability to disperse in water (at a concentration of 1 mg/mL), having Z-average hydrodynamic diameters of less than 1000 nm and PDI values of less than 0.5.

(104) It was observed that there is a clear correlation between the log P of the prodrug compound and the number of prodrug nanoparticle “hits” formed from the 42 binary combinations of polymer/surfactant. With being bound theory, the data suggest that as the carbon chain length of the carbonate/carbamate groups of the prodrug compounds 1-8 increases, the number of prodrug nanoparticle “hits” formed from the 42 binary combinations of polymer/surfactant increases. FIG. 17 illustrates the relationship between the log P of carbonate/carbamate prodrug compounds 1-8 and the number of prodrug nanoparticle “hits” formed from the 42 binary combinations of polymer/surfactant.

(105) Prodrug nanoparticles based on the carbonate/carbamate prodrug compounds 1-8 having narrow size distributions with PDI values of less than (<0.2) were selected. It was also observed that at a minimum carbon chain length of 4 of the carbonate/carbamate groups there was an enhanced quality of prodrug nanoparticles produced. This is illustrated in FIG. 18 where prodrug nanoparticles of the present invention show narrow hydrodynamic size distributions.

(106) FIG. 19 illustrates a heat map showing the frequency of “hits” produced for each binary combination of polymer/surfactant. The data indicates that the prodrug nanoparticles containing sodium deoxycholate (NDC) surfactant had an increased likelihood of being identified as a “hit” as compared to the other surfactants.

(107) TABLE-US-00010 TABLE 10 Z-average hydro- Prodrug Prodrug dynamic nanoparticle com- diameter composition pound Polymer Surfactant (nm) PDI (i) 4 PVA Tween ™ 80 177 0.176 (ii) 4 Pluronics ® NDC 180 0.181 F-68 (iii) 4 Kollicoat ™ NDC 250 0.173 (iv) 5 PVP k30 Tween ™ 20 184 0.073 (v) 5 PVA Tween ™ 20 193 0.010 (vi) 5 Pluronics ® Tween ™ 20 184 0.060 F-127 (vii) 6 Kollicoat ™ AOT 226 0.013 (viii) 7 PVP k30 Tween ™ 20 197 0.143 (ix) 7 Kollicoat ™ Tween ™ 80 192 0.197 (x) 8 Pluronics ® NDC 165 0.187 F-68

(108) Table 10 shows a selection of prodrug nanoparticles (i)-(x) identified from the initial screening approach at 10 wt % prodrug compound loading which demonstrate narrow size distributions.

(109) Screening of Prodrug Particles Containing Prodrug Compounds at 50 wt % and 70 Wt % Loadings

(110) Similar screening approaches were performed for the prodrug nanoparticles having prodrug compound loadings of 50 wt % and 70 wt %. The prodrug nanoparticles were prepared according to the procedures described below.

(111) Procedure for the Preparation of 50 wt % Loaded Prodrug Nanoparticles

(112) Stock solutions of polymer were dissolved in water at a concentration of 13.3 mg/mL, with surfactants dissolved in water at a concentration of 10 mg/mL and left to roll overnight to ensure full dissolution. Prodrugs compounds were dissolved in chloroform at a concentration of 50 mg/mL, using a magnetic stirrer bar and magnetic stirrer plate, at room temperature, to ensure full dissolution. The resultant prodrug solutions were not left rolling overnight, due to the likely hydrolysis of the modified carbamate and/or carbonate groups.

(113) In a 4 mL glass vial, 300 μL of polymer solution was added, along with 100 μL of surfactant solution. To this total of 400 μL aqueous phase, 100 μL of prodrug compound in solution of chloroform as added, giving a ratio of 1:4 organic to aqueous phase. The samples were then processed using sonication and freeze drying as for the 10 wt % samples described above. The dried nanoparticle monoliths had a composition of 5 mg drug, 1 mg surfactant, and 4 mg polymer.

