Cidofovir Diphosphate Research Articles

Peripheral blood mononuclear cells isolation from mouse and rabbit blood to quantify active nucleotide and define brincidofovir dose for smallpox.

Aim: Brincidofovir has been approved by the US FDA for the treatment of smallpox via the "Animal Rule". The active moiety, cidofovir diphosphate (CDV-PP), was measurable in human peripheral blood mononuclear cells (PBMCs), but quantitation in animals was challenging given their limited blood volume. The aim of this study was to optimize PBMC isolation from rabbit and mouse blood to allow quantitation of CDV-PP.Materials & methods: PBMC isolation methods using various separation media were evaluated using blood from naive and brincidofovir-dosed animals. CDV-PP was quantified using liquid chromatography tandem mass spectrometry.Results & conclusion: PBMC isolation yields were increased by >fourfold compared with yields obtained using human methods, allowing cross-species exposure comparisons.

Structural basis of human mpox viral DNA replication inhibition by brincidofovir and cidofovir

Mpox virus has wildly spread over 108 non-endemic regions in the world since May 2022. DNA replication of mpox is performed by DNA polymerase machinery F8-A22-E4, which is known as a great drug target. Brincidofovir and cidofovir are reported to have broad-spectrum antiviral activity against poxviruses, including mpox virus in animal models. However, the molecular mechanism is not understood. Here we report cryogenic electron microscopy structures of mpox viral F8-A22-E4 in complex with a DNA duplex, or dCTP and the DNA duplex, or cidofovir diphosphate and the DNA duplex at resolution of 3.22, 2.98 and 2.79 Å, respectively. Our structural work and DNA replication inhibition assays reveal that cidofovir diphosphate is located at the dCTP binding position with a different conformation to compete with dCTP to incorporate into the DNA and inhibit DNA synthesis. Conformation of both F8-A22-E4 and DNA is changed from the pre-dNTP binding state to DNA synthesizing state after dCTP or cidofovir diphosphate is bound, suggesting a coupling mechanism. This work provides the structural basis of DNA synthesis inhibition by brincidofovir and cidofovir, providing a rational strategy for new therapeutical development for mpox virus and other pox viruses.

Anti-Monkeypox Infection Approaches: From Prevention to Therapeutic Lines.

Anti-Monkeypox Infection Approaches: From Prevention to Therapeutic Lines.

A quantitative LC–MS/MS method for the determination of tissue brincidofovir and cidofovir diphosphate in a MuPyV‐infected mouse model

Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C18 (50 × 2.1 mm, 3.5 μm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 μm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.

A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2′ or 3′ modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3′-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2′-OMe-UTP), and 3 did not terminate the polymerase reaction (2′-F-dUTP, 2′–NH2–dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2′-OH at the 3′-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2′-OH, have a blocked 2′-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.

Cidofovir Diphosphate Inhibits Adenovirus 5 DNA Polymerase via both Nonobligate Chain Termination and Direct Inhibition, and Polymerase Mutations Confer Cidofovir Resistance on Intact Virus.

Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC50) for BCV and a 1.9-fold increased EC50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity.

The efficacy and pharmacokinetics of brincidofovir for the treatment of lethal rabbitpox virus infection: A model of smallpox disease

Brincidofovir (BCV) has broad-spectrum in vitro activity against dsDNA viruses, including smallpox, and is being developed as a treatment for smallpox as well as infections caused by other dsDNA viruses. BCV has previously been shown to be active in multiple animal models of smallpox. Here we present the results of a randomized, blinded, placebo-controlled study of the efficacy and pharmacokinetics of a novel, “humanized” regimen of BCV for treatment of New Zealand White rabbits infected with a highly lethal inoculum of rabbitpox virus, a well characterized model of smallpox. Compared with placebo, a dose-dependent increase in survival was observed in all BCV-treatment groups. Concentrations of cidofovir diphosphate (CDV-PP), the active antiviral, in rabbit peripheral blood mononuclear cells (PBMCs) were determined for comparison to those produced in humans at the dose proposed for treatment of smallpox. CDV-PP exposure in PBMCs from rabbits given BCV scaled to human exposures at the dose proposed for treatment of smallpox, which is also currently under evaluation for other indications. The results of this study demonstrate the activity of BCV in the rabbitpox model of smallpox and the feasibility of scaling doses efficacious in the model to a proposed human dose and regimen for treatment of smallpox.

(399) - Emerging Combination Activity and Resistance Profile for Brincidofovir (CMX001) in CMV Prevention and Treatment

Brincidofovir (CMX001, BCV) is a broad-spectrum oral antiviral in Phase 3 development for prevention of CMV in hematopoietic cell transplantation. BCV is converted intracellularly to cidofovir diphosphate which acts as an alternative substrate inhibitor for the CMV viral DNA polymerase (UL54, pol). The combination activity and resistance profile of BCV was evaluated in vitro and in two completed clinical trials; Study 201enrolled CMV antiviral naïve subjects and Study 350 enrolled antiviral therapy experienced patients.

First Pharmacokinetic and Safety Study in Humans of the Novel Lipid Antiviral Conjugate CMX001, a Broad-Spectrum Oral Drug Active against Double-Stranded DNA Viruses

CMX001 is a novel, broad-spectrum lipid antiviral conjugate (LAC) that produces high intracellular levels of the active antiviral agent cidofovir diphosphate (CDV-PP). Study CMX001-102 was a randomized, double-blind, placebo-controlled, parallel group, dose-escalating study in healthy volunteers. The objectives of the study were to evaluate the safety and pharmacokinetic parameters of CMX001 after single and multiple doses. Single doses ranging from 0.25 to 2.0 mg/kg of body weight and multiple doses ranging from 0.1 to 1.0 mg/kg (3 total doses, administered every 6 days) were given orally. Safety was assessed using comprehensive clinical and laboratory evaluations, including enhanced monitoring for potential gastrointestinal (GI) effects using wireless capsule endoscopy (WCE). Serial plasma and pooled urine samples were collected to estimate pharmacokinetic parameters for both CMX001 and cidofovir (CDV). No adverse events occurred that prevented dose escalation. No clinically significant drug-related changes in blood chemistry, hematology, renal function, or intraocular pressure were observed. No CMX001-related gastrointestinal mucosal changes were observed by WCE. CMX001 was absorbed rapidly, with maximum plasma concentrations observed 2 to 3 h postdose. Maximum plasma drug concentration and systemic exposure of CMX001 increased approximately in proportion to dose following single and multiple doses; no significant accumulation of CMX001 or CDV was observed following multiple doses. We conclude that CMX001 is orally bioavailable and well tolerated in healthy volunteers at doses up to 2 mg/kg, approximately 140 mg in a typical adult. This is the first demonstration of the use of phospholipid conjugation technology to achieve plasma drug exposures that are expected to result in activity against multiple double-stranded DNA viruses.

Intraocular Safety and Pharmacokinetics of Hexadecyloxypropyl-Cidofovir (HDP-CDV) as a Long-lasting Intravitreal Antiviral Drug

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. 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. 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. 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.

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.

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.

Mechanism of Inhibition of Vaccinia Virus DNA Polymerase by Cidofovir Diphosphate

Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primer-template pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV + 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3' terminus by the 3'-to-5' proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3' residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.

Expression and purification of recombinant Kaposi’s sarcoma-associated herpesvirus DNA polymerase using a Baculovirus vector system

The DNA polymerase (POL) of Kaposi’s sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72 h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA–cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.

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.

Characterization of wild-type and cidofovir-resistant strains of camelpox, cowpox, monkeypox, and vaccinia viruses.

Cidofovir ([(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] [HPMPC])-resistant forms of camelpox, cowpox, monkeypox, and vaccinia viruses were developed by prolonged passage in Vero 76 cells in the presence of drug. Eight- to 27-fold-higher concentrations of cidofovir were required to inhibit the resistant viruses than were needed to inhibit the wild-type (WT) viruses. Resistant viruses were characterized by determining their cross-resistance to other antiviral compounds, examining their different replication abilities in two cell lines, studying the biochemical basis of their drug resistance, and assessing the degrees of their virulence in mice. These viruses were cross resistant to cyclic HPMPC and, with the exception of vaccinia virus, to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. Three of the four resistant cowpox and monkeypox viruses exhibited reduced abilities to infect and replicate in 3T3 cells compared to their abilities in Vero 76 cells. Compared to the WT virus polymers the resistant cowpox virus DNA polymerase was 8.5-fold less sensitive to inhibition by cidofovir diphosphate, the active form of the drug. Intracellular phosphorylation of [3H]cidofovir was not stimulated or inhibited by infection with resistant cowpox virus. In intranasally infected BALB/c mice, WT cowpox virus was 80-fold more virulent than the resistant virus. Cidofovir treatment (100 mg/kg of body weight, given one time only as early as 5 min after virus challenge) of a resistant cowpox virus infection could not protect mice from mortality. However, the drug prevented mortality in 80 to 100% of the mice treated with a single 100-mg/kg dose at 1, 2, 3, or 4 days after WT virus challenge. By application of these results to human orthopoxvirus infections, it is anticipated that resistant viruses may be untreatable with cidofovir but their virulence may be attenuated. Studies will need to be conducted with cidofovir-resistant monkeypox virus in monkeys to further support these hypotheses.

Interactions of 1-[(S)-3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine (Cidofovir) Diphosphate with DNA Polymerases α, δ and ε*

The inhibitory and/or substrate activity of 1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC, cidofovir, Vistide™] diphosphate towards eukaryotic DNA polymerases α, δ and ε* was examined. Cidofovir diphosphate is a weak competitive inhibitor of the above enzymes, approximately 3 to 7 times weaker than its adenine analogue (S)-HPMPApp. The enzymes also catalyze incorporation of (S)-HPMPC into DNA; after insertion of one (S)-HPMPC residue into DNA, another dNMP residue may incorporate. DNA polymerase δ and ε* can successively accommodate in the growing chain two (S)-HPMPC residues at the maximum, whereas pol α up to three residues.

Isolation and identification of a metabolite of cidofovir from rat kidney

Cidofovir is an acyclic nucleotide analog with potent and broad-spectrum antiviral activity against adenoviruses and herpesviruses including cytomegalovirus (CMV). Cidofovir undergoes intracellular phosphorylation by host enzymes to cidofovir phosphate and cidofovir diphosphate (the active form). An unidentified metabolite has been observed previously in rat tissues and in urine of rabbits, rats and monkeys dosed with cidofovir. In the present study, this metabolite was isolated from rat kidney following an intravenous dose of 100 mg kg −1 cidofovir. The metabolite (metabolite I) was separated from cidofovir and impurities using extraction on anion-exchange resin followed by preparative normal and reversed-phase high-performance liquid chromatography (HPLC). The isolated metabolite I was subjected to proton, 13C and phosphorus nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization mass spectroscopy, and confirmed to be cidofovir–phosphocholine. The uptake of cidofovir by rat kidney was saturated at an intravenous dose of 100 mg kg −1, probably as a result of saturation of the renal tubular secretion pathway. However, the relative abundance of cidofovir phosphocholine was not affected by dose.

Expression of the Catalytic Subunit (UL54) and the Accessory Protein (UL44) of Human Cytomegalovirus DNA Polymerase in a Coupledin VitroTranscription/Translation System

The catalytic subunit (UL54) and accessory protein (UL44) of human cytomegalovirus (HCMV) DNA polymerase have been cloned and expressed in anin vitro-coupled transcription/translation reticulocyte lysate system. The influence of the 5′-untranslated region (5′-UTR) on the efficiency of expression from the circular plasmids has been investigated. For expression of both UL54 and UL44, a truncated form of the alfalfa mosaic virus (AMV) RNA4 5′-UTR was found to be superior to the full-length AMV 5′-UTR or the original HCMV 5′-UTRs of different lengths. Protein products withMr≈ 140 and 55 kDa were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis in the UL54 and UL44in vitroexpression reactions, respectively. The properties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3′–5′ exonuclease activities of the expressed UL54/UL44 complex were found to be dependent on salt concentration in the same manner as the activities of the native enzyme. Thein vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidofovir diphosphate, ganciclovir triphosphate, and foscarnet). This straightforward method for protein expression may also be applicable to other enzymes where reproducible generation of fully functional products is desirable.

Metabolism of ganciclovir and cidofovir in cells infected with drug-resistant and wild-type strains of murine cytomegalovirus

Murine cytomegalovirus (MCMV) has been used extensively as an animal model for human cytomegalovirus (HCMV). Understanding drug resistance and its treatment in MCMV may lead to more effective treatments of HCMV disease. Most ganciclovir-resistant HCMV clinical isolates exhibit a decreased capacity to induce ganciclovir phosphorylation (to its biologically active form) in infected cells. Using an MCMV strain resistant to both ganciclovir and cidofovir, the intracellular metabolism of these drugs was studied to determine if MCMV resistance correlates with decreases in drug phosphorylation. The wild-type (WT) MCMV used for comparison was inhibited in plaque reduction assays, by ganciclovir and cidofovir by 50% at 5.1 and 0.24 μM, respectively; the resistant strain was inhibited at 72 and 2.7 μM, respectively. In uninfected, WT, or resistant virus-infected cells, the extent of metabolism of 10 μM ganciclovir or 1 μM cidofovir to intracellular triphosphorylated species was similar. Phosphorylation and catabolism (following drug removal) rates over time were also similar. Intracellular levels of ganciclovir triphosphate and cidofovir diphosphate increased less than two-fold with increasing multiplicity of virus infection. Because few differences in drug phosphorylation between WT and resistant virus-infected cells were found, virus resistance to ganciclovir and cidofovir apparently is not linked to altered drug phosphorylation. Since the viral DNA polymerase is the antiviral target for these compounds, the resistant MCMV is most likely a DNA polymerase mutant.