Safety, Pharmacokinetics, and Antiretroviral Activity of Intravenous 9-[2-(R)-(Phosphonomethoxy)propyl]adenine, a Novel Anti-Human Immunodeficiency Virus (HIV) Therapy, in HIV-Infected Adults

9-[2-(R)-(Phosphonomethoxy)propyl]adenine (PMPA) is a nucleotide analogue with potent antiretroviral activity in vitro and in simian models. A randomized, double-blind, placebo-controlled, dose-escalation clinical trial of intravenous PMPA monotherapy was conducted in 20 human immunodeficiency virus (HIV)-infected adults with CD4 cell counts of ≥200 cells/mm³ and plasma HIV RNA levels of ≥10,000 copies/ml. Two dose levels were evaluated (1 and 3 mg/kg of body weight/day). Ten subjects were enrolled at each dose level (eight randomized to receive PMPA and two randomized to receive placebo). On day 1, a single dose of PMPA or placebo was administered by intravenous infusion. Beginning on study day 8, PMPA or placebo was administered once daily for an additional 7 consecutive days. All subjects tolerated dosing without significant adverse events. Mean peak serum PMPA concentrations were 2.7 ± 0.9 and 9.1 ± 2.1 μg/ml in the 1- and 3-mg/kg cohorts, respectively. Serum concentrations declined in a biexponential fashion, with a terminal half-life of 4 to 8 h. At 3 mg/kg/day, a single infusion of PMPA resulted in a 0.4 log₁₀ median decline in plasma HIV RNA by study day 8. Following 7 consecutive days of study drug administration thereafter, the median changes in plasma HIV RNA from baseline were −1.1, −0.6, and 0.1 log₁₀ in the 3-mg/kg/day, 1-mg/kg/day, and placebo dose groups, respectively. Following the final dose in the 3-mg/kg/day cohort, the reduction in HIV RNA was sustained for 7 days before returning toward baseline. Further studies evaluating an oral prodrug of PMPA are under way.     

Hydroxyurea Enhances the Activities of Didanosine, 9-[2-(Phosphonylmethoxy)ethyl]adenine, and 9-[2-(Phosphonylmethoxy)propyl]adenine against Drug-Susceptible and Drug-Resistant Human Immunodeficiency Virus Isolates

We assessed the effects of hydroxyurea (HU) at a concentration of 50 microM on the in vitro activities of 2',3'-dideoxyinosine (ddI), 9-[2-(phosphonylmethoxy)ethyl]adenine (PMEA), and 9-[2-(phosphonylmethoxy)propyl]adenine (PMPA) against a wild-type human immunodeficiency virus (HIV) type 1 (HIV-1) laboratory isolate and a panel of five well-characterized drug-resistant HIV isolates. Fifty micromolar HU significantly increased the activities of ddI, PMEA, and PMPA against both the wild-type and the drug-resistant HIV-1 isolates. In fixed combinations, both ddI and PMEA were synergistic with HU against wild-type and drug-resistant viruses.     

The Antiherpesvirus Activity of H2G [(R)-9-[4-Hydroxy-2-(Hydroxymethyl)Butyl]Guanine] Is Markedly Enhanced by the Novel Immunosuppressive Agent Mycophenolate Mofetil

Mycophenolate mofetil (MMF) has been approved as an immunosuppressive agent in kidney transplant recipients and may thus be used concomitantly with antiherpetic agents, which are used for the treatment of intercurrent herpesvirus infections. We have recently demonstrated that MMF and its parent compound mycophenolic acid (MPA), which is a potent inhibitor of IMP dehydrogenase, potentiate the antiherpesvirus activity of acyclovir, ganciclovir, and penciclovir. We have now evaluated the antiviral efficacy of the combination of MPA and the novel antiherpesvirus agent H2G [(R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine]. When combined with H2G, MPA (at concentrations ranging from 0.25 to 10 μg/ml, which are readily attainable in human plasma) markedly potentiated the antiviral efficacy of H2G against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), as reflected by a 10- to 150-fold decrease in the 50% effective concentration. Moreover, the activity of H2G against a thymidine kinase-deficient strain of HSV-1 (TK⁻ HSV-1) was increased more than 2,500-fold when combined with MPA. MPA by itself had little or no effect on the replication of these viruses. Similar observations were made for varicella-zoster virus. Also, ribavirin (another inhibitor of IMP dehydrogenase) caused a marked enhancement of the activity of H2G against HSV-1 (10-fold), HSV-2 (10-fold), and TK⁻ HSV-1 (>185-fold). Exogenously added guanosine reversed the potentiating effects of MPA on the antiviral activity of H2G, indicating that this potentiating effect resulted from a depletion of the endogenous dGTP pools, thus favoring the inhibitory action of the H2G triphosphate on the viral DNA polymerase.     

Clinical Pharmacokinetics of 1-[((S)-2-Hydroxy-2-Oxo-1,4,2-Dioxaphosphorinan-5-yl)methyl]cytosine in Human Immunodeficiency Virus-Infected Patients

The pharmacokinetics and bioavailability of 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine (cyclic HPMPC) were examined at four doses in 22 patients with human immunodeficiency virus infection. Two groups of six patients received a single dose of cyclic HPMPC at 1.5 or 3.0 mg/kg of body weight by each of the oral and intravenous routes in a random order with a 2-week washout period between administrations. Additional patients received single intravenous doses of cyclic HPMPC at 5.0 mg/kg (n = 6) or 7.5 mg/kg (n = 4). Serial serum and urine samples were collected at intervals over 24 h after dosing. The concentrations of cyclic HPMPC and cidofovir in serum and urine samples were determined by validated reverse-phase ion-pairing high-performance liquid chromatography methods with derivatization and fluorescence detection. After intravenous administration of cyclic HPMPC, concentrations of cyclic HPMPC declined in a biexponential manner, with a mean ± standard deviation half-life of 1.09 ± 0.12 h (n = 22). The pharmacokinetics of cyclic HPMPC were independent of dose over the dose range of 1.5 to 7.5 mg/kg. The total clearance of cyclic HPMPC from serum and the volume of distribution of intravenous cyclic HPMPC were 198 ± 39.6 ml/h/kg and 338 ± 65.1 ml/kg, respectively (n = 22). The renal clearance of cyclic HPMPC (132 ± 27.3 ml/h/kg; n = 22) exceeded the creatinine clearance (86.2 ± 16.3 ml/h/kg), indicating active tubular secretion. The cyclic HPMPC excreted in urine in 24 h accounted for 71.3% ± 16.0% of the administered dose. Cidofovir was formed from cyclic HPMPC in vivo with a time to the maximum concentration in serum of 1.64 ± 0.23 h (n = 22). Cidofovir levels declined in an apparent monoexponential manner, with a mean terminal half-life of 3.98 ± 1.26 h (n = 22). The cidofovir excreted in urine in 24 h accounted for 9.40% ± 2.33% of the administered cyclic HPMPC dose. Exposure to cidofovir after intravenous administration of cyclic HPMPC was dose proportional and was 14.9% of that from an equivalent dose of cidofovir. The present study suggests that intravenous cyclic HPMPC also has a lower potential for nephrotoxicity in humans compared to that of intravenous cidofovir. The oral bioavailabilities of cyclic HPMPC were 1.76% ± 1.48% and 3.10% ± 1.16% with the administration of doses of 1.5 and 3.0 mg/kg, respectively (n = 6 per dose). The maximum concentrations of cyclic HPMPC in serum were 0.036 ± 0.021 and 0.082 ± 0.038 μg/ml after the oral administration of doses of 1.5 and 3.0 mg/kg, respectively. Cidofovir reached quantifiable levels in the serum of only one patient for each of the 1.5- and 3.0-mg/kg oral cyclic HPMPC doses.     

Anti-Human Immunodeficiency Virus Activity and Cellular Metabolism of a Potential Prodrug of the Acyclic Nucleoside Phosphonate 9-R-(2-Phosphonomethoxypropyl)adenine (PMPA), Bis(isopropyloxymethylcarbonyl)PMPA

Bis(isopropyloxymethylcarbonyl) 9-R-(2-phosphonomethoxypropyl)adenine [bis(POC)PMPA] has been identified as a novel prodrug of PMPA. The anti-human immunodeficiency virus activity of bis(POC)PMPA was >100-fold greater than that of PMPA in both an established T-cell line and primary peripheral blood lymphocytes. This improved efficacy was shown to be due to a rapid intracellular uptake of the prodrug resulting in an increased intracellular accumulation of PMPA diphosphate (PMPApp), the pharmacologically active metabolite. PMPApp levels in bis(POC)PMPA-treated cells exceeded by >1,000-fold the levels seen in cells treated with unmodified PMPA in both resting and activated peripheral blood lymphocytes. Significant differences in the intracellular catabolism of PMPA metabolites were noted between the resting and activated lymphocytes. The half-life for the disappearance of PMPApp, derived from either bis(POC)PMPA or PMPA, was 12 to 15 h in the activated lymphocytes and 33 to 50 h in the resting lymphocytes. This long persistence of PMPApp, particularly in resting lymphocytes, may be unique to the nucleoside phosphonate analogs and indicates that effective levels of the active metabolite can be achieved and maintained with relatively infrequent administration of the parent drug.     

Antiherpesvirus Activities of (1′S,2′R)-9-{[1′,2′-Bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) in Cell Culture

Antiherpetic activity of (1′S,2′R)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) was compared with those of acyclovir (ACV) and penciclovir (PCV) in cell cultures. In a plaque reduction assay using a selection of human cells, A-5021 showed the most potent activity in all cells. Against clinical isolates of herpes simplex virus type 1 (HSV-1, n = 5) and type 2 (HSV-2, n = 6), mean 50% inhibitory concentrations (IC₅₀s) for A-5021 were 0.013 and 0.15 μg/ml, respectively, in MRC-5 cells. Corresponding IC₅₀s for ACV were 0.22 and 0.30 μg/ml, and those for PCV were 0.84 and 1.5 μg/ml, respectively. Against clinical isolates of varicella-zoster virus (VZV, n = 5), mean IC₅₀s for A-5021, ACV, and PCV were 0.77, 5.2, and 14 μg/ml, respectively, in human embryonic lung (HEL) cells. A-5021 showed considerably more prolonged antiviral activity than ACV when infected cells were treated for a short time. The selectivity index, the ratio of 50% cytotoxic concentration to IC₅₀, of A-5021 was superior to those of ACV and PCV for HSV-1 and almost comparable for HSV-2 and VZV. In a growth inhibition assay of murine granulocyte-macrophage progenitor cells, A-5021 showed the least inhibitory effect of the three compounds. These results show that A-5021 is a potent and selective antiviral agent against HSV-1, HSV-2, and VZV.     

Mode of Action of (1′S,2′R)-9-{[1′,2′-Bis(hydroxymethyl) cycloprop-1′-yl]methyl}guanine (A-5021) against Herpes Simplex Virus Type 1 and Type 2 and Varicella-Zoster Virus

The mode of action of (1′S,2′R)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2′ site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.     

Amidate Prodrugs of 9-[2-(Phosphonomethoxy)Ethyl]Adenine as Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC₅₀] = 22 and 46 nM) than the phosphonodiamidates (IC₅₀ = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC₅₀ = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca²⁺]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.     

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.Schistosomiasis is the second most prevalent parasitic disease worldwide after malaria, with about 200 million human beings infected in 74 countries. It is estimated that 779 million people are at risk of contracting schistosomiasis and more than 200 million individuals are infected, with more than half of them suffering from disease-associated symptoms (18, 29, 34). Severe disease manifestations are exhibited in about 20 million individuals (30). The annual mortality rate due to schistosomiasis in sub-Saharan Africa might be as high as 280,000 (31). Chemotherapeutic measures have been the mainstay for control of schistosomiasis (12), and since the 1970s, praziquantel (PZQ) has become the drug of choice against the three major human species of schistosomes, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum (10, 13). PZQ is a relatively safe, orally administered drug that leads to reduction in the prevalence of schistosomiasis (3, 28). Mass drug administration programs currently rely heavily on PZQ for the control of schistosome-induced morbidity. However, with only one drug of choice for treatment and with the possibility of development of parasite resistance, the present situation is dangerous. There is a real and pressing need for discovering alternatives to the only available antischistosomal drug worldwide (5).Acyclic nucleoside phosphonates are a group of biologically active compounds which have been developed primarily as antivirals (15). The S-enantiomer of 9-(3-hydroxy-2-phosphonyl-methoxypropyl)adenine [(S)-HPMPA] is of particular interest because it has a broad spectrum of antiviral activity (8) as well as in vivo activity against Plasmodium falciparum and Plasmodium berghei (murine models for human malaria) (27). The compound showed a trypanocidal activity against all extracellular trypanosomes and some of the intracellular hemoflