Quenelle DC;Collins DJ;Wan WB;Beadle JR;Hostetler KY;Kern ER; Antimicrobial Agents and Chemotherapy. 2004 Feb; 48(2):404-412.

Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log(10). In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.

Aldern KA;Ciesla SL;Winegarden KL;Hostetler KY; Molecular Pharmacology 2003 Mar; 63(3):678-681.

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.

Kern ER;Hartline C;Harden E;Keith K;Rodriguez N;Beadle JR;Hostetler KY; Antimicrobial Agents and Chemotherapy. 2002 Apr; 46(4):991-995.

The nucleotide phosphonates cidofovir (CDV) and cyclic cidofovir (cCDV) are potent antiviral compounds when administered parenterally but are not well absorbed orally. These compounds have been reported to have activity against orthopoxvirus replication in vitro and in animal models when administered parenterally or by aerosol. To obtain better oral activity, we synthesized a novel series of analogs of CDV and cCDV by esterification with two long-chain alkoxyalkanols, 3-hexadecyloxy-1-propanol (HDP-CDV; HDP-cCDV) or 3-octadecyloxy-1-ethanol (ODE-CDV; ODE-cCDV). Their activities were evaluated and compared with those of CDV and cCDV in human foreskin fibroblast (HFF) cells infected with vaccinia virus (VV) or cowpox virus (CV) using a plaque reduction assay. The 50% effective concentrations (EC(50)s) against VV in HFF cells for CDV and cCDV were 46.2 and 50.6 microM compared with 0.84 and 3.8 microM for HDP-CDV and HDP-cCDV, respectively. The EC(50)s for ODE-CDV and ODE-cCDV were 0.20 and 1.1 microM, respectively. The HDP analogs were 57- and 13-fold more active than the parent nucleotides, whereas the ODE analogs were 231- and 46-fold more active than the unmodified CDV and cCDV. Similar results were obtained using CV. Cytotoxicity studies indicated that although the analogs were more toxic than the parent nucleotides, the selective index was increased by 4- to 13-fold. These results indicate that the alkoxyalkyl esters of CDV and cCDV have enhanced activity in vitro and need to be evaluated for their oral absorption and efficacy in animal models.

Ciesla SL, Trahan J, Wan WB, Beadle JR, Aldern KA, Painter GR, Hostetler KY; Antiviral Research 2003 Aug; 59(3):163-171.

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.

Lebeau, I; Andrei, A; Dal Pozzo, F; Beadle, JR; Hostetler, KY; De Clerq, E; van den Oord, J; and Snoeck, R; Antimicrobial Agents and Chemotherapy 2006 July; 50:2525-29.

The potencies of several alkoxyalkyl esters of acyclic nucleoside phosphonates against vaccinia virus and cowpox virus were evaluated in cell monolayers and tree-dimensional epithelial raft cultures. Prodrugs were at least 20-fold more active than their parent compounds. Octadecyloxyethyl-(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine emerged as the most potent derivative.

George Painter, Bernhard Lampert, Lawrence C Trost, Neil Frazer, and Alice Robertson ICAR April 13—17, 2008 Montreal, Quebec, Canada

Cidofovir (CDV), which is approved for treatment of cytomegalovirus induced retinitis in AIDS patients, is active in vitro against all five families of human dsDNA viruses. However, the clinical utility of CDV is limited by the requirement for administration by intravenous infusion and the frequency of acute nephrotoxicity. The hexadecyl-oxypropyl lipid conjugate increases in vitro antiviral activity up to a thousand-fold, promotes high oral bioavailability and has shown no indication of nephrotoxicity. CMX001 is currently in Phase I clinical trials for the prophyaxis and treatment of smallpox infection and is entering development for treatment of
dsDNA viral infections that cause graft loss in transplant patients. In vitro activity (EC50) against variola major and monkeypox virus ranged from 0.04 to 0.10 ?M and from 0.013 to 0.07 ?M, respectively, depending on cell line. The EC50 for ectromelia virus was 0.5 ?M. Human CMV EC50s ranged from 0.9-20 nM for a variety of wild-type, CDV, and ganciclovir resistant
isolates. Representative EC50 values for the other human dsDNA viruses include AdV5 (0.009 ?M), HPV18 (0.42 ?M), and BK virus (0.13 ?M). The activity of CMX001 has been extensively characterized in mice infected with cowpox, vaccinia, ectromelia, and CMV viruses, and in rabbits infected with rabbitpox virus. Notably, one 20 mg/kg oral dose of CMX001 administered four days post infection provided 100% protection against lethal ectromelia infection in mice. Treatment experiments in rabbits have also shown that CMX001 can be given late into the infection cycle after signs of disease are apparent (fever and lesions) and still prevent mortality. A Phase I placebo controlled study to evaluate, single-dose, dose-escalation, safety and pharmacokinetics of CMX001 (CMX001-102) in healthy volunteers is underway.
The first six cohorts of the study (25, 50, 100, 200, 400, and 600 ?g/kg) have been completed.
The blinded pharmacokinetic data suggest that increases in plasma levels of CMX001 are dose proportional; Cmax ranged from 2.36 ng/ml at the lowest dose to 114.73 ng/ml at the highest dose, while AUC 0-00 ranged from 18.51 ng.hr/ml to 728.8 ng.hr/ml. No serious adverse events have been observed. The study is ongoing and currently enrolling subjects into multiple dose cohorts.

Beadle JR;Hartline C;Aldern KA;Rodriguez N;Harden E;Kern ER;Hostetler KY; Antimicrobial Agents and Chemotherapy. 2002 Aug; 46(8):2381-2386.

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.

Deborah J. Bidanset, James R. Beadle, W. Brad Wan, Karl Y. Hostetler, and Earl R. Kern

Infection with human cytomegalovirus (HCMV) can cause serious complications in bone-marrow and solid organ transplant recipients, and current therapies are not optimal. We evaluated 2 orally active ether lipidester analogues of cidofovir (CDV)—hexadecyloxypropyl-CDV (HDP-CDV) and octadecyloxyethyl-CVD(ODECDV)— in severe combined immunodeficient mice in which either human fetal retinal tissue or human fetal thymus and liver tissue had been implanted and was later infected with HCMV. Our results indicate that orally administered treatment with either HDP-CDV or ODE-CDV is 4–8-fold more active, on a molar basis, than is intraperitoneally administered CDV. These data suggest that HDP-CDV and ODE-CDV should be further evaluated as potential antiviral agents for treatment of HCMV infection.

Parmjeet Randhawa, Noush Afarin Farasati, Ron Shapiro, and Karl Y. Hostetler

Polyomavirus BK is a significant pathogen in transplant recipients, but no effective antiviral therapy is available. We show that cidofovir can inhibit BK virus replication in vitro. Esterification of cidofovir with hexadecyloxypropyl, octadecyloxyethyl, and oleyloxyethyl groups results in up to a 3-log lowering of the 50% effective concentration and an increased selectivity index.

Stephanie L. Williams-Aziz, Caroll B. Hartline, Emma A. Harden, Shannon L. Daily,
Mark N. Prichard, Nicole L. Kushner, James R. Beadle, W. Brad Wan,
Karl Y. Hostetler, and Earl R. Kern

Cidofovir (CDV) is an effective therapy for certain human cytomegalovirus (HCMV) infections in immunocompromised patients that are resistant to other antiviral drugs, but the compound is not active orally. To improve oral bioavailability, a series of lipid analogs of CDV and cyclic CDV (cCDV), including hexadecyloxypropyl- CDV and -cCDV and octadecyloxyethyl-CDV and -cCDV, were synthesized and found to have multiple-log-unit enhanced activity against HCMV in vitro. On the basis of the activity observed with these analogs, additional lipid esters were synthesized and evaluated for their activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus, murine cytomegalovirus, varicella-zoster virus (VZV), Epstein- Barr virus (EBV), human herpesvirus 6 (HHV-6), and HHV-8. Using several different in vitro assays, concentrations of drug as low as 0.001 _M reduced herpesvirus replication by 50% (EC50) with the CDV analogs, whereas the cCDV compounds were generally less active. In most of the assays performed, the EC50 values of the lipid esters were at least 100-fold lower than the EC50 values for unmodified CDV or cCDV. The lipid analogs were also active against isolates that were resistant to CDV, ganciclovir, or foscarnet. These results indicate that the lipid ester analogs are considerably more active than CDV itself against HSV, VZV, CMV,EBV, HHV-6, and HHV-8 in vitro, suggesting that they may have potential for the treatment of infections caused by a variety of herpesviruses.

