Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses

Definitions

• Adenovirus (ADV) infection results in significant morbidity and mortality in both immunocompetent and immunosuppressed hosts.
• Adenoviruses have been recovered from human immunodeficiency virus (HJN) positive patients since the beginning of the AIDS epidemic (Khoo et al. J. Infect. Dis 1996, 172:629-637).
• HJN human immunodeficiency virus
• adenoviruses are responsible for a broad range of clinical diseases that may be associated with high mortality, including pneumonia, hepatitis, encephalitis, hemorrhagic cystitis, nephritis, and gastroenteritis in immunocompromised patients.
• Bosol J.C., J. Med.
• Adenovirus colitis is a common cause of diarrhea in HIN-infected patients and may facilitate enteric infection with cytomegalovirus (CMN) (Thomas et al, HIV Med., 1999, 1:19-24). Gastrointestinal adenovirus excretion occurs at an advanced stage of HIV disease (Sabin et al., J. Med. Virol, 1999, 58:280-285). The median survival of HIN-infected patients with adenovirus-positive diarrhea is 1 year compared with 2.4 years for those without adenoviruses (Sabin et al., J. Med. Virol, 1999, 58:280-285).
• nucleoside analogs including 2',3'- dideoxynucleoside 5'-triphosphates and 3'-fluoro-2'-deoxythymidine (FTdR), have been discovered as having antiviral activity against adenovirus. These nucleoside analogs inhibit the adenovirus DNA polymerase-mediated DNA replication in adenovirus-infected cells (Mentel et al., Med. Mircobiol.
• anti-HIN agents Of the currently available anti-HIN agents, none have been reported to have anti-ADV activity. Moreover, anti-ADN agents such as ribavarin cannot be administered long term in HIN-infected patients because of the associated side effects, such as anemia and severe hematologic toxicity (De Clercq, E., Perb. K. Acad. Geneeskd. Belg , 1996, 58:19-47; Reefschlager et al., Antiviral Res _ 1982,
• the present invention is directed to aryl phosphate derivatives of 2',3'- didehydro-2',3'-dideoxythymidine (hereinafter "d4T") that have anti-ADN activity in host cells or anti-ADN and anti-HIN activity in hosts co-infected with ADN and fflN.
• d4T aryl phosphate derivatives of 2',3'- didehydro-2',3'-dideoxythymidine
• One aspect of the invention provides methods for treating ADN infections or
• ADN/HIN co-infections by administering an aryl phosphate derivative of d4T having an electron withdrawing substituent on the aryl group and an amino acid substituent on the phosphate group as in Formula I:
• R ⁇ is an aryl group substituted with an electron withdrawing group and R 2 is an amino acid or an ester of an amino acid.
• Ri is a phenyl substituted with an electron withdrawing group and R 2 is an ester of an ⁇ - amino acid.
• Formula I is d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] where Ri is phenyl group substituted with bromo at the para position and R 2 is the methyl ester of alanine or Formula I is d4T-5'-[p-chlorophenyl methoxyalaninyl phosphate] where Ri is phenyl group substituted with chloro at the para position and R 2 is the methyl ester of alanine.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] or d4T-5'-[p-chlorophenyl methoxyalaninyl phosphate] results in the formation of two key metabolites: alaninyl-d4T-monophosphate (Ala- d4T-MP) and d4T.
• d4T-5'-[ ⁇ -bromophenyl methoxyalaninyl phosphate] or d4T-5'-[p-chlorophenyl methoxyalaninyl phosphate] results in more prolonged systemic exposure to Ala-d4T-MP as well as d4T than administration of an equimolar dose of either metabolite.
• Each metabolite has a significantly longer elimination half life when formed from the administration of d4T-5'-[p- bromophenyl methoxyalaninyl phosphate] or d4T-5'-[p-chlorophenyl methoxyalaninyl phosphate] than when administered directly.
• Another aspect of the invention provides a method for treating ADN infections or ADN/HIN co-infections comprising administering an effective amount of a compound of Formula IN:
• R 2 is an amino acid or amino acid ester residue.