(114) The number of “hits” (as described above) for the prodrug nanoparticles containing prodrug compounds at 50 wt % was lower compared to the number of “hits” identified during the 10 wt % loading screen. Prodrug nanoparticles containing prodrug compound 1 at 50 wt % loading together with the NDC/PVP k30 and NDC/Kollicoat™ combinations produced “hits”. Prodrug nanoparticles containing prodrug compound 4 at 50 wt % loading together with the following surfactant/polymer combinations produced “hits” (TPGS/Pluronics® F-127, TPGS/Kollicoat™, TPGS/PVA, Tween™ 20/PVA and Tween™ 80/PVA). Prodrug nanoparticles containing prodrug compound 8 at 50 wt % loading together with the following surfactant/polymer combinations produced “hits” (TPGS/Pluronics® F-68, TPGS/HMPC, TPGS/Pluronics® F-127, TPGS/Kollicoat™ TPGS/PVA, Tween™ 20/HMPC, Tween™ 20/PVA, Tween™ 80/HMPC, Tween™ 80/Kollicoat™, NDC/HMPC, NDC/Pluronics® F-127, NDC/Kollicoat™, NDC/PVA, AOT/HMPC, AOT/PVA, Solutol™ HS/Pluronics® F-68 and Solutol™ HS/HMPC).

(115) Procedure for the Preparation of 70 wt % Loaded Prodrug Nanoparticles Stock solutions of polymer were dissolved in water at a concentration of 10 mg/mL, with surfactants dissolved in water at a concentration of 5 mg/mL and left to roll overnight to ensure full dissolution. Prodrugs compounds were dissolved in chloroform at a concentration of 70 mg/mL, using a magnetic stirrer bar and magnetic stirrer plate, at room temperature, to ensure full dissolution. The resultant prodrug solutions were not left rolling overnight, due to the likely hydrolysis of the modified carbamate and/or carbonate groups.

(116) In a 4 mL glass vial, 200 μL of polymer solution was added, along with 200 μL of surfactant solution. To this total of 400 μL aqueous phase, 100 μL of pro drug in solution of chloroform was added, giving a ratio of 1:4 organic to aqueous phase. The samples were then processed using sonication and freeze drying as for 10 wt % samples described above. The dried nanoparticle monoliths had a composition of 7 mg drug, 1 mg surfactant, and 2 mg polymer

(117) The number of “hits” (as described above) for the prodrug nanoparticles containing prodrug compounds at 70 wt % was lower compared to the number of “hits” identified during the 50 wt % loading screen. Prodrug nanoparticles containing prodrug compound 4 at 70 wt % loading together with the Tween™ 20/PVA and AOT/HMPC surfactant/polymer combinations produced “hits”. Prodrug nanoparticles containing prodrug compound 8 at 70 wt % loading together with TPGS/Pluronics® F-127 and NDC/Polyethylene glycol k1 surfactant/polymer combinations also produced “hits”.

(118) Summary of Initial Screening Results

(119) Based on the physicochemical properties determined during the initial screening studies performed on the prodrug nanoparticles having prodrug compound loadings of 10 wt %, 50 wt % and 70 wt %, the most promising surfactant/polymer combinations were identified. These were prodrug nanoparticles containing prodrug compound 4 (at both 50 wt % and 70 wt % loadings) and Tween™ 20/PVA as well as prodrug nanoparticles containing prodrug compound 8 (at both 50 wt % and 70 wt % loadings) and TPGS/Pluronics® F-127.

(120) Table 11 shows that these prodrug nanoparticles (xi)-(xiv) possess narrow size distributions. FIG. 20 illustrates the hydrodynamic size distributions for prodrug nanoparticles containing prodrug compound 4 (10 wt %, 50 wt % and 70 wt %) and Tween™ 20/PVA. FIG. 21 illustrates the hydrodynamic size distributions for prodrug nanoparticles containing prodrug compound 8 (10 wt %, 50 wt % and 70 wt %) and TPGS/Pluronics® F-127.

(121) TABLE-US-00011 TABLE 11 Z-average hydro- Prodrug Prodrug dynamic nano- Prodrug loading diameter particle compound (wt %) Polymer Surfactant (nm) PDI xi 4 50 PVA Tween ™ 291 0.240 20 xii 4 70 PVA Tween ™ 412 0.437 20 xiii 8 50 Pluronics ® TPGS 177 0.279 F-127 xiv 8 70 Pluronics ® TPGS 240 0.415 F-127

(122) In Vivo Pharmacokinetic Studies

(123) In vivo pharmacokinetic studies were performed to determine the plasma concentrations of long-acting emtricitabine prodrug nanoparticles following intramuscular administration. The prodrug nanoparticles consist of novel emtricitabine prodrugs loaded into solid prodrug nanoparticles. The effects 12 solid prodrug nanoparticles compositions intramuscularly administered over a period of 7 days was investigated. Compositions containing a polymer/surfactant combination of Pluronics® F-127/TPGS and PVA/Tween™ 20 were selected based on their excellent performance during the initial screening studies described above. The compositions containing a polymer/surfactant combination of HMPC/NDC and HMPC/AOT were selected in order to further investigate the effect of the polymer/surfactant combination on the pharmacokinetic profile of the compositions. Table 12 provides details of the solid prodrug nanoparticles compositions selected for the study.