Karl Y. Hostetler, Steffney Rought, Kathy A. Aldern, Julissa Trahan, James R. Beadle, and Jacques Corbeil

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 activethan unmodified cidofovir in vitro against various doublestranded DNA viruses, including cytomegalovirus, herpes simplex virus, adenoviruses, cowpox, vaccinia, and variola viruses. We compared the activity of octadecyloxyethylcidofovir (ODE-CDV) and oleyloxyethyl-cidofovir (OLECDV) 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. ODECDV 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.

George R. Painter, Merrick R. Almond, Lawrence C. Trost, Bernhard M. Lampert, Johan Neyts, Erik De Clercq, Brent E. Korba, Kathy A. Aldern, James R. Beadle, and Karl Y. Hostetler

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 (EC50s) 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 EC50, 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.

ER Lanier B Lampert,  Trost, G Painter and M Almond, Chimerix Inc, Research Triangle Park, NC, USA

AUTHOR CONCLUSIONS/DISCUSSION

CMX157 is designed to improve bioavailability and cellular penetration of tenofovir while decreasing peripheral exposure to TFV per se.  The greatly diminished IC₅₀s for CMX157 versus TFV suggested that higher intracellular levels of the active anabolite, TFV-PP, were being made following exposure to similar molar equivalents.  We tested this in human PBMCs using dilutions of both drugs which bracketed the Cmax of TFV in humans following standard dosing of TDF⁴.  The TFV Cmax is approximately 1 µM (1167 nM) following 300 mg dosing of TDF.  PHA/IL-2 activated human PBMCs exposed to 10 nM CMX157 contained more TFV-PP (70 fmol/10⁶) than those exposed to 1000 nM TFV (50 fmol/10⁶).  Notably, these levels were similar to those seen in patients taking TDF (median 76 fmol/10⁶). These data in combination with a favorable toxicology profile support further development of CMX157 for HIV.

1) CMX157 is >300 fold more potent than TFV in vitro against wt HIV and against clinically relevant HIV mutants

 2) The amount of active drug produced in human PBMCs is much higher following equimolar exposure to CMX157 vs TFV

3) Toxicology and TK data reveal high exposure to CMX157 in plasma with minimal toxicity (rodent and non-rodent)

 INTRODUCTION

CMX157 is a lipid (1-0-hexadecyloxypropyl, HDP) conjugate of the acyclic nucleotide analogue tenofovir. Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir (TFV) is one of the most widely used nucleoside/tide reverse transcriptase inhibitors (NRTIs).  However, it loses activity against specific HIV mutants, including those with K65R, multiple thymidine analog mutations (TAMs) or multi-NRTI resistant (MNR) mutations and has been associated with nephrotoxicity.  Our goal is to increase efficacy and decrease toxicity of TFV.  The approach we have taken is to create a prodrug that is more stable in plasma, but effectively penetrates cells and yields high intracellular levels of the active anabolite, TFV-diphosphate (TFV-PP). Unlike tenofovir-DF and most prodrugs, CMX157 is not efficiently cleaved to free tenofovir in vivo.   This should both increase the levels of active TFV-PP in target cells for HIV and decrease the levels of free TFV in blood, potentially lowering the apparent IC₅₀ and reducing the rate of secretion into the kidney.

Chimerix's lipid conjugate technology employs covalent coupling of a synthetic lipid to drugs, in this case via a phosphonate moiety². The goal of lipid conjugation is to increase oral bioavailability, increase potency and target delivery to specific tissues. The novel molecules are designed to resemble natural lipids that are readily absorbed from the small intestine and distributed to tissues via lymph and/or plasma. Hydrophobic lipid conjugates may be incorporated into chylomicrons released into the lymphatic system and ultimately venous blood while hydrophilic conjugates may be absorbed and pass directly into portal blood. Once in the cell the drug is cleaved from the lipid carrier by hydrolytic action of phospholipases on the phosphorous-O-alkyl bond³.