• R is the methyl ester of alanine.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] and 12 structurally similar d4T derivatives were substantially more potent against ADV than d4T and inhibited ADN-induced plaque formation at nanomolar IC 50 values.
• Compounds with halo substitutions in the phenyl ring as well as the unsubstituted compound 607 were more potent against ADN than compounds with methoxy, methyl, or cyano substitutions.
• administering refers to providing to a mammal in any manner including: orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles, and other known modes of drug delivery.
• amino acid refers to any of the naturally occurring amino acids, as well as their opposite enantiomers or racemic mixture of both enantiomers, synthetic analogs, and derivatives thereof.
• the term includes, for example, ⁇ -, ⁇ -, ⁇ - , ⁇ -, and ⁇ -amino acids.
• Suitable naturally occurring amino acids include glycine, alanine, valine, leucine, isoleucine, proline, threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, phenylalanine, tryptophan, tyrosine, and histidine.
• amino acids such as, for example, trifluoroleucine, p-fluorophenylalanine, and 3-triethylalanine can be used.
• the term amino acid includes esters of the amino acids. Esters include lower alkyl esters in which the alkyl group has one to seven carbon atoms, preferably one to four carbon atom such as, for example, methyl, ethyl, propyl, and butyl. The amino group of the amino acid or ester thereof is attached to the phosphate group in Formula I.
• animal includes, but is not limited to mammals, such as humans.
• aryl includes aromatic groups such as, for example, phenyl, naphthyl, and anthryl.
• electro-withdrawing groups includes groups such as halo (- ⁇ O 2 , -CN, -SO 3 H, -COOH, -CHO, -COR (where R is a (Ci to C 4 ) alkyl), and the like.
• halo or “halogen” is used to describe an atom selected from the group of Bromine (Br), Chlorine (CI), Fluorine (F) and Iodine (I).
• protecting or “preventing” refers to taking advance measures against a possible or probable infection to prevent the morbidity and mortality normally associated with a disease causing agent.
• host in the context of this invention means a mammal, i.e., any class of higher vertebrates that nourish their young with milk secreted by mammary glands, or a cell or cells from a mammal.
• cell in the context of this invention means the smallest structural unit of an organism that is capable of independent functioning, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable membrane.
• coinfected in the context of this invention means a host simultaneously infected with both ADN and HIV.
• Inhibiting ADN activity in the context of this invention means inhibiting ADN replication, inhibiting ADN induced plaque formation, killing ADN, or inhibiting ADN D ⁇ A polymerase.
• Inhibiting HIV activity in the context of this invention means inhibiting HIN replication, killing HIN, or inhibiting HIV reverse transcriptase.
• the invention is directed to methods of using aryl phosphate derivatives of
• 2 l ,3'-didehydro-2',3'-dideoxythymidine (derivatives of d4T) to inhibit the effects of infection by adenovirus in a cell, in vitro or in vivo. More particularly, the present invention provides methods to inhibit the effects of infection by adenovirus in a cell, in a mammal by administering an aryl phosphate derivative of d4T having an electron withdrawing substituent on the aryl group and an amino acid substituent on the phosphate group as in Formula I:
• Ri is an aryl group substituted with an electron withdrawing group and R 2 is an amino acid or an ester of an amino acid.
• the compounds of Formula I can also be in the form of pharmaceutically acceptable salts.
• Pharmaceutically acceptable salts are formed with organic and inorganic acids.
• suitable acids for salt formation with the amino group of the amino acid or amino acid ester residue of a compound of Formula I include, but are not limited to hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, gluconic, fumaric, succinic, asorbic, maleic, methanesulfonic, and the like.
• the salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce either a mono or di, etc. salt in the conventional manner.
• Suitable bases for the formation of a salt with the carboxylate group of the amino acid residue of a compound of Formula I include, for example, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, and potassium bicarbonate.
• R ⁇ is a phenyl group substituted with an electron withdrawing group and R 2 is an ⁇ -amino acid or ester thereof as shown in Formula II:
• X is an electron-withdrawing group such as halo -NO , -CN, -SO 3 H, - COOH, -CHO, -COR (where R is a (d to C 4 ) alkyl), and the like.
• R 3 is hydrogen or an alkyl of one to seven carbon atoms, preferably an alkyl of one to four carbon atoms, such as, for example, methyl, ethyl, propyl, and butyl.
• Rj is hydrogen (e.g., as in glycine), an alkyl (e.g.
• a substituted alkyl e.g., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginie, and lysine
• an arylalkyl e.g., as in phenylalanine and tryptophan
• a substituted arylalkyl e.g., as in tyrosine
• a heteroalkyl e.g., as in histidine.
• the compound of Formula II is d4T-5'-[p-bromophenyl methoxyalaninyl phosphate], (d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]) where X is bromo attached to the phenyl group in the para position, Ri is methyl, and R 3 is methyl.
• X is bromo attached to the phenyl group in the para position
• Ri is methyl
• R 3 is methyl.
• the structure of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] is shown in Formula III:
• the anti- viral agent d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] (referred to in Scheme 1 below as HI-113) is quickly metabolized in vivo to form two metabolites: 2',3'-didehydro-3'-deoxythymidine (d4T) and alaninyl-d4T- monophosphate (Ala-d4T-MP) as shown in Scheme 1. Ala-d4T-MP can also be metabolized further to yield d4T. The metabolite d4T had not been found in earlier in vitro studies with cells.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] readily metabolizes in either plasma or whole blood to form Ala-d4T-MP and a small amount of d4T (see Figure 2).
• Ala-d4T-MP is stable both in plasma and in whole blood.
• other enzymes e.g., liver enzymes
• This hypothesis is consistent with the formation of a significant amount of d4T after incubation of d4T-5'- p-bromophenyl methoxyalaninyl phosphate] with a liver homogenate (see Figure 4).
• EDTA an inhibitor of arylesterase, did not affect the hydrolysis of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate], indicating that arylesterase is probably not involved in the hydrolysis of d4T-5'-[ ⁇ -bromophenyl methoxyalaninyl phosphate] (see Figure 3C).
• the elimination half-life of intravenously administered d4T is fairly similar to the elimination half-life of d4T formed after intravenous administration of Ala- d4T-MP (ti 2 of 30.3 minutes vs. 34.0 minutes) as shown in the Examples below.
• the elimination half-life for d4T formed after intravenous administration of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] was significantly prolonged (t ⁇ /2 of 114.8 minutes).
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] results in prolonged systemic exposure to both Ala- d4T-MP and d4T compared to administration of equimolar dose of Ala-d4T-MP or d4T due to apparently longer elimination half-lives of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] -derived metabolites.
• d4T- 5'-[p-bromophenyl methoxyalaninyl phosphate] was converted to the active metabolites Ala-d4T-MP (23%) and d4T (24%).
• the t max values for Ala-d4T-MP and d4T formed from intravenously administered d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] were 5.9 minutes and 18.7 minutes, respectively.
• Ala-d4T-MP can also be used as a d4T prodrug.
• the invention provides a method for inhibiting the effects of infection by adenovirus in a cell, in vitro or in vivo by administering an effective amount of a compound of Formula IV:
• R 2 is an amino acid or esterified thereof.
• Salts The compounds of Formula I to TV can also be in the form of pharmaceutically acceptable salts.
• Pharmaceutically acceptable salts can be formed with organic and inorganic acids.
• suitable acids for salt formation with the amino group of the amino acid or amino acid ester residue of Formula IN include, but are not limited to, hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, gluconic, fumaric, succinic, asorbic, maleic, methanesulfonic, and the like.
• the salts can be prepared by contacting the free base form with a sufficient amount of the desired acid to produce either a mono or di, etc. salt in the conventional manner.
• Suitable bases for the formation of a salt with the carboxylate group of the amino acid residue of Formula IV include, for example, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, and potassium bicarbonate.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] also yielded Ala-d4T-MP and d4T as the major metabolites.
• No parent d4T-5'-[p- bromophenyl methoxyalaninyl phosphate] was detectable in the blood after oral administration.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] is stable in gastric fluid and can be absorbed in the stomach, it can quickly hydrolyze in blood.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] decomposes readily in intestinal fluid to form Ala-d4T-MP.
• This metabolite can be absorbed in the intestine and then further metabolized to yield d4T in the blood.
• the t max and t ⁇ /2 values for d4T in mice were longer when derived from orally administered d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] (42.4 minutes and 99.0 minutes, respectively) than from orally administered d4T (5 minutes and 18 minutes, respectively).
• the t max value is higher but the t ⁇ /2 value is lower for orally administered d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] compared to intravenously administered d4T-5 ' -[p-bromophenyl methoxyalaninyl phosphate] .
• the estimated bioavailabilities of Ala-d4T-MP and d4T were approximately 12% and 48%, respectively, after oral administration of d4T-5'-[j->-bromophenyl methoxyalaninyl phosphate].
• the bioavailability of d4T metabolized from d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] (48%) was lower than that of orally administered D4T (98%).
• Compounds of Formulas I to IV can be formulated as pharmaceutical compositions and administered to a mammalian host, including a human patient in a variety of forms adapted to the chosen route of administration.
• the compounds are typically administered in combination with a pharmaceutically acceptable carrier, and can be combined with specific delivery agents, including targeting antibodies or cytokines.
• the administered dose is that effective to have the desired effect, such as sufficient to reduce or eliminate one or more symptoms of adenovirus.
• Appropriate amounts can be determined by those skilled in the art, extrapolating using known methods and relationships, from the in vivo animal model data provided in the Specification and Examples.
• the dose of the aryl phosphate derivatives of d4T effective to achieve therapeutic treatment of adenovirus infection, including reduction of symptoms or effects of adenovirus infection such as increased survival time is in the approximate range of about 1-500 mg/kg body weight/dose, preferably about 10-100 mg/kg body weight/dose, and approximately 800-1000 mg/kg body weight per week of a cumulative dose.
• the effective dose to be administered will vary with conditions specific to each patient. In general, factors such as the viral burden, host age, metabolism, sickness, prior exposure to drugs, and the like, contribute to the expected effectiveness of a drug. One skilled in the art will use standard procedures and patient analysis to calculate the appropriate dose, extrapolating from the data provided in the Examples. In general, a dose which delivers about 1-100 mg/kg body weight is expected to be effective, although more or less may be useful.
• the compositions of the invention may be administered in combination with other therapies. In such combination therapy, the administered dose of the compounds may be less than for single drug therapy.
• the compounds can be administered orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non- toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
• Pharmaceutical compositions of the invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.
• compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
• the compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents.
• the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
• compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
• the compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents generally known in the art.
• suitable dispersing or wetting and suspending agents such as sterile oils, including but not limited to, synthetic mono- or diglycerides, and fatty acids, including oleic acid.
• the compositions can be prepared by known methods, for example, by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, that are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
• a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, that are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
• Solutions or suspensions of the compounds can be prepared in water, isotonic saline (PBS), and the like, and optionally can be mixed with a nontoxic surfactant.
• Dispersions may also be prepared by known methods, for example in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin, and mixtures thereof. Under ordinary conditions of storage and use, these preparations may contain a preservative, for example, to prevent the growth of microorganisms
• the pharmaceutical dosage form suitable for injection or infusion use can include sterile, aqueous solutions or dispersions, sterile powders comprising an active ingredient, and the like, that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions.
• the ultimate dosage form is preferable be sterile, fluid, and stable under the conditions of manufacture and storage.
• the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid polyethylene glycols and the like, vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size, in the case of dispersion, or by the use of nontoxic surfactants.
• the prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, buffers, or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption—for example, aluminum monosterate hydrogels and gelatin.
• Sterile injectable solutions are prepared by incorporating the conjugates in the required amount in the appropriate solvent with various other ingredients as enumerated above and, as required, followed by filter sterilization.
• the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
• the HPLC system used for these studies was a Hewlett Packard (Palo Alto, CA) series 1100 instrument equipped with a quaternary pump, an autosampler, an automatic electronic degasser, an automatic thermostatic column compartment, a diode array detector and a computer with Chemstation software for data analysis (Chen et al., J. Chromatogr. B., 724, 157 (1999); Chen et al., J. Chromatogr. B., 727, 205 (1999); and Chen et al., J. Liq. Chromatogr., 22, 1771 (1999)).
• the analytical column used was a Zobax SB-Phenyl (5 ⁇ m, Hewlett Packard, Inc.) column attached to a guard column (Hewlett Packard, Inc.). The column was equilibrated prior to data collection.
• the detection wavelength was 268 nm, the peakwidth was less than 0.03 minutes, the response time was 0.5 seconds, and the slit was 4 nm.
• HPLC-grade reagents and deionized, distilled water were used in this study.
• Acetonitrile was purchased from Burdick & Jackson (Allied Signal Inc., Muskegon, MI).
• Acetic acid was purchased from Fisher Chemicals (Fair Lawn, NJ).
• Ammomum phosphate and phosphoric acid were purchased from Sigma- Aldrich (St. Louis, MO).
• Plasma samples 200 ⁇ L were mixed 1 :4 with acetone (800 ⁇ L ) and vortexed for at least 30 seconds. Following centrifugation, the supernatant was transferred into a clean tube and was dried under nitrogen. A 50 ⁇ L solution of 50% methanol in 200 mM HCl was used to reconstitute the extraction residue, and 40 ⁇ L was injected into the HPLC.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]A and d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]B are diastereomers of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]. At these retention times, no significant interference peaks from the blank plasma were observed ( Figures 1 A and IB).
• Plasma samples were pre-incubated with the esterase inhibitors paraoxon (final concentration of 0.1 mM), physostigmine (final concentration of 0.1 mM), and EDTA (final concentration of 1M) at 37°C for 30 minutes. Then d4T-5'-[p- bromophenyl methoxyalaninyl phosphate] was added to yield final concentrations of 250 ⁇ M. At a predetermined time, an aliquot (100 ⁇ l) of spiked plasma was extracted by adding 400 ⁇ l of acetone to induce precipitation of proteins, as described above.
• esterase inhibitors paraoxon final concentration of 0.1 mM
• physostigmine final concentration of 0.1 mM
• EDTA final concentration of 1M
• Fresh mouse liver was obtained from Balb/c mice and homogenated in lx PBS (1:1, W/V) using a Polytron (PT-MR2000) homogenizer (Kinematical AG, Littau, Switzerland). d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] was added to the liver homogenate to yield a final concentration of 100 ⁇ M. At a predetermined time, an aliquot (100 ⁇ l) of spiked liver homogenate was extracted by adding 400 ⁇ l of acetone to induce precipitation of proteins, as described above.
• PT-MR2000 Polytron homogenizer
• Simulated gastric and intestinal fluids were prepared following United States Pharmacopia methods and were spiked with d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] and Ala-d4T-MP to yield a solution with a final concentration of 100 ⁇ M of each compound.
• the spiked fluids were then placed in a 37°C water bath.
• 100 ⁇ l aliquots of the spiked gastric or intestinal fluid were extracted by adding 400 ⁇ l of acetone as discussed above.
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] is relatively stable in gastric fluid for 8 hours, but it is not stable in intestinal fluid ( Figures 5A and 5B).
• mice Female Balb/c mice (6-8 weeks old) from Taconic (Germantown, NY) were housed in a controlled environment (12-hours of light/ 12-hours of dark, 22 ⁇ 1°C, 60 + 10% relative humidity), which is fully accredited by the USD A. All rodents were housed in microisolator cages (Lab Products, Inc., NJ) containing autoclaved bedding. The mice were allowed free access to autoclaved pellet food and tap water throughout the study. All animal care procedures conformed to the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington DC 1996).
