Octadecyloxyethyl benzyl tenofovir: A novel tenofovir diester provides sustained intracellular levels of tenofovir diphosphate.

Pre-exposure prophylaxis (PrEP) with topically or systemically administered antiretroviral agents can prevent acquisition of human immunodeficiency virus type 1 (HIV-1) infection. However, in clinical trials using tenofovir-containing agents, HIV-1 acquisition is reduced but not eliminated. Incomplete adherence remains the major contributor to failure. Sustained release or long-acting antiretroviral agents may provide better HIV-1 protection by reducing the clinical impact of incomplete adherence. To reduce dosing frequency, we synthesized a novel tenofovir prodrug, octadecyloxyethyl benzyl tenofovir (ODE-Bn-TFV), that is designed to release TFV slowly in tissues, and showed potent anti-HIV activity in vitro (EC50 = 1.7 nM). In cells exposed to 14C labeled TFV, ODE-Bn-TFV or the quickly activated monoester ODE-TFV, rapid cellular uptake for both lipophilic analogs was noted, achieving 50-fold higher levels than unmodified TFV after 48 h. Following exposure to ODE-[8-14C]TFV, the intracellular diphosphate levels were approximately four-fold higher than with ODE-Bn-TFV. However, intracellular TFVpp drug levels fell rapidly yielding a half-life of about two days. TFVpp levels in ODE-Bn-TFV treated cells decreased much more slowly and reached half-maximal levels in about seven days. These results suggest early accumulation of ODE-Bn-TFV followed by sustained intracellular release following cleavage of the ester bonds linking the ODE and benzyl moieties to the active molecular precursor, thereby potentially allowing for less frequent administration than with more rapidly activated forms of tenofovir.

Synthesis and Antiviral Evaluation of Octadecyloxyethyl Benzyl 9-[(2-Phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG), a Potent Inhibitor of Transient HPV DNA Amplification.

Human papillomavirus (HPV) high-risk genotypes such as HPV-16 and HPV-18 cause the majority of anogenital tract carcinomas, including cervical cancer, the second most common malignancy in women worldwide. Currently there are no approved antiviral agents that reduce or eliminate HPV and reverse virus-associated pathology. We synthesized and evaluated several alkoxyalkyl acyclic nucleoside phosphonate diesters and identified octadecyloxyethyl benzyl 9-[(2-phosphonomethoxy)ethyl]guanine (ODE-Bn-PMEG) as an active compound which strongly inhibited transient amplification of HPV-11, -16, and -18 origin-containing plasmid DNA in transfected cells at concentrations well below its cytotoxic concentrations. ODE-Bn-PMEG demonstrated increased uptake in human foreskin fibroblast cells and was readily converted in vitro to the active antiviral metabolite, PMEG diphosphate. The P-chiral enantiomers of ODE-Bn-PMEG were obtained and appeared to have equivalent antiviral activities against HPV. ODE-Bn-PMEG is a promising candidate for the local treatment of HPV-16 and HPV-18 and other high-risk types, an important unmet medical need.

A novel cytarabine crystalline lipid prodrug: hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate for proliferative vitreoretinopathy.

PURPOSE: The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection. METHODS: Hexadecyloxypropyl cytarabine 5'-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-ß-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology. RESULTS: HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 µM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes (generalized estimating equations, p=0.039 and 0.01). CONCLUSIONS: The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes.

Intraocular safety and pharmacokinetics of hexadecyloxypropyl-cidofovir (HDP-CDV) as a long-lasting intravitreal antiviral drug.

PURPOSE: To evaluate the intraocular safety and pharmacokinetics of hexadecyloxypropyl-cidofovir (HDP-CDV), the hydrolysis product of HDP-cyclic-CDV, a long-lasting intravitreal cidofovir prodrug for cytomegalovirus (CMV) retinitis. METHODS: HDP-cyclic-CDV was suspended in phosphate-buffered saline (PBS) at 37°C and formation of HDP-CDV was monitored by high-performance liquid chromatography (HPLC) analysis for 30 weeks. The safety and pharmacokinetics of HDP-CDV intravitreal injections were studied using New Zealand Red rabbits and (14)C labeled HDP-CDV. Ocular tissues from five time points (1, 3, 7, 14, and 35 days) were analyzed by scintillation counting and HPLC to characterize the pharmacokinetics. RESULTS: During the hydrolysis study, approximately 35% of the HDP-cyclic-CDV was converted to HDP-CDV. Evaluation of safety found no toxicity after intravitreal injection of HDP-CDV up to 28 µg/eye. Intravitreal pharmacokinetics of HDP-CDV in the retina, choroid, and vitreous followed a two-phase elimination process and elimination half-lives of 8.4 days (retina), 6.9 days (choroid), and 6.2 days (vitreous). In the retina, cidofovir and an unknown metabolite were detected in the first 2 weeks, and the maximum metabolite concentrations were present 48 hours after the maximum HDP-CDV concentration. CONCLUSIONS: HDP-cyclic CDV, under simulated physiologic conditions, slowly converts to HDP-CDV, another potent anti-CMV prodrug that may be taken up by retinal cells and metabolized further to the active antiviral metabolite, cidofovir diphosphate. Taken together, these observations help to explain the ability of a single intravitreal dose of HDP-cyclic-CDV to prevent viral retinitis for up to 68 days in a rabbit model.

Antiproliferative effects of octadecyloxyethyl 9-[2-(phosphonomethoxy)ethyl]guanine against Me-180 human cervical cancer cells in vitro and in vivo.

BACKGROUND/AIMS: 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) is one of the most active antiproliferative compounds in a series of acyclic nucleoside phosphonates and is active in intraperitoneal P388 tumors in mice. METHODS: We synthesized octadecyloxyethyl (ODE) and hexadecyloxypropyl esters of PMEG and compared their antiproliferative activity with unmodified PMEG in primary human fibroblasts and CaSki, Me-180 and HeLa human cervical cancer cell lines in vitro. RESULTS: ODE-PMEG had excellent antiproliferative activity in vitro in this panel of human cervical cancers. We compared the effects of ODE-PMEG and ODE-cidofovir (ODE-CDV) in a solid tumor model using Me-180 human cervical cancer cell lines in athymic nude mice. Intratumoral injection of 25 microg of ODE-PMEG or 100 microg of ODE-CDV daily for 21 days followed by observation for 20-35 days resulted in near-complete disappearance of measurable cervical cancers. CONCLUSION: ODE-PMEG may be suitable for local or topical treatment of cervical dysplasia.

Cidofovir and (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine are highly effective inhibitors of vaccinia virus DNA polymerase when incorporated into the template strand.

The acyclic nucleoside phosphonate drug (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]adenine [(S)-HPMPA], is a broad-spectrum antiviral and antiparasitic agent. Previous work has shown that the active intracellular metabolite of this compound, (S)-HPMPA diphosphate [(S)-HPMPApp], is an analog of dATP and targets DNA polymerases. However, the mechanism by which (S)-HPMPA inhibits DNA polymerases remains elusive. Using vaccinia virus as a model system, we have previously shown that cidofovir diphosphate (CDVpp), an analog of dCTP and a related antiviral agent, is a poor substrate for the vaccinia virus DNA polymerase and acts to inhibit primer extension and block 3'-to-5' proofreading exonuclease activity. Based on structural similarities and the greater antiviral efficacy of (S)-HPMPA, we predicted that (S)-HPMPApp would have a similar, but more pronounced effect on vaccinia polymerase than CDVpp. Interestingly, we found that (S)-HPMPApp is a good substrate for the viral enzyme, exhibiting K(m) and V(max) parameters comparable to those of dATP, and certainly not behaving like CDVpp as a functional chain terminator. Metabolic experiments indicated that (S)-HPMPA is converted to (S)-HPMPApp to a much greater extent than CDV is converted to CDVpp, although both drugs cause identical effects on virus DNA replication at their 50% effective concentration. Subsequent studies showed that both compounds can be faithfully incorporated into DNA, but when CDV and (S)-HPMPA are incorporated into the template strand, both strongly inhibit trans-lesion DNA synthesis. It thus appears that nucleoside phosphonate drugs exhibit at least two different effects on DNA polymerases depending upon in what form the enzyme encounters the drug.

Evaluation of hexadecyloxypropyl-9-R-[2-(Phosphonomethoxy)propyl]- adenine, CMX157, as a potential treatment for human immunodeficiency virus type 1 and hepatitis B virus infections.

9-R-[2-(Phosphonomethoxy)propyl]-adenine (tenofovir) is an acyclic nucleoside phosphonate with antiviral activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). Tenofovir is not orally bioavailable but becomes orally active against HIV-1 infection as the disoproxil ester (tenofovir disoproxil fumarate [Viread]). We have developed an alternative strategy for promoting the oral availability of nucleoside phosphonate analogs which involves esterification with a lipid to form a lysolecithin mimic. This mimic can utilize natural lysolecithin uptake pathways in the gut, resulting in high oral availability. Since the mimic is not subject to cleavage in the plasma by nonspecific esterases, it remains intact in the circulation and facilitates uptake by target cells. Significant drops in apparent antiviral 50% effective concentrations (EC(50)s) of up to 3 logs have been observed in comparison with non-lipid-conjugated parent compounds in target cells. We have applied this technology to tenofovir with the goal of increasing oral availability, decreasing the apparent EC(50), and decreasing the potential for nephrotoxicity by reducing the exposure of the kidney to the free dianionic tenofovir. Here we report that, in vitro, the hexadecyloxypropyl ester of tenofovir, CMX157, is 267-fold more active than tenofovir against HIV-1 and 4.5-fold more active against HBV. CMX157 is orally available and has no apparent toxicity when given orally to rats for 7 days at doses of 10, 30, or 100 mg/kg/day. Consequently, CMX157 represents a second-generation tenofovir analog which may have an improved clinical profile.

Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir.

Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40-100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6-40 times more active against HCMV and 7-30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.

Oral 1-O-octadecyl-2-O-benzyl-sn-glycero-3-cidofovir targets the lung and is effective against a lethal respiratory challenge with ectromelia virus in mice.

Hexadecyloxypropyl-cidofovir (HDP-CDV) has been shown to be orally active against lethal infection with orthopoxviruses including, mousepox, cowpox, vaccinia and rabbitpox. The alkoxyalkyl group provides oral absorption and reduces greatly the amount of drug reaching the kidney, the site of CDV's dose limiting toxicity. However, the amount of HDP-CDV detected in lung, an important site of early poxvirus replication, is low and the reduction of viral titers in surviving animals is reduced moderately compared with the liver where poxvirus titers are virtually undetectable. We synthesized a novel glycerol ester of CDV, 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV), and compared its oral pharmacokinetics with that of HDP-CDV. Surprisingly, ODBG-CDV levels in lung are much higher and liver levels are reduced, suggesting that the compound is transported in small intestinal lymph instead the portal vein. ODBG-CDV has excellent in vitro activity in cells infected with ectromelia virus (ECTV). In mice infected with a lethal aerosol or intranasal challenge of ECTV, HDP-CDV and ODBG-CDV are equally effective in preventing death from disease. Other drugs esterified to 1-O-octadecyl-2-O-benzyl-sn-glycerol or 1-O-octadecyl-2-O-benzyl-sn-glycerol-3-phosphate may provide lung targeting for treatment of microbial or neoplastic diseases while reducing first pass removal by the liver during oral absorption.

Antiviral activities of novel 5-phosphono-pent-2-en-1-yl nucleosides and their alkoxyalkyl phosphonoesters.

Three acyclic nucleoside phosphonates are currently approved for clinical use against infections caused by cytomegalovirus (Vistide), hepatitis B virus (Hepsera), and human immunodeficiency virus type 1 (Viread). This important antiviral class inhibits viral polymerases after cellular uptake and conversion to their diphosphates, bypassing the first phosphorylation, which is required for conventional nucleoside antivirals. Small chemical alterations in the acyclic side chain lead to marked differences in antiviral activity and the spectrum of activity of acyclic nucleoside phosphonates against various classes of viral agents. We synthesized a new class of acyclic nucleoside phosphonates based on a 5-phosphono-pent-2-en-1-yl base motif in which the oxygen heteroatom usually present in acyclic nucleoside phosphonates has been replaced with a double bond. Since the intrinsic phosphonate moiety leads to low oral bioavailability and impaired cellular penetration, we also prepared the hexadecyloxypropyl esters of the 5-phosphono-pent-2-en-1-yl nucleosides. Our earlier work showed that this markedly increases antiviral activity and oral bioavailability. Although the 5-phosphono-pent-2-en-1-yl nucleosides themselves were not active, the hexadecyloxypropyl esters were active against DNA viruses and hepatitis B virus, in vitro. Notably, the hexadecyloxypropyl ester of 9-(5-phosphono-pent-2-en-1-yl)-adenine was active against hepatitis B virus mutants resistant to lamivudine, emtricitabine, and adefovir.

Synthesis and antiviral evaluation of alkoxyalkyl esters of phosphonopropoxymethyl-guanine and phosphonopropoxymethyl-diaminopurine.

Phosphonopropoxymethyl-guanine is the methylene phosphonate analogue of acyclovir. Although not highly active against HSV, 4-38 microM of phosphonopropoxymethyl-guanine has been reported to be active against human and murine cytomegalovirus. Recently we found that cidofovir, when esterified with alkoxyalkyl moieties, showed greatly increased antiviral activity against cytomegalovirus, herpes simplex virus and orthopoxviruses, in vitro. The alkoxyalkyl esters of cidofovir are orally active in murine models of human and murine cytomegalovirus and orthopoxviruses in vivo. To see if the antiviral activity of phosphonopropoxymethyl-guanine, phosphonopropoxymethyl-diaminopurine and phosphonopropoxymethyl-N6-cyclopropyl-diaminopurine could be increased by this approach, we synthesized their hexadecyloxypropyl- and octadecyloxyethyl- esters and evaluated antiviral activity and cytotoxicity in cells infected with HSV-1 and HCMV, in vitro. Marked increases in antiviral activity were noted in the alkoxyalkyl esters of phosphonopropoxymethyl-guanine. Alkoxyalkyl esters of diaminopurine and N6-cyclopropyl-diaminopurine showed slight increases in activity against HSV-1 and marked increases in activity against HCMV. The results suggest that esterification with alkoxyalkyl moieties may be a generally useful way to increase antiviral activity of nucleoside phosphonates.

Alkoxyalkyl esters of (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine are potent inhibitors of the replication of wild-type and drug-resistant human immunodeficiency virus type 1 in vitro.

(S)-9-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA], is an effective broad-spectrum antiviral against many DNA viruses but has been reported to be inactive against human immunodeficiency virus (HIV). We synthesized several alkoxyalkyl esters of (S)-HPMPA and now report that hexadecyloxypropyl-(S)-HPMPA [HDP-(S)-HPMPA] and octadecyloxyethyl-(S)-HPMPA [ODE-(S)-HPMPA]had 50% effective concentrations of 0.4 to 7.0 nanomolar and were nearly fully active against HIV variants having reverse transcriptase mutations M184V and K103N and against a zidovudine-resistant variant with mutations D67N, K70R, T215Y, and K219Q. Resistance to HDP-(S)-HPMPA and ODE-(S)-HPMPA was noted for a mutant with mutation K65R. HDP-(S)-HPMPA is also active against herpes simplex virus type 1, human cytomegalovirus, hepatitis B virus, adenoviruses, and orthopoxviruses and is worthy of further evaluation as a possibly therapy for HIV infection.

Synthesis and antiviral evaluation of alkoxyalkyl esters of acyclic purine and pyrimidine nucleoside phosphonates against HIV-1 in vitro.

Alkoxyalkyl esters of cidofovir, an acyclic nucleoside phosphonate, have been shown to have antiviral activities several orders of magnitude greater than unmodified cidofovir against cytomegalovirus, herpes simplex virus, vaccinia, cowpox, ectromelia and adenoviruses in vitro. Hexadecyloxypropyl-cidofovir is orally bioavailable and active in lethal animal models of vaccinia, cowpox, ectromelia and cytomegalovirus. To see if this strategy is also applicable to other acyclic nucleoside phosphonates, we have converted several phosophonomethoxyethyl purines and pyrimidines to their hexadecyloxypropyl, octadecyloxyethyl and oleyloxyethyl esters and compared their activity against HIV-1 with the activity of the respective unmodified acyclic nucleoside phosphonates. The hexadecyloxypropyl esters of phosphonomethoxyethyl-adenine, phosphonomethoxyethyl-2,6-diaminopurine and phosphonomethoxyethyl-N(6)-cyclopropyl-diaminopurine were 3-5 orders of magnitude more active against HIV-1 in vitro than the parent nucleotides. The EC(50) values for these compounds were in the 10-20 pM range with selective indexes of 1,250 to >4,000. The acyclic pyrimidine phosphonates were generally inactive against HIV-1 in vitro. Phosphonomethoxyethyl-cytosine and phosphonomethoxyethyl-5-fluorocytosine were inactive against HIV-1. Surprisingly, hexadecyloxypropyl-phosphonomethoxyethyl-5-fluorocytosine was active against HIV-1 with a submicromolar EC(50) and a selective index of 174. Esterification of acyclic nucleoside phosphonates with alkoxyalkyl moieties may represent a general approach for increasing antiviral activity and selectivity of this class of antivirals.

Enhanced antiproliferative effects of alkoxyalkyl esters of cidofovir in human cervical cancer cells in vitro.

Nearly all cervical cancers are associated with the high-risk subtypes of human papillomavirus (HPV) expressing the E6 and E7 oncoproteins. The E6 and E7 oncoproteins reduce cellular levels of the p53 and the retinoblastoma (pRb) tumor suppressors, respectively, and represent an important component of the malignant phenotype. Several groups have shown that treatment with cidofovir suppresses levels of E6 and E7, restoring cellular p53 and pRb levels, in turn slowing cell replication and increasing the susceptibility of the cancer cells to radiation and apoptosis. Recently, our group synthesized alkoxyalkyl esters of cidofovir, which were found to be >100 times more active than unmodified cidofovir in vitro against various double-stranded DNA viruses, including cytomegalovirus, herpes simplex virus, adenoviruses, cowpox, vaccinia, and variola viruses. We compared the activity of octadecyloxyethyl-cidofovir (ODE-CDV) and oleyloxyethyl-cidofovir (OLE-CDV) with that of unmodified cidofovir against both HPV-negative and HPV-positive cervical cancer cells. We compared the antiproliferation activity in CaSki, HeLa, and Me-180 cells, prototypical HPV-positive cell lines bearing the HPV-16, HPV-18, and HPV-68 high-risk subtypes, with the activity in C33A cells, a cervical cancer cell line lacking HPV, and in nonmalignant primary human foreskin fibroblast cells. OLE-CDV and ODE-CDV were several logs more potent than cidofovir in CaSki, Me-180, HeLa, and C33A cervical cancer cells as determined by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt proliferation assay. Cell cycle analysis indicates that the cidofovir analogues interfere with passage of dividing cells through the S phase. ODE-CDV and OLE-CDV were 500 to 17,000 times more active than cidofovir in inhibiting the growth of cervical cancer cells. ODE-CDV and OLE-CDV showed selectivity for cervical cancer cells versus nonmalignant human foreskin fibroblast cells and warrant further investigation as potential therapies for cervical cancer.

Esterification of cidofovir with alkoxyalkanols increases oral bioavailability and diminishes drug accumulation in kidney.

Smallpox was eradicated by vaccination in the 1970s. However, concerns have arisen about the potential use of variola virus as a biological weapon. Most of the world's population has little residual immunity because systematic vaccination against smallpox ceased in the early 1970s. Vaccination of key elements of the population against smallpox is again being considered. However, there are now large numbers of persons who cannot be safely vaccinated with the current vaccine because of AIDS, immunosuppressive drugs, and certain common skin disorders. It would be useful to have a potent orally active drug as an alternative for these persons in case of an outbreak of smallpox. Alkoxyalkyl esters of cidofovir (CDV) have been shown to be highly active and selective against poxviruses in vitro with activities several logs greater than the activity of unmodified CDV. This is due in large part to increased cellular penetration and conversion to CDV-diphosphate, the active antiviral. In this paper, the oral pharmacokinetics of 14C-labeled hexadecyloxypropyl-cidofir (HDP-CDV), octadecyloxyethyl-cidofir (ODP-CDV), and oleyloxypropyl-cidofir (OLP-CDV) are examined and oral bioavailability and tissue distribution assessed and compared with parenteral CDV. The alkoxyalkyl CDVs are highly orally bioavailable and do not concentrate in kidney, the site of the dose-limiting toxicity of CDV. Plasma and tissue drug levels are many times greater than the in vitro EC(50s) for variola, cowpox, and vaccinia viruses. Thus, the compounds are good candidates for further development for prevention and treatment of smallpox infection and the complications of vaccination.

Ganciclovir release rates in vitreous from different formulations of 1-O-hexadecylpropanediol-3-phospho-ganciclovir.

PURPOSE: To determine the optimal formulation of lipid prodrug, 1-O-hexadecyloxypropyl-phospho-ganciclovir (HDP-P-GCV), for intravitreal delivery. METHODS: Equal concentrations of crystalline or liposomal HDP-P-GCV were exposed to rabbit whole vitreous, core vitreous, peripheral vitreous, human plasma, and heat inactivated rabbit vitreous, and the samples were incubated at 37 degrees C for one week. Aliquots were taken at day 1, 2, 3, and 7 and subjected to HPLC analysis for conversion to GCV. RESULTS: The resultant concentration of GCV from crystalline HDP-P-GCV in vitreous was 198 +/- 49 microM (n = 3) at day 1 and 1253 +/- 248 microM (n = 3) at day 7. The resultant concentration of GCV from the liposomal formulation of HDP-P-GCV in vitreous was much lower, yielding a concentration of 66 +/- 7 microM (n = 3) at day 1 and 243 +/- 39 microM (n = 3) at day 7 (P < 0.001, t Test). When the crystalline HDP-P-GCV was incubated with heat-inactivated vitreous, the detectable GCV concentrations were low (22 microM) and did not increase over time. The concentration of GCV detected from the crystalline HDP-P-GCV in the core vitreous was 19.69 +/- 3.84 microM (n = 3) at day 1 and 1537.36 +/- 177.14 microM (n = 3) at day 7. The concentration of GCV released from crystalline HDP-P-GCV in peripheral vitreous was 32.86 +/- 5.07 microM (n = 3) at day 1 and 1805.78 +/- 327.94 microM (n = 3) at day 7. Detectable GCV concentration from both core and peripheral vitreous samples increased over time, however, the magnitude of GCV release from peripheral vitreous samples was higher (P < 0.05, t Test). CONCLUSION: In vitreous, HDP-P-GCV as a crystalline formulation was converted to GCV more rapidly than liposomal formulation of HDP-P-GCV. Vitreous cells may play an important role in the metabolism of either formulation of HDP-P-GCV delivered into vitreous.

Increased antiviral activity of 1-O-hexadecyloxypropyl-[2-(14)C]cidofovir in MRC-5 human lung fibroblasts is explained by unique cellular uptake and metabolism.

Recently, there has been renewed interest in finding orally active drugs against smallpox. Cidofovir (CDV) given by parenteral injection has been shown to protect against lethal poxvirus infection. We have been interested in the synthesis and evaluation of orally active derivatives of CDV. Previous studies showed that the CDV and cyclic cidofovir (cCDV) analogs 1-O-hexa-decyloxypropyl-CDV (HDP-CDV) and 1-O-hexadecyloxypropyl-cCDV (HDP-cCDV), show >100-fold increases in antiviral activity versus the unmodified nucleosides against cells infected with orthopoxviruses, cowpox, and vaccinia virus. In contrast to CDV, HDP-CDV is orally bioavailable and has been reported to be orally active in lethal cowpox virus infection in mice. To assess the metabolic basis for the increased antiviral activity of HDP-CDV in vitro, we studied the cellular uptake and anabolic metabolism of (14)C-labeled CDV, cCDV, and their alkoxyalkanol esters HDP-CDV and HDP-cCDV. HDP-CDV and HDP-cCDV were taken up rapidly by MRC-5 human lung fibroblasts in vitro, but uptake of CDV and cCDV was much slower. Analysis of cellular metabolites showed that levels of cidofovir diphosphate (CDV-DP), the active antiviral compound, were >100 times greater with HDP-CDV than levels observed with CDV. When cells were exposed to HDP-CDV, the intracellular half-life of CDV-DP was 10 days versus 2.7 days reported when cells are exposed to CDV. HDP-CDV seems to circumvent poor cellular uptake by rapid association with cellular membrane phospholipids, whereas CDV uptake proceeds via the slow process of fluid endocytosis.

Alkoxyalkyl esters of cidofovir and cyclic cidofovir exhibit multiple-log enhancement of antiviral activity against cytomegalovirus and herpesvirus replication in vitro.

The incidence of cytomegalovirus (CMV) retinitis is declining in AIDS patients but remains a significant clinical problem in patients with organ transplants and bone marrow transplants. Prophylaxis with ganciclovir (GCV) or valganciclovir reduces the incidence of CMV disease but may lead to the emergence of drug-resistant virus with mutations in the UL97 or UL54 gene. It would be useful to have other types of oral therapy for CMV disease. We synthesized hexadecyloxypropyl and octadecyloxyethyl derivatives of cyclic cidofovir (cCDV) and cidofovir (CDV) and found that these novel analogs had 2.5- to 4-log increases in antiviral activity against CMV compared to the activities of unmodified CDV and cCDV. Multiple-log increases in activity were noted against laboratory CMV strains and various CMV clinical isolates including GCV-resistant strains with mutations in the UL97 and UL54 genes. Preliminary cell studies suggest that the increase in antiviral activity may be partially explained by a much greater cell penetration of the novel analogs. 1-O-Hexadecyloxypropyl-CDV, 1-O-octadecyloxyethyl-CDV, and their corresponding cCDV analogs are worthy of further preclinical evaluation for treatment and prevention of CMV and herpes simplex virus infections in humans.