In vitro and in vivo activity of 1-O-hexadecylpropanediol-3-phospho-ganciclovir and 1-O-hexadecylpropanediol-3-phospho-penciclovir in cytomegalovirus and herpes simplex virus infections.

Human cytomegalovirus (HCMV) and herpes simplex virus (HSV) can cause a wide variety of clinical manifestations in man. Ganciclovir (GCV) is effective against HCMV infection when administered by the intravenous route and may be used orally in large doses for prophylaxis of HCMV infections in organ transplantation patients and in AIDS patients. In previous studies with acyclovir (ACV), we found that covalent attachment of an alkyl glycerol phosphate moiety greatly increased oral bioavailability and increased antiviral activity against hepatitis B virus. Adducts of ACV with alkyl propanediol phosphate were more active than the alkyl glycerol phosphate analogue in vitro in 2.2.15 cells, which constitutively produce hepatitis B virus. To see if this strategy would work for two other poorly absorbed nucleoside analogues, we synthesized 1-O-hexadecylpropanediol-3-phospho-GCV (HDP-P-GCV) and 1-O-hexadecyl-propanediol-3-phospho-penciclovir (HDP-P-PCV), and evaluated the in vitro antiviral activity, selectivity and oral antiviral activity of both compounds versus GCV or PCV in mice infected with HSV-1 or HDP-P-GCV versus murine cytomegalovirus (MCMV). HDP-P-GCV is orally active in both MCMV and HSV-1 infection in mice with antiviral activity equivalent to (HSV-1) or greater than oral GCV (MCMV). Oral HDP-P-PCV was more active than PCV orally versus intranasal HSV-1 infection in mice.

Alkylglycerol prodrugs of phosphonoformate are potent in vitro inhibitors of nucleoside-resistant human immunodeficiency virus type 1 and select for resistance mutations that suppress zidovudine resistance.

Phosphonoformate (foscarnet; PFA) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), but its use for the treatment of HIV-1 infection is limited by toxicity and the lack of an orally bioavailable formulation. Alkylglycerol-conjugated prodrugs of PFA (1-O-octadecyl-sn-glycero-3-PFA [B-PFA]) having sn-2 substituents of hydrogen (deoxybatyl-PFA [DB-PFA]), methyl (MB-PFA), or ethyl (EB-PFA) are more-potent inhibitors of wild-type HIV-1 in vitro than unmodified PFA and are orally bioavailable in mice. We have evaluated the activities of these compounds against a panel of nucleoside-resistant HIV-1 variants and have characterized the resistant variants that emerge following in vitro selection with the prodrugs. Except for an HIV-1 variant encoding the K65R mutation in RT that exhibited 3.3- to 8.2-fold resistance, the nucleoside-resistant viruses included in the panel were sensitive to the PFA prodrugs (<3-fold increase in 50% inhibitory concentration), including multinucleoside-resistant variants encoding the Q151M complex of mutations or the T69S[SA] insert. Viruses resistant to the PFA prodrugs (>10-fold) were selected in vitro after 15 or more serial passages of HIV-1 in MT-2 cells in escalating prodrug concentrations. Mutations detected in the resistant viruses were S117T, F160Y, and L214F (DB-PFA); M164I and L214F (MB-PFA); and W88G and L214F (EB-PFA). The S117T, F160Y, and M164I mutations have not been previously identified. Generation of recombinant viruses encoding the single and double mutations confirmed their roles in prodrug resistance, including 214F, which generally increased the level of resistance. When introduced into a zidovudine (AZT)-resistant background (67N 70R 215F 219Q), the W88G, S117T, F160Y, and M164I mutations reversed AZT resistance. This suppression of AZT resistance is consistent with the effects of other foscarnet resistance mutations that reduce ATP-dependent removal of AZT monophosphate from terminated template primers. The favorable activity and resistance profiles of these PFA prodrugs warrant their further evaluation as clinical candidates.

In vitro anti-HIV-1 activity of sn-2-substituted 1-O-octadecyl-sn-glycero-3-phosphonoformate analogues and synergy with zidovudine.

Monoalkyl ether lipid analogues of foscarnet (phosphonoformate, PFA) exhibit substantially greater in vitro antiviral activity than unmodified PFA against human immunodeficiency virus type 1 (HIV-1). Our previous studies indicate that the length of the alkyl chain must be 14-22 carbons for optimal antiviral activity. To further evaluate the structure-activity relationship, we prepared 1-O-octadecyl-sn-glycerol analogues of PFA with various substitutions at the sn-2 position of glycerol and determined the effect of structure on in vitro antiviral activity and selectivity against HIV-1 in MT-2 and CD4-expressing HeLa cells (HT4-6C). We also studied combinations of zidovudine with PFA, 1-O-octadecyl-2-O-methyl-sn-glycero-3-PFA, or 1-O-octadecyl-sn-glycero-3-PFA and calculated their combination index values against HIV-1 in HT4-6C cells. Alkyl substitutions of one to four carbons at the sn-2 position of glycerol showed optimal antiviral activity. Both alkyl ether lipid analogues were strongly synergistic with zidovudine over a wide range of drug ratios and concentrations. 1-O-octadecyl-sn-glycerol analogues of PFA have selective antiviral properties and warrant further evaluation as potential antiretroviral drugs.

Antiviral activities of oral 1-O-hexadecylpropanediol-3-phosphoacyclovir and acyclovir in woodchucks with chronic woodchuck hepatitis virus infection.

Acyclovir triphosphate is a potent inhibitor of hepatitis B virus DNA polymerase, but acyclovir treatment provides no benefit in patients with hepatitis B virus infection. This is due in part to the fact that hepatitis B virus, unlike herpes simplex virus, does not code for a viral thymidine kinase which catalyzes the initial phosphorylation of acyclovir. We synthesized 1-O-octadecyl-sn-glycero-3-phospho (3-P)-acyclovir and found that it was highly active in reducing hepatitis B virus replication in 2.2. 15 cells, while acyclovir was inactive. The greater antiviral activity of 1-O-octadecyl-sn-glycero-3-P-acyclovir appeared to be due to liver cell metabolism of the compound to acyclovir monophosphate (K. Y. Hostetler et al., Biochem. Pharmacol. 53:1815-1822, 1997). However, a closely related compound without a hydroxyl group at the sn-2 position of glycerol, 1-O-hexadecylpropanediol-3-P-acyclovir, was more active and selective in 2.2.15 cells in vitro. In this study, we treated woodchucks chronically infected with woodchuck hepatitis virus with increasing oral doses of 1-O-hexadecylpropanediol-3-P-acyclovir and assessed the response to therapy versus acyclovir or a placebo. At a dosage of 10 mg/kg of body weight twice a day, the test compound significantly inhibited viral replication in vivo, as indicated by a 95% reduction in serum woodchuck hepatitis virus DNA levels and by a 54% reduction in levels of woodchuck hepatitis virus replicative intermediates in the liver. Higher doses were somewhat less effective. In contrast, 20 mg of acyclovir/kg twice daily, a 5. 3-fold-higher molar dosage, had no demonstrable activity against woodchuck hepatitis virus. Oral 1-O-hexadecylpropanediol-3-P-acyclovir appeared to be safe and effective in chronic woodchuck hepatitis virus infection.

Intravitreal toxicology and duration of efficacy of a novel antiviral lipid prodrug of ganciclovir in liposome formulation.

PURPOSE: To evaluate the intraocular safety and antiviral treatment efficacy of the sustained lipid prodrug of ganciclovir, 1-O-hexadecylpropanediol-3-phospho-ganciclovir (HDP-P-GCV), as an intravitreal injectable drug system for viral retinitis. METHODS: HDP-P-GCV was synthesized by coupling 1-O-hexadecyl-propanediol-3-phosphate to either free hydroxyl of ganciclovir in pyridine with dicyclohexylcarbodiimide as catalyst. The compound was formulated into liposomes. The antiviral activity was assessed by DNA reduction in vitro, and intraocular safety was assessed by ophthalmoscopy, electrophysiology, and histology after intravitreal injections, with resultant intravitreal concentrations of 0.2, 0.632, 1.12, and 2 mM. The treatment efficacy was evaluated by simultaneous intravitreal injection of HDP-P-GCV and herpes simplex virus type 1 (HSV-1) or by intravitreal injection of HDP-P-GCV at various times before HSV-1 intravitreal inoculation. Retinitis was scored with ophthalmoscopy and compared with controls. RESULTS: In vitro, the IC50 of HDP-P-GCV against HSV-1 and human cytomegalovirus (HCMV) infected cells was 0.02 and 0.6 microM, respectively. In rabbits in vivo, HDP-P-GCV dispersed evenly and maintained a good vitreous clarity at all doses except 2 mM final intravitreal concentration. Although cataracts were observed in some eyes at the higher doses, they were not observed in eyes with 0.2 mM final intravitreal concentration. No other indications of ocular toxicity were observed. Intravitreal injection of HDP-P-GCV with resultant 0.2 mM intravitreal concentration in the HSV-1 retinitis rabbit model demonstrated a complete protection of the retina with the simultaneous treatment strategy and a 4 (P = 0.03) to 6-(P = 0.058) week significant protection of retina with the pretreatment strategies when compared with ganciclovir or blank liposome controls. CONCLUSIONS: In the rabbit model of HSV-1 retinitis HDP-P-GCV acts as a long-lasting intravitreal injectable anti-CMV or anti-HSV compound. This self-assembling liposome system could be applicable for many compounds available for intraocular diseases.