• a solution (50 ⁇ l) of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] (100 mg/kg) dissolved in DMSO was administered intravenously via the tail vein.
• This volume of DMSO is well-tolerated by mice when administrated by rapid intravenous or extravascular injection (Rosenkrantz et al., Cancer Chemother. Rep., 31, 7 (1963); Wilson et al., Toxicol Appl Pharmacol, 7, 104 (1965)).
• Blood samples ( ⁇ 500 ⁇ L) were obtained from the ocular venous plexus by retro-orbital venipuncture at 0, 2, 5, 10, 15, 30, 45, 60, 120, 240 and 360 minutes after intravenous injection.
• mice were injected with 75 mg/kg Ala- d4T-MP and 40 mg/kg d4T, respectively (these doses are equimolar to the 100 mg/kg d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]).
• mice In order to determine the pharmacokinetics of d4T-5'-
• Sampling time points were 0, 2, 5, 10, 15, 30, 45, 60, 120, 240 and 360 minutes after the gavage.
• the metabolic clearance of the parent drug, the formation clearance of the metabolite, the clearance elimination of the metabolite, and the distribution clearance of the metabolite were estimated by simultaneous fitting of the concentration of parent drug and metabolites as a function of time curve to pharmacokinetic models (see Figures 7 A & 8 A) specified as a system of differential equations (Gabrielsson & Weiner, Phamacokinetic / Phamacodynamic Data Analysis: Concepts and Applications, Swedish Pharmaceutical Press (1997)).
• Example 7 Metabolism and Pharmacokinetic Profile of d4T-5'-
• d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] had a moderate size volume of distribution with a V ss of 819.9 ml/kg, which is roughly equal to the total volume of water in the body.
• the diastereomers of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] were separated using the HPLC conditions described above (the retention times were 28.7 and 28.9 minutes).
• d4T-5'-[j-.-bromophenyl methoxyalaninyl phosphate]-B also had a slightly longer elimination half-life than the d4T-5'-[p- bromophenyl methoxyalaninyl phosphate]-A diastereomer (4.1 minutes vs. 2.8 minutes), which may be due to faster clearance of d4T-5'- . -bromophenyl methoxyalamnyl phosphate]-A relative to that of d4T-5'- .
• FIG. 7A and 7B The model depicted in Figures 7A and 7B describes the metabolite pharmacokinetics of Ala-d4T-MP and d4T after intravenous injection of d4T-5'-[p- 5 bromophenyl methoxyalaninyl phosphate] .
• d4T-5 ' -p- bromophenyl methoxyalaninyl phosphate] is biotransformed to produce Ala-d4T- MP (CLm and d4T (CL ⁇ ⁇ ), respectively.
• Ala-d4T-MP derived from d4T-5'-[p- bromophenyl methoxyalaninyl phosphate] can be further metabolized to form D4T (CLm 3 ) or distributed to the extravascular compartment (Cl ml d).
• D4T produced from either d4T-5'-[p-bromophenyl methoxyalamnyl phosphate] or Ala-d4T-MP is finally eliminated from the body (CL me2 ).
• the pharmacokinetic parameters estimated for these two metabolites are presented in Table 3.
• the values in parenthesis are the C.N. of modeling.
• the metabolic clearance of d4T-5'-[p-bromophenyl methoxyalaninyl phosphate] and the formation clearance of the metabolites were 83.9 ml/minute/kg for Ala-d4T-MP and 87.4 ml/minute/kg for d4T, respectively.
• the metabolic clearance of Ala-d4T-MP and the formation clearance of its metabolite, d4T were 36.1 ml/minute/kg, and a small portion of Ala- d4T-MP was distributed to the extravascular compartment with a CL ⁇ ud of 47.1 ml minute/kg.
• d4T was eliminated with a CL rne2 of 62.0 ml/minute/kg.
• concentration of Ala-d4T-MP as a function of time can be described using a two-compartment model, while a one-compartment model best fits the concentration of its metabolite, d4T, as a function of time ( Figure 8B).
• the C max values for Ala-d4T-MP and d4T were
• Ala-d4T-MP 1206.6 ⁇ M and 35.2 ⁇ M, respectively.
• the AUC was 11648.7 ⁇ M»minute for Ala- d4T-MP and 1888.0 ⁇ M»minute for d4T.
• the systemic clearance of Ala-d4T-MP was only 15.8 mL/minute/kg (Table 4), which is much less than the blood flow to either the kidney or the liver (Davies et al., Pharm. Res., 10, 1093 (1993)).
• Example 10 Pharmacokinetic Profile of d4T-5'-
• a one-compartment pharmacokinetic model was used to describe both the Ala-d4T-MP and the d4T concentration changes as a function of time ( Figures 10A and 10B).
• the estimated values for the pharmacokinetic parameters are presented in Table 7.
• the maximum concentrations (C max ) for Ala-d4T-MP and D4T are 12.7 ⁇ M and 30.7 ⁇ M, respectively.
• the elimination half-lives were 66.4 minutes and 99.0 minutes for Ala-d4T-MP and d4T, respectively.
• HPLC purification was achieved using a reverse-phase Lichrospher column (250 x 4 mm, Hewlett-Packard, RP-18, Cat. # 79925) and an isocratic flow (1 ml/minute) consisting of water (70%) and acetonitrile (30%). Melting points were determined using a Melt John's apparatus and are uncorrected. Column chromatography was performed using silica gel obtained from Baker Company. The octanol/water partition coefficient was determined by the shake flask method. The phosphoramidate analogs were added to 2 ml of water and 2 ml of octanol in a glass vial. The mixture was shaken for 4 hours at room temperature. The two phases were carefully separated and filtered through a Millipore filter and analyzed by HPLC. The partition coefficient was calculated using the ratio of the area under the curve for octanol and water, respectively.
• d4T-5' [para- bromophenyl methoxyalanininyl phosphate], have been previously described in detail by Vig et al., 1992 Antiviral Chem. and Chemother., 9:445-447. Zidovudine was obtained from Toronto Research Chemicals Inc, Missassagua, Ontario, Canada. d4T was prepared from thymidine following the procedure of Mansuri et al., 1989
• HIV-l strain used in this study was HTLV-IIIB (Uckun et al., 1998 Antimicro. Agents and Chemother., 42:383-388).
• the non-HIV viruses included in the present study were the gancyclovir-sensitive cytomegalo virus
• CMV strain AD169 ATCC VR-538
• HSV herpes simplex virus
• HSV Type I strain HF ATCC VR-260
• acyclovir-sensitive HSV Type II strain G ATCC VR-734
• adenovirus strain Type 5, strain adenoid 75 ATCC VR-5
• respiratory syncytial virus RSV
• EIA enzyme immunoassay
• Plaque formation assays Plaque assays were used to examine the activity of the compounds against non-HIN viruses.
• the skin fibroblast cell line SF (ATCC CRL-2097) was used as a target for AD 169, Adenovirus Type 5.
• the ECHO 30, VERO cell line (ATCC CCL-81) was used as a target for HSV HF and G.
• the HEP-2 cell line (ATCC CCL-23) was used as a target for the RSV strain Long. These cell lines were cultured at 1 x 10 5 cells/well in 24-well (all but ECHO 30- infected SF) or 6-well (ECHO 30-infected SF) tissue culture plates with 0.9% methylcellulose or 0.4% SeaPlaque agarose semisolid support.
• MEM Minimum Essential Medium
• Gibco Earle's salts
• L-glutamine L-glutamine
• non-essential amino acids 2% heat-inactivated fetal bovine serum
• 1% penicillin/streptomycin 0.05% gentamicin served as the culture medium.
• the incubation times were 3 days for HF, G, ECHO 30, and Long; and 7 days for AD169 and Adenovirus Type 5.
