Rethinking Remdesivir: Synthesis, Antiviral Activity, and Pharmacokinetics of Oral Lipid Prodrugs.

Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

Octadecyloxyethyl Adefovir Exhibits Potent in vitro and in vivo Cytotoxic Activity and Has Synergistic Effects with Ara-C in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) continues to be a deadly disease, with only 50-70% of patients achieving complete remission and less than 30% of adults having sustained long-term remissions. In order to address these unmet medical needs, we carried out a high-throughput screen of an in-house library of on- and off-patent drugs with the OCI/AML-2 cell line. Through this screen, we discovered adefovir dipi-voxil (adefovir-DP) as being active against human AML. In addition to adefovir-DP, there are second-generation formulations of adefovir, including octadecyloxyethyl adefovir (ODE-adefovir) and hexadecyloxypropyl adefovir (HDP-adefovir), which were designed to overcome the pharmacokinetic problems of the parent compound adefovir. Given the known clinical benefit of nucleoside analogs for the treatment of AML, we undertook studies to evaluate the potential benefit of adefovir-based molecules. In AML cell lines and patient samples, adefovir-DP and ODE-adefovir were highly potent, whereas HDP-adefovir was significantly less active. Interestingly, ODE-adefovir was remarkably less toxic than adefovir-DP towards normal hematopoietic cells. In addition, ODE-adefovir at a dose of 15 mg/kg/day showed potent activity against human AML in a NOD/SCID mouse model, with a reduction of human leukemia in mouse bone marrow of > 40% in all mice tested within 20 days of treatment. Based on its chemical structure, we hypothesized that the cytotoxicity of ODE-adefovir toward AML was through cell cycle arrest and DNA damage. Indeed, ODE-adefovir treatment induced cell cycle arrest in the S phase and increased levels of pH2Ax, indicating the induction of DNA damage. Furthermore, there was an increase in phospho-p53, transactivation of proapoptotic genes and activation of the intrinsic apoptotic pathway. Subsequent investigation unveiled strong synergism between ODE-adefovir and ara-C, making their coadministration of potential clinical benefit. Expression of MRP4, a nucleoside transporter, appeared to influence the response of AML cells to ODE-adefovir, as its inhibition potentiated ODE-adefovir killing. Taken together, our findings indicate that clinical development of ODE-adefovir or related compounds for the treatment of AML is warranted.

Novel mutations in the gene encoding very long-chain acyl-CoA dehydrogenase identified in patients with partial carnitine palmitoyltransferase II deficiency.

INTRODUCTION: Twenty-six patients with clinical symptoms of adult onset carnitine palmitoyltransferase II (CPTII) deficiency were examined. All patients had skeletal muscle CPTII enzyme activity levels indicative of heterozygosity for CPT2 mutations, however sequence analysis identified no pathogenic mutations within the CPT2 gene. METHODS: Because the reaction product of CPTII is the substrate for very long-chain acyl-CoA dehydrogenase (VLCAD), we examined the ACADVL gene in these patients by sequence analysis. RESULTS: Missense mutations within the ACADVL gene were identified in 3 of the patients. CONCLUSIONS: The locations of the altered amino acid residues within the crystal structure of VLCAD are on the surface of the molecule and may be involved in interactions with neighboring molecules. These findings support the importance of considering that mutations may be present in the ACADVL gene when a significant partial deficiency is found in CPTII activity, but no mutations in the CPT2 gene can be identified.

Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs.

Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.

1-O-Octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-GS-441524 (V2043). Evaluation of Oral V2043 in a Mouse Model of SARS-CoV-2 Infection and Synthesis and Antiviral Evaluation of Additional Phospholipid Esters with Enhanced Anti-SARS-CoV-2 Activity.

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.

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.

Solution structure of a DNA duplex containing the potent anti-poxvirus agent cidofovir.

Cidofovir (1(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that, when CDV is incorporated into a growing primer strand, it can inhibit both the 3'-to-5' exonuclease and the 5'-to-3' chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase, but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3'-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used (1)H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18 and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which were confirmed using NMR-monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower T(m) for the CDV DNA duplex (46 °C) compared to the control (58 °C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than that of the control, suggesting increased dynamics around the CDV.

Oral hexadecyloxypropyl-cidofovir therapy in pregnant guinea pigs improves outcome in the congenital model of cytomegalovirus infection.