Synthesis and antiviral evaluation of 1-O-hexadecylpropanediol-3-P-acyclovir: efficacy against HSV-1 infection in mice.

We synthesized, 1-O-hexadecylpropanediol-3-P-acyclovir, an orally bioavailable lipid prodrug of acyclovir and evaluated it for in vitro and in vivo activity against herpes simplex virus infections. Although 1-O-hexadecylpropanediol-3-P- acyclovir was less active in vitro than acyclovir, on a molar basis it was 2.4 times more active orally in preventing mortality from acute HSV-1 infection in mice. In vitro, 1-O-hexadecylpropanediol-3-P-acyclovir was also more active than acyclovir in a thymidine kinase negative mutant strain of HSV-1 (DM21) and had somewhat higher activity in cytomegalovirus infection in vitro due to it's ability to bypass thymidine kinase.

Efficacy of topical acyclovir monophosphate, acyclovir, or penciclovir in orofacial HSV-1 infections of mice and genital HSV-2 infections of guinea pigs.

The purpose of these studies was to compare the efficacy of acyclovir monophosphate (ACVMP), acyclovir (ACV), or penciclovir (PCV) against HSV-1 in an orofacial infection of mice and against ACV sensitive and resistant genital HSV-2 infections of guinea pigs. Treatment was initiated 24, 48, or 72 hours post inoculation with 5% ACVMP, 5% ACV (Zovirax) or 1% PCV (Denavir). In all experiments, similar efficacy was obtained for ACVMP and ACV, whereas PCV was considerably less effective.

Intravitreal toxicology and therapeutic efficacy of the carboxymethyl ester of the 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA-O-Me), a novel lipid antiviral prodrug for intraocular drug delivery.

This study was conducted to evaluate the vitreous clarity and intraocular therapeutic index of three preparations ofthe carboxymethyl ester of 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA-O-Me), a long acting lipid derivative of foscarnet with potent anti-CMV activity. Twenty-six New Zealand white rabbits were intravitreally injected with one of three preparations of ODG-PFA-O-Me or control diluent. The vitreous clarity was graded after injection using indirect ophthalmoscopy and fundus photography. Drug intraocular toxicity was evaluated by electroretinography and by post-sacrifice tissue pathology using light and electron microscopy. Intravitreal injection of micellar ODG-PFA-O-Me showed variable local retinal toxicity and vitreal compound aggregates in eyes with the middle and high doses. The intraocular therapeutic index was lower than 465:1. Intravitreal injection of liposomal ODG-PFA-O-Me, either free acid or sodium salt, revealed clear vitreous for the 0.632 and 0.84 mM final intravitreal concentrations. No retinal toxicity was confirmed for the 1.12 mM final intravitreal concentration at the eight week observation following injection. The intraocular therapeutic index was between 585-1037:1. ODG-PFA-O-Me possesses better vitreous compatibility than ODG-PFA. Liposomal ODG-PFA-O-Me can be intravitreally injected with a resulting clear vitreous and high intraocular therapeutic index. Liposomal ODG-PFA-O-Me could be a long acting nontoxic intravitreous injectable drug for CMV retinitis.

Treatment of herpes retinitis in an animal model with a sustained delivery antiviral drug, liposomal 1-O-octadecyl-SN-glycerol-3-phosphonoformate.