(124) TABLE-US-00012 TABLE 12 Prodrug Prodrug Prodrug loading nanoparticle compound (wt %) Polymer Surfactant xiv 8 70 Pluronics ® TPGS F-127 xii 4 70 PVA Tween ™ 20 xv 4 50 Pluronics ® TPGS F-127 xvi 6 50 Pluronics ® TPGS F-127 xiii 8 50 Pluronics ® TPGS F-127 xi 4 50 PVA Tween ™ 20 xvii 6 50 PVA Tween ™ 20 xviii 8 50 PVA Tween ™ 20 xix 6 50 HMPC NDC xx 8 50 HMPC NDC xxi 6 50 HMPC AOT xxii 8 50 HMPC AOT

(125) General Procedure for In Vivo Studies

(126) Adult male Wistar rats (˜300 g) were divided into 12 groups (1 rat per group). Groups were dosed with the prodrug nanoparticles (as shown in Table 12 above) (10 mg/Kg of emtricitabine, adjusted for mz of emtricitabine in each prodrug) via intramuscular injection in the musculus biceps femoris. Food and water was provided ad libitum throughout the procedure.

(127) Following habituation (7 days) the rats received a single dose of the prodrug nanoparticles via intramuscular injection (0.1 mL as a suspension in distilled water) as outlined above. The rats were anesthetised during injection (isoflurane), EMLA was given topically and buprenorphine provided subcutaneously (0.3 mg/mL). Blood samples were then collected (500 μl) post dosing from the tail vein (detailed in Table 13 below). The weight of each rat was determined prior to sampling and used as an estimation of healthiness. At the point of termination the rats were sacrificed using the rising gradient of CO.sub.2 followed by cervical dislocation.

(128) Analysis of In Vivo Data

(129) Bioanalysis was performed on a TSQ Endura (Thermoscientific) using a validated assay for emtricitabine in plasma. A calibration curve of emtricitabine was prepared in rat plasma via serial dilution, ranging from 1.9 to 500 ng/ml. Extraction was performed using protein precipitation. Linearity was then assessed by three independent preparations of the standard curve. Maximum allowed deviation of standards was set at 15% of the stated value, excluding the LLOQ where deviation was set at no more than 20%.

(130) Inter and intra assay accuracy and precision was assessed by preparation of three concentrations (in the range of the standard curve 5, 200 and 400 ng/mL) with each preparation in triplicate. The mean value of each concentration was be within 15% of the stated concentration (except the lower concentration, where deviation was <20%).

(131) Plasma concentrations of emtricitabine following administration of nanoparticle prodrugs were then plotted using GraphPad Prism (v7.0a) and pharmacokinetic parameters (C.sub.max, T.sub.max and AUC) were calculated using the PKsolver plugin. The data can be seen in Table 13.

(132) TABLE-US-00013 TABLE 13 Prodrug Prodrug C.sub.max Cmin AUC.sub.144 T.sub.max nanoparticle compound (ng/mL) (ng/mL) (ng/h/mL) (h) xiv 8 2.36 <2.00 170.50 72 xii 4 1.34 <2.00 89.04 24 xv 4 1.57 <2.00 86.00 24 xvi 6 19.83 <2.00 802.34 24 xiii 8 6.04 <2.00 305.90 48 xi 4 3.57 <2.00 236.05 24 xvii 6 12.11 <2.00 443.18 24 xviii 8 3.31 <2.00 169.09 72 xix 6 42.8 22.116 3449.71 24 xx 8 33.96 24.075 3731.00 24 xxi 6 88.57 2.452 3960.91 24 xxii 8 47.1 16.867 3695.12 24

(133) Table 14 below shows that solid prodrug nanoparticle compositions of the present invention demonstrated sustained release over 144 h. Solid prodrug nanoparticle compositions having polymer/surfactant combinations of HMPC/NDC and HMPC/AOT performed particularly well in the in vivo studies.

(134) TABLE-US-00014 TABLE 14 Emtricitabine concentration at 144 h Prodrug nanoparticle (ng/mL) xix 22.12 xx 24.10 xxii 16.87