Cytomegalovirus (CMV) infection is the leading cause of congenital infection, producing both sensorineural hearing loss and mental retardation. We evaluated the in vivo efficacy of an orally bioavailable analog of cidofovir, hexadecyloxypropyl-cidofovir (HDP-CDV), against guinea pig CMV (GPCMV) in a guinea pig model of congenital CMV infection. HDP-CDV exhibited antiviral activity against GPCMV with a 50% effective concentration (EC(50)) of 0.004 µM ± 0.001 µM. To evaluate in vivo efficacy, pregnant Hartley guinea pigs were inoculated with GPCMV during the late second/early third trimester of gestation. Animals were administered 20 mg HDP-CDV/kg body weight orally at 24 h postinfection (hpi) and again at 7 days postinfection (dpi) or administered 4 mg/kg HDP-CDV orally each day for 5 days or 9 days. Virus levels in dam and pup tissues were evaluated following delivery, or levels from dam, placenta, and fetal tissues were evaluated following sacrifice of dams at 10 dpi. All HDP-CDV regimens significantly improved pup survival, from 50 to 60% in control animals to 93 to 100% in treated animals (P ≤ 0.019). Treatment with 20 mg/kg HDP-CDV significantly reduced the viral load in pup spleen (P = 0.017) and liver (P = 0.029). Virus levels in the placenta were significantly reduced at 10 dpi following daily treatment with 4 mg/kg HDP-CDV for 5 or 9 days. The 9-day treatment also significantly reduced the viral levels in the dam spleen and liver. Although the 4-mg/kg treatment improved pup survival, virus levels in the fetal tissues were similar to those in control tissues. Taken together, HDP-CDV shows potential as a well-tolerated antiviral candidate for treatment of congenital human CMV (HCMV) infection.

Inhibition of HIV-1 by octadecyloxyethyl esters of (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] nucleosides and evaluation of their mechanism of action.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC [cidofovir]) and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of a variety of DNA viruses. These drugs possess a 3'-hydroxyl equivalent which could support chain extension from an incorporated drug molecule. HPMPC and HPMPA were initially reported to lack activity against human immunodeficiency virus type 1 (HIV-1); more recent results have shown that the octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of HPMPA are potent inhibitors of the virus. We have synthesized the ODE esters of a series of (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] (HPMP) nucleosides, including HPMPC, HPMP-guanine (HPMPG), HPMP-thymine (HPMPT), and HPMP-diaminopurine (HPMPDAP), as well as the ODE ester of the obligate chain terminator (S)-9-[3-methoxy-2-(phosphonomethoxy)-propyl]adenine (MPMPA). All compounds except ODE-HPMPT were inhibitors of HIV-1 replication at low nanomolar concentrations. These compounds were also inhibitors of the replication of HIV-1 variants that are resistant to various nucleoside reverse transcriptase (RT) inhibitors at concentrations several times lower than would be expected to be achieved in vivo. To investigate the mechanism of the antiviral activity, the active metabolites of HPMPC and HPMPA were studied for their effects on reactions catalyzed by HIV-1 RT. Incorporation of HPMPC and HPMPA into a DNA primer strand resulted in multiple inhibitory effects exerted on the enzyme and showed that neither compound acts as an absolute chain terminator. Further, inhibition of HIV-1 RT also occurred when these drugs were located in the template strand. These results indicate that HPMPC and HPMPA inhibit HIV-1 by a complex mechanism and suggest that this class of drugs has a broader spectrum of activity than previously shown.

Antiproliferative property of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) for unwanted ocular proliferation.

PURPOSE: To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) on ocular cell proliferation and collagen matrix contraction. METHODS: For the antiproliferation studies, various ocular cell monolayers were exposed to HDP-PMEG, PMEG, 5-fluorouracil (5-FU), and daunorubicin (DNB). For the collagen contraction studies, retinal pigment epithelium (RPE) cells seeded onto type I collagen lattices were exposed for a single 5- or 50-min period to various concentrations of HDP-PMEG or 5-FU. For the cytotoxicity study, trypan blue exclusion tests were performed using a human Müller cell line. Cytotoxicity was determined up to 4 days after treatment. RESULTS: The proliferation of RPE cells, scleral fibroblasts, vessel endothelial cells, and ocular melanoma cells can all be significantly inhibited by HDP-PMEG. Its inhibitory effects on those cells were uniformly stronger than that of 5-FU. Contraction of the collagen matrix containing RPE cells was significantly inhibited by HDP-PMEG and by 5-FU at concentrations of 20 µM and 2,000 µM, respectively, as compared with controls (p<0.05). The safety profile of HDP-PMEG was significantly better than 5-FU and daunorubicin. The ocular therapeutic index is 1,100 for HDP-PMEG, 17.2 for 5-FU, and 1.25 for daunorubicin. CONCLUSIONS: HDP-PMEG possesses a significant inhibitory effect on the proliferation of RPE, retinal glial cells, scleral fibroblasts, and ocular melanoma cells. HDP-PMEG is also genotoxic and may be used as a single short application for the modulation of unwanted ocular proliferation.

Synthesis and antiviral evaluation of 9-(S)-[3-alkoxy-2-(phosphonomethoxy)propyl]nucleoside alkoxyalkyl esters: inhibitors of hepatitis C virus and HIV-1 replication.

We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This is surprising because acyclic nucleoside phosphonates have been regarded as having antiviral activity only against double stranded DNA viruses, HIV and HBV. We synthesized octadecyloxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]-adenine and found it to be active in genotype 1b and 2a HCV replicons with EC50 values of 1-2 µM and a CC50 of > 150 µM. Analogs with substitutions at the 3'-hydroxyl larger than methyl or ethyl, or with other purine bases were less active but most compounds had significant antiviral activity against HIV-1 in vitro. The most active anti-HIV compound was octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]guanine with an EC50 < 0.01 nanomolar and a selectivity index of > 4.4 million.

Synthesis, metabolic stability and antiviral evaluation of various alkoxyalkyl esters of cidofovir and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine.

Alkoxyalkyl esters of cidofovir (CDV) are orally active agents which inhibit the replication of a variety of double stranded DNA (dsDNA) viruses including variola, vaccinia, ectromelia, herpes simplex virus, cytomegalovirus, adenovirus and others. One of these compounds, hexadecyloxypropyl-CDV (HDP-CDV, CMX001) is in clinical development for prevention and treatment of poxvirus infection, vaccination complications, and for infections caused by cytomegalovirus, adenovirus, herpesviruses and other dsDNA viruses. This class of lipid analogs is potentially prone to undergo omega oxidation of the alkyl moiety which can lead to a short chain carboxylic acid lacking antiviral activity. To address this issue, we synthesized a series of alkoxyalkyl or alkyl glycerol esters of CDV and (S)-HPMPA having modifications in the structure of the alkyl residue. Antiviral activity was assessed in cells infected with vaccinia, cowpox or ectromelia viruses. Metabolic stability was determined in S9 membrane fractions from rat, guinea pig, monkey and human liver. All compounds had substantial antiviral activity in cells infected with vaccinia, cowpox or ectromelia. Metabolic stability was lowest in monkey liver S9 incubations where rapid disappearance of HDP-CDV and HDP-(S)-HPMPA was noted. Metabolic stability in monkey preparations increased substantially when a ω-1 methyl group (15-methyl-HDP-CDV) or a terminal cyclopropyl residue (14-cyclopropyl-tetradecyloxypropyl-CDV) was present in the alkyl chain. The most stable compound was 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV) which was not metabolized extensively by monkey liver S9. In rat, guinea pig or human liver S9 incubations, most of the modified antiviral compounds were considerably more stable.

Broad-Spectrum In Vitro Antiviral Activity of ODBG-P-RVn: An Orally-Available, Lipid-Modified Monophosphate Prodrug of Remdesivir Parent Nucleoside (GS-441524).

The necessity for intravenous administration of remdesivir confines its utility for treatment of coronavirus disease 2019 (COVID-19) to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524), against viruses that cause diseases of human public health concern, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had activity nearly equivalent to that of remdesivir in primary-like human small airway epithelial cells. Our results warrant in vivo efficacy evaluation of ODBG-P-RVn. IMPORTANCE While remdesivir remains one of the few drugs approved by the FDA to treat coronavirus disease 2019 (COVID-19), its intravenous route of administration limits its use to hospital settings. Optimizing the stability and absorption of remdesivir may lead to a more accessible and clinically potent therapeutic. Here, we describe an orally available lipid-modified version of remdesivir with activity nearly equivalent to that of remdesivir against emerging viruses that cause significant disease, including Ebola and Nipah viruses. Our work highlights the importance of such modifications to optimize drug delivery to relevant and appropriate human tissues that are most affected by such diseases.

Broad-spectrum in vitro antiviral activity of ODBG-P-RVn: an orally-available, lipid-modified monophosphate prodrug of remdesivir parent nucleoside (GS-441524).

The intravenous administration of remdesivir for COVID-19 confines its utility to hospitalized patients. We evaluated the broad-spectrum antiviral activity of ODBG-P-RVn, an orally available, lipid-modified monophosphate prodrug of the remdesivir parent nucleoside (GS-441524) against viruses that cause diseases of human public health concern, including SARS-CoV-2. ODBG-P-RVn showed 20-fold greater antiviral activity than GS-441524 and had near-equivalent activity to remdesivir in primary-like human small airway epithelial cells. Our results warrant investigation of ODBG-P-RVn efficacy in vivo.

Rethinking Remdesivir: Synthesis, Antiviral Activity and Pharmacokinetics of Oral Lipid Prodrugs.

Remdesivir (RDV, GS-5734) is currently the only FDA-approved antiviral drug for the treatment of SARS CoV-2 infection. The drug is approved for use in adults or children 12-years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2 infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed and several including molnupiravir and PF-07321332 are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of Remdesivir nucleoside (RVn, GS-441524) that are processed to RVn-monophosphate, the precursor of the active RVn-triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells and Huh7.5 cells. In Syrian hamsters oral treatment with ODBG-P-RVn was well tolerated and achieved therapeutic levels in plasma above the EC90 for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.

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.

Antischistosomal activity of hexadecyloxypropyl cyclic 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine and other alkoxyalkyl esters of acyclic nucleoside phosphonates assessed by schistosome worm killing in vitro.

9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] has been reported to have antischistosomal activity. Ether lipid esters of (S)-HPMPA and cidofovir (CDV) have greatly increased activities in antiviral assays and in lethal animal models of poxvirus diseases. To see if ether lipid esters of CDV and (S)-HPMPA enhance antischistosomal activity, we tested their alkoxyalkyl esters using Schistosoma mansoni worm killing in vitro. Hexadecyloxypropyl (HDP)-cyclic-(S)-HPMPA and HDP-cyclic-CDV exhibited significant in vitro antischistosomal activities and may offer promise alone or in combination with praziquantel.

The octadecyloxyethyl ester of (S)-9-[3-hydroxy-2-(phosphonomethoxy) propyl]adenine is a potent and selective inhibitor of hepatitis C virus replication in genotype 1A, 1B, and 2A replicons.

The octadecyloxyethyl (ODE) and hexadecyloxypropyl (HDP) esters of (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) are potent inhibitors of orthopoxvirus, herpesvirus, human immunodeficiency virus type 1, and hepatitis B virus replication in vitro. HDP and ODE esters of (S)-HPMPA and (R)-HPMPA were evaluated for their activity in hepatitis C virus (HCV) replicon assays using luciferase (1B and 2A replicons) or RNA (1B) quantification. The ODE ester of (S)-HPMPA [ODE-(S)-HPMPA] was the most active compound, with 50% effective concentrations (EC(50)s) in the 0.69 to 1.31 microM range. HDP and ODE esters of (R)-HPMPA were severalfold less active, while (S)-HPMPA and (R)-HPMPA were inactive. In genotype 1A and 1B replicons analyzed by HCV RNA analysis, ODE-(S)-HPMPA was the most active compound, with EC(50)s of 1.8 and 2.1 microM, respectively.

Alkoxyalkyl esters of 9-(s)-(3-hydroxy-2-phosphonomethoxypropyl) adenine are potent and selective inhibitors of hepatitis B virus (HBV) replication in vitro and in HBV transgenic mice in vivo.

Alkoxyalkyl esters of acyclic nucleoside phosphonates have previously been shown to have increased antiviral activity when they are administered orally in animal models of viral diseases, including lethal infections with vaccinia virus, cowpox virus, ectromelia virus, murine cytomegalovirus, and adenovirus. 9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] was previously shown to have activity against hepatitis B virus (HBV) in vitro. To assess the effect of alkoxyalkyl esterification of (S)-HPMPA, we prepared the hexadecyloxypropyl (HDP), 15-methyl-hexadecyloxypropyl (15M-HDP), and octadecyloxyethyl (ODE) esters and compared their activities with the activity of adefovir dipivoxil in vitro and in vivo. Alkoxyalkyl esters of (S)-HPMPA were 6 to 20 times more active than unmodified (S)-HPMPA on the basis of their 50% effective concentrations in 2.2.15 cells. The increased antiviral activity appeared to be due in part to the increased uptake and conversion of HDP-(S)-HPMPA to HPMPA diphosphate observed in HepG2 cells in vitro. HDP-(S)-HPMPA retained full activity against HBV mutants resistant to lamivudine (L180M, M204V), but cross-resistance to a mutant resistant to adefovir (N236T) was detected. HDP-(S)-HPMPA is orally bioavailable and provides excellent liver exposure to the drug. Oral treatment of HBV transgenic mice with HDP-(S)-HPMPA, 15M-HDP-(S)-HPMPA, and ODE-(S)-HPMPA for 14 days reduced liver HBV DNA levels by roughly 1.5 log units, a response equivalent to that of adefovir dipivoxil.

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.