PURPOSE: To evaluate the clinical treatment efficacy of a long-lasting intravitreous injectable anti-cytomegalovirus (CMV) liposomal drug, 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA). METHODS: Sixty-four pigmented rabbits were used for evaluation of the potency and duration of action of ODG-PFA after intravitreal injection using a herpes simplex virus (HSV)-1 retinitis model. For the potency evaluation, liposomal ODG-PFA was injected into rabbit vitreous at the same time that HSV-1 virus was inoculated onto the retina (simultaneous treatment). For the duration evaluation, ODG-PFA was injected days or weeks before inoculation (pretreatment). Retinitis was clinically graded by indirect ophthalmoscopy, and the retinitis scores were compared across the treatment and control groups. RESULTS: Simultaneous treatment study revealed that ODG-PFA was much more potent than its parent compound, foscarnet (P = 0.0027). Pretreatment study indicated that ODG-PFA possesses a much longer antiviral effect (at least 2 weeks) than foscarnet after a single intravitreal injection. CONCLUSION: Liposomal ODG-PFA is a potent long-lasting intravitreal injectable antiviral compound that may be an ideal alternative for treatment of CMV retinitis in patients with acquired immunodeficiency syndrome.

Intravitreal toxicology in rabbits of two preparations of 1-O-octadecyl-sn-glycerol-3-phosphonoformate, a sustained-delivery anti-CMV drug.

PURPOSE: To determine intraocular toxicity and efficacy of the lipid prodrug of foscarnet, 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA), as a long-acting, nontoxic intravitreous injectable drug delivery system for cytomegalovirus (CMV) retinitis. METHODS: ODG-PFA was synthesized by coupling the phosphonate residue of PFA to the 3 hydroxyl of 1-O-octadecyl-sn-glycerol and formulated as micelles and liposomes at concentrations so that, after injection into the rabbit vitreous, the resultant intravitreal concentrations were 0.2 mM, 0.63 mM, and 2 mM in micellar formulation and 0.02 mM, 0.063 mM, 0.2 mM, and 0.63 mM for liposomal formulation. The compounds were injected, and toxicology evaluations were performed. RESULTS: Intravitreal injections of micellar ODG-PFA resulted in aggregation of the material in vitreous and variable local retinal damage. Intravitreal injections of the liposomal ODG-PFA revealed even dispersion of the compounds and a clear vitreous, using final concentration in the vitreous of 0.2 mM. No intraocular toxicity was found with the 0.632 mM final concentration. The 50% inhibitory concentration (IC50) for CMV of ODG-PFA was 0.43+/-0.27 microM, and the therapeutic index of ODG-PFA after intravitreal injection was estimated to be 1470:1. CONCLUSIONS: Lipid-derivatized foscarnet liposome formulations may be a useful long-acting delivery system for the therapy of CMV retinitis.

Intravitreal pharmacokinetics in rabbits of the foscarnet lipid prodrug: 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA).

PURPOSE: To evaluate the intraocular distribution and metabolism of the lipid prodrug of foscarnet, 1-O-octadecyl-sn-glycerol-3- phosphonoformate (ODG-PFA), following intravitreal administration. METHODS: Twenty rabbits received ODG-[14C]PFA intravitreal injection, yielding 0.632 mM resultant intravitreal concentration. Two animals per group were sacrificed at different intervals post-injection. The drug levels in ocular tissues were determined with counting the radioactivity by Tracor Mark III Liquid Scintillation Counter. Four rabbits were used for analysis of the drug metabolism in vitreous by lipid extraction technique. RESULTS: The drug level in vitreous was 526 microM at day one and 227 microM at the fifth week. The vitreous half life was approximately four to five weeks. The retinal level of the drug was 292 microM at day one, 75 microM at the fifth week and 32 microM at the tenth week, which was still more than ten times higher than the IC90 against HCMV. Lipid extraction analysis showed that, in vivo, both ODG-PFA and PFA were present in vitreous, but in in vitro incubations with vitreous, ODG-PFA conversion to PFA was negligible. CONCLUSION: ODG-PFA possesses a long vitreous half life and sustained high drug level in retina. The vitreous did not metabolize drug but acted as a drug reservoir. Intravitreal liposomal ODG-PFA may be expected to be a long acting potent local therapy for CMV retinitis.

Alkylthioglycerol prodrugs of foscarnet: synthesis, oral bioavailability and structure-activity studies in human cytomegalovirus-, herpes simplex virus type 1- and human immunodeficiency virus type 1-infected cells.

In a previous study, we reported that 1-O-octadecyl-sn-glycero-3-foscarnet (ODG-PFA) was 40 to 93 times more potent than free foscarnet (PFA) in human cytomegalovirus (HCMV)-, herpes simplex virus type 1 (HSV-1)- and human immunodeficiency virus type 1 (HIV-1)-infected cells. To evaluate the effect of substituting a 1-S-alkyl thioether for a 1-O-alkyl ether, we synthesized a series of PFA conjugates of 1-S-alkyl-sn-thioglycerols with varied 1-S-alkyl chain lengths. To establish structure-activity relationships we measured the in vitro antiviral activity of liposomal formulations of the drugs in cells infected with HCMV, HSV-1 or HIV-1. The optimum 1-S-alkyl chain length in the series was 16 to 18 carbon atoms. We compared the antiviral activity of 16- and 18-carbon alkyl thioglycerol versus alkylglycerol prodrugs and did not observe any significant differences in their antiviral activities. The series' most active member, 1-S-octadecyl-sn-glycero-3-foscarnet (ODSG-PFA) was 56-, eight- and 45-fold more active than PFA in HCMV-, HSV-1- and HIV-1-infected cells in vitro. The oral absorption of PFA and 1-S-octadecyl-sn-thioglycero-3-PFA was compared in mice by measuring plasma levels of 14C after oral administration of radiolabelled compounds. The peak plasma level of 14C was sevenfold higher following administration of [14C]ODSG-PFA than following an equimolar dose of [14C]PFA. Area-under-the-curve was 23-fold greater for ODSG-PFA than for PFA. Like previously reported alkyloxyether-lipid PFA conjugates, alkylthioether conjugates provided enhanced antiviral activity and oral bioavailability. However, S-ether conjugates may be metabolized differently than O-ether conjugates. More detailed in vivo pharmacokinetic evaluation of the alkyl-thioether-PFA conjugates is required.

Cardiolipin synthase from mammalian mitochondria.

Cardiolipin was first isolated from beef heart and was shown to contain an unusually high content of linoleic acid ester residues. Cardiolipin is found throughout the eukaryotes including animals, plants and fungi. In mammalian tissue and in yeast, cardiolipin is found exclusively in mitochondria. Mitochondrial synthesis of cardiolipin utilizes phosphatidylglycerol and CDP-diacylglycerol as substrates in a reaction which requires a divalent cation (Mg2+, Mn2+ or Co2+). Cardiolipin synthase has been purified to near-homogeneity from rat liver by solubilization with Zwittergent 3-14 followed by FPLC anion exchange, gel permeation and chromatofocusing steps. Cardiolipin synthase has a molecular mass of 50 kDa, a pH optimum of 8.0, and requires added phospholipids (phosphatidylethanolamine and cardiolipin) and 4 mM Co2+ for optimal activity. Except for the effects of divalent cations and the requirement for phospholipids, little is known about the regulation of cardiolipin synthase. Cardiolipin deficiency in aging mitochondria has been linked to decreased metabolite transport across the inner membrane. Both cardiolipin levels and cardiolipin synthase activity are increased in hyperthyroidism and decreased in hypothyroidism suggesting regulation by thyroid hormone. Mammalian cardiolipin synthase has not been sequenced or cloned and its biological role in mitochondria is not yet fully understood.

Alkoxy propane prodrugs of foscarnet: effect of alkyl chain length on in vitro antiviral activity in cells infected with HIV-1, HSV-1 and HCMV.

The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy would be useful. We recently synthesized 1-O-octadecyl-sn-glycero-3-phosphonoformic acid (ODG-PFA) and noted a 93-fold increase in its anti-HCMV activity relative to PFA. In addition, the antiviral activity of ODG-PFA in herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1) infected cells was increased 40-fold relative to PFA (Hostetler et al., 1996. Antiviral Res. 31, 59). To evaluate structure-activity relationships further, we synthesized alkoxypropyl esters of foscarnet with varying alkyl chain lengths and degrees of saturation. These compounds were tested in vitro for activity and selectivity in comparison with PFA and ODG-PFA in cells infected with HCMV, HSV-1 or HIV-1. Antiviral activity was strongly dependent on chain length with alkyl ethers 14-18 carbon atoms long exhibiting the greatest antiviral activity against HCMV and HSV-1. In HIV-infected HT4-6C cells, optimal activity was observed at 18-22 carbon chain lengths. The antiviral activities of 1-octadecyloxypropane-3-PFA and 1-docosyloxypropane-3-PFA were 135- and 338-fold greater than that of PFA in HT4-6C cells infected with HIV-1. This also represents a 2.6-6-fold improvement in antiviral activity over ODG-PFA, the previously reported analog.

Synthesis and in vitro activity of long-chain 5'-O-[(alkoxycarbonyl)phosphinyl]-3'-azido-3'-deoxythymidines against wild-type and AZT- and foscarnet-resistant strains of HIV-1.

Lipophilic esters of 3'-azido-3'-deoxy-5'-O-(carboxyphosphinyl)thymidine (PFA-AZT) were synthesized and tested for antiretroviral activity in CD4+ HT4-6C cells infected with either wild-type HIV-1LAI, a PFA-resistant strain encoding a single-point mutation in reverse transcriptase (E89K), or an AZT-resistant clinical isolate (A018-post). Arbuzov condensation of 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroformate with trimethyl phosphite yielded the corresponding dimethyl long-chain alkyl triesters of PFA. Selective removal of one methyl group from the triesters with sodium iodide yielded monosodium salts, whereas treatment with bromotrimethylsilane cleaved both methyl groups while leaving the long-chain alkyl group intact. Neutralization of the resulting [(alkyloxy)carbonyl]phosphonic acids with 2 equiv of sodium methoxide afforded disodium salts of the phosphonic acid moiety. Similar chemistry was used to obtain the mono- and disodium salts of the cholesterol ester of PFA. Reaction of the triesters with phosphorous pentachloride, followed by coupling with AZT and O-demethylation with sodium iodide, afforded 3'-azido-3'-deoxy-5'-O-[[(1-octadecyloxy)carbonyl]phosphinyl ]thymidine (9a), 3'-azido-3'-deoxy-5'-O-[[(1-eicosanyloxy)carbonyl]phosphinyl ]thymidine (9b), 3'-azido-3'-deoxy-5'-O-[[(1-docosanyloxy)carbonyl]phosphinyl ]thymidine (9c), and 3'-azido-3'-deoxy-5'-O-[[(3 beta-cholest-5-enyloxy)carbonyl]phosphinyl]thymidine (9d). Concentrations of 9a-d found to inhibit replication of wild-type HIV-1LAI by 50% (EC50 values) as measured in a plaque reduction assay were in the 0.1-0.3 microM range as compared with 0.013 microM for AZT and 133 microM for PFA. The concentration at which toxicity was observed in 50% of the host cells (TC50 values) as measured by a visual grading scale of cellular morphology was 10 microM for 9a and 9d, 32 microM for 9b, and 320 microM for 9c. Thus, the TC50/EC50 ratio or selectivity index (SI) was 100 for 9a, 230 for 9b, and 1000 for 9c but only 33 for 9d, suggesting that the straight-chained fatty alcohol esters were more therapeutically selective. Similar TC50 and SI values were obtained for rapidly dividing CEM lymphoblasts as for HT4-6C cells. In assays against E89K, 9a-c had mean EC50 values of 0.13, 0.009, and 0.17 microM, whereas the EC50 of PFA was > 1000 microM and that of AZT was 0.009 microM; thus, E89K was highly resistant to PFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. In viral replication assays against the A018C-post isolate, the mean EC50 values of 9a-c were 0.30, 0.53, and 0.77 microM as compared with 2.9 microM for AZT and 65 microM for PFA; thus, the virus recovered from a patient pretreated with AZT was not cross-resistant to either PFA or 9a-c. A notable feature of these results was that, in addition to being > 1000-fold more potent than PFA against the PFA-resistant mutant, the lipophilic PFA-AZT conjugates were more potent than PFA, as well as AZT, against AZT-resistant HIV-1.

Enhanced oral absorption and antiviral activity of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir and related compounds in hepatitis B virus infection, in vitro.

Acyclovir (ACV) triphosphate and azidothymidine (AZT) triphosphate inhibit the DNA polymerase of human hepatitis B virus (HBV) by 50% at submicromolar concentrations, but no effects of ACV or AZT treatment have been noted on the clinical manifestations of hepatitis B. We synthesized 1-O-octadecyl-sn-glycero-3-phospho-acyclovir (ODG-P-ACV), 1-O-hexadecylpropanediol-3-phospho-acyclovir (HDP-P-ACV), and 1-O-octadecyl-sn-glycero-3-phospho-azidothymidine (ODG-P-AZT), and evaluated their antiviral activity in human hepatoma cells that constitutively produce HBV (2.2.15 cells). ACV and AZT up to 100 microM caused only slight inhibition of HBV replication in 2.2.15 cells. However, HDP-P-ACV and ODG-P-ACV inhibited viral replication by 50% at 0.5 and 6.8 microM, respectively. ODG-P-AZT also showed increased antiviral activity, with a 50% reduction in HBV replication at 2.1 microM. Based on the EC50, HDP-P-ACV, ODG-P-ACV, and ODG-P-AZT were > 200, > 14.7, and > 48 times more active than their free nucleosides in reducing HBV replication in 2.2.15 cells. To evaluate the biochemical basis for the increased antiviral activity, we studied the uptake and metabolism of 1-O-octadecyl-sn-glycero-3-phospho-[3H]acyclovir (ODG-P-[3H]ACV) in HepG2 cells. Cellular uptake of ODG-P-[3H]ACV was found to be substantially greater than that of [3H]ACV, and cellular levels of ACV-mono-, -di-, and -triphosphate were much higher with ODG-P-ACV. ODG-P-[3H]ACV was well absorbed orally. Based on urinary recovery of tritium after oral or parenteral administration of the radiolabeled compounds, oral absorption of ODG-P-ACV in mice was 100% versus 37% for ACV. ODG-P-ACV plasma area under the curve was more than 7-fold greater than that of ACV. Lipid prodrugs of this type may be useful orally in treating viral diseases.

Evaluation of a novel lipid prodrug for intraocular drug delivery: effect of acyclovir diphosphate dimyristoylglycerol in a rabbit model with herpes simplex virus-1 retinitis.

BACKGROUND: Acyclovir diphosphate dimyristoylglycerol is a lipid prodrug of acyclovir that forms liposomes and provides substantial activity against herpes simplex virus, acyclovir-resistant strains of herpes simplex virus, and human cytomegalovirus. We therefore tested this promising new drug in a rabbit model of herpes simplex retinitis. METHODS: A total of 22 pigmented rabbits were pretreated with either acyclovir diphosphate dimyristoylglycerol, ganciclovir, acyclovir, or buffer. Retinae then were inoculated with herpes simplex virus-1 or buffer 1 week after the injection of drug. In another experiment we compared the effects of acyclovir diphosphate dimyristoylglycerol and acyclovir diphosphate dioleoylglycerol on the optical clarity of vitreous. RESULTS: Animals injected intravitreally with acyclovir diphosphate dimyristoylglycerol showed retinitis that was less severe than that in animals injected with ganciclovir, acyclovir, and buffer; differences in grading scores of the retinitis between animals injected with acyclovir diphosphate dimyristoylglycerol and those injected with buffer were statistically significant (P = 0.0015). Vitreous and optical media became clear 4 days after acyclovir diphosphate dioleoylglycerol injection compared with 10 days after with acyclovir diphosphate dimyristoylglycerol injections. CONCLUSION: Acyclovir diphosphate dimyristoylglycerol had prolonged antiviral activity against herpes simplex virus-1 retinitis in a rabbit model. This drug delivery system, modified to improve optical clarity, may allow long-acting intravitreal treatment of cytomegalovirus retinitis and other retinal diseases.

Lipid prodrugs of phosphonoacids: greatly enhanced antiviral activity of 1-O-octadecyl-sn-glycero-3-phosphonoformate in HIV-1, HSV-1 and HCMV-infected cells, in vitro.

Phosphonoformate (PFA) effectively inhibits viral polymerases but is relatively ineffective in virus-infected cells in tissue culture. A lipid prodrug of phosphonoformate was synthesized by coupling the phosphonate residue of phosphonoformate to the sn-3 hydroxyl of 1-O-octadecyl-sn-glycerol. This prodrug, 1-O-octadecyl-sn-glycero-3-phosphonoformate (ODG-PFA), was 93-fold more active than phosphonoformate in cells infected with the AD169 strain of cytomegalovirus (CMV), and 111-147-fold more active in cells infected with three human clinical isolates of CMV. The compound was also 44-fold more active in human immunodeficiency virus-1 (HIV-1) infected cells and 43-fold more active in cells infected with herpes simplex virus (HSV). Studies of the mechanisms of increased antiviral activity indicate that 1-O-octadecyl-sn-glycero-3-[14C]phosphonoformate is taken up more extensively than the free drug by the host MRC-5 human lung fibroblasts. Intracellular enzymes convert 1-O-octadecyl-sn-glycero-3-phosphonoformate to phosphonoformate. This conversion does not occur in the tissue culture medium containing fetal bovine serum (FBS) or in MRC-5-conditioned medium. In view of its greatly increased in vitro potency and selectivity, 1-O-octadecyl-sn-glycero-3-phosphonoformate may be useful in treating viral diseases.

Liver-targeted antiviral nucleosides: enhanced antiviral activity of phosphatidyl-dideoxyguanosine versus dideoxyguanosine in woodchuck hepatitis virus infection in vivo.

It would be desirable to develop antiviral agents that can be targeted to liver to enhance their antiviral effects and reduce nonhepatic toxicity. 2',3'-Dideoxyguanosine (ddG) has been found to be a potent and selective antihepatitis B agent both in vitro and in vivo. To evaluate ddG and its liver-targeted analog, we synthesized a series of phosphatidyl-ddGs and incubated them with 2.2.15 cells, which chronically produce hepatitis B virus. 1,2-Dipalmitoylphosphatidyl-dideoxyguanosine (DPP-ddG) inhibited the production of hepatitis B virus (HBV) DNA in the culture medium by 90% at 4.5 mumol/L versus 9.1 mumol/L for ddG, while the liposome vehicle itself had no effect. To compare the efficacy of free ddG with its lipid prodrug in vivo, we treated woodchucks that were experimentally infected with woodchuck hepatitis virus (WHV) for 4 weeks by intraperitoneal injection of 2.6 mumol/kg/d of free ddG or liposomes containing 2.6 mumol/kg/d of DPP-ddG. Liposomal DPP-ddG reduced serum WHV DNA by 23- to 46-fold at the end of the fourth week, while free ddG reduced serum WHV DNA by 2.2- to 10.4-fold. Treatment with small unilamellar liposomes containing DPP-ddG is substantially more effective than free ddG in reducing WHV-DNA levels in serum in WHV-infected woodchucks. The data suggest that the use of lipid prodrugs to target the liver may be useful in enhancing antiviral therapy of hepatitis.

Comparative evaluation of amiodarone-induced phospholipidosis and drug accumulation in Fischer-344 and Sprague-Dawley rats.

Amiodarone (AD) and its major metabolite, desethylamiodarone (desethylAD), are both phospholipogenic. The present study was undertaken to evaluate the comparative susceptibilities of male Fischer-344 and Sprague-Dawley rats to AD-induced phospholipidosis in alveolar macrophages (AMs), liver and kidney tissue and the concomitant accumulation of AD and desethylAD in these cells, tissues and plasma. Rats were administered AD (100 mg/kg/day, p.o.) for 1 week. Plasma concentrations of AD and desethylAD were approximately 4- and 12-fold higher, respectively, in Fischer-344s compared to Sprague-Dawleys 24 h after the last dose. AD and desethylAD levels in AMs were approximately 12- and 25-fold higher, respectively, in Fischer-344s than Sprague-Dawleys. In the liver and kidney, levels of both compounds were also significantly higher in Fischer-344s than Sprague-Dawleys. Ultrastructural features indicative of phospholipidosis were not observed consistently in any tissue except AMs from treated Fischer-344s. AM total phospholipid increased nearly 5-fold in Fischer-344s, while Sprague-Dawleys showed no increase over control. AMs from both strains incubated with 10 microM AD or desethylAD in vitro were not significantly different in their accumulation of the compounds. When incubated with AD or desethylAD, the lysosomal phospholipases A1 partially purified from AMs of both strains were equally sensitive to inhibition as measured by the drug concentration giving 50% inhibition in activity (IC50). The results of this study indicate that at the same administered dose, AD and desethylAD, accumulate to higher tissue levels and are more phospholipogenic in male Fischer-344 rats than in male Sprague-Dawley rats. The basis for the high susceptibility of Fischer-344 rats to AM-induced phospholipidosis is unknown at present but appears not to be related to biochemical or cellular features of the AMs.