• Plaque counting was performed with a 20X dissecting microscope for AD 169, Type 5, ECHO 30, and RSV Long, and with a light box for HF and G.
• the fixative agent was crystal violet for all viruses, except for the CMV strain AD 169 for which methylene blue was used.
• Percent inhibition of plaque formation was calculated by comparing the plaque numbers from the test substance-treated infected cells with the plaque numbers from untreated infected cells (i.e., virus controls).
• the IC 50 - values were determined using the Statview statistics program (SAS Institute, Inc., Cary, NC). Statistical analysis. Each drug was tested at 6-7 different concentrations ranging from 0.0001 ⁇ M to 100 ⁇ M. Each assay was set up in triplicate wells and repeated 1-3 times. An IC 50 value was calculated from each set of triplicate wells using nonlinear regression modeling of the exponential form of the linearized equation (Statview, SAS Institute, Inc., Cary, NC).
• Hydrolysis rates were determined by fitting single exponential decay equations to the disappearance of the compound in alkali conditions (all R values greater than 0.85). The IC 50 values obtained were correlated to the log transformed hydrolysis rate constants by fitting a linear model (JMP Software, SAS Institute Inc., Cary, NC). P-values less than 0.05 were deemed significant. Identification of compound 113 and compound 609 as dual function anti-HIV agents with potent and selective antiviral activity against ADV. The antiviral activity of stavudine and 13 aryl phosphoramidate derivatives of stavudine against human adenovirus (ADV strain Type 5) were first examined.
• Stavudine inhibited the cytopathic effects of ADV with an IC 50 value of 12.3+0.3 ⁇ M. All 13 derivatives of stavudine were substantially more potent than stavudine and inhibited ADV-induced plaque formation with nanomolar IC 50 values (Table 8).
• Compounds with halo substitutions in the phenyl ring as well as the unsubstituted compound 607 were more potent than compounds with methoxy, methyl, or cyano substitutions.
• Compound 113 (d4T-5'-[p-bromophenyl methoxyalaninyl phosphate]) with a 4-Br substitution and compound 609 with a 4- Cl substitution were identified as the most potent lead anti-ADV agents.
• Compound 113 inhibited ADV-induced plaque formation in skin fibroblasts in a concentration- dependent fashion, with a mean ( ⁇ SEM) IC 50 value of 0.022 ⁇ 0.009 ⁇ M without any evidence of cytotoxicity even at 100 ⁇ M (Table 8).
• compound 609 inhibited ADV-induced plaque formation with an IC 50 value of 0.0027+0.003 ⁇ M
• HPMC HPMC
• compound 113 and compound 609 were selectively active against adenovirus only, which suggests a higher susceptibility of adenovirus D ⁇ A polymerase to these nucleoside analogs.
• a selective antiviral activity is not unprecedented for nucleoside antiviral drugs.
• a number of novel 5- substituted 2'deoxypyrimidine nucleosides exhibited antiviral activity against he ⁇ es simplex virus type 1 and type 2 strains V3, but not against adenovirus (Reefschlager et al., 1982 Antiviral Res., 2:41-52).
• compound 113 and compound 609 are the first nucleoside analogs to be identified as dual-function anti-HIV agents with selective anti-ADV activity.
• HIV-1 HIV-1, HTLV-IIIB O.OOl ⁇ O.OOO O.OOl ⁇ O.OOO 0.023+0.008
• Adenovirus Type 5 0.022+0.009 0.027+0.003 12.3+0.3
• Plaque assays were used to examine the activity of compound 113 and compound 609 against non-HIN viruses.
• the skin fibroblast cell line SF (ATCC CRL-2097) was used as a target for AD 169, Adenovirus Type 5, and ECHO 30; the VERO cell line (ATCC CCL-81) was used as a target for HF and G; and the HEP-2 cell line (ATCC CCL-23) was used as target for the RSV strain Long. Results are expressed as the average IC 50 values in ⁇ M. Stavudine (STV) was included as a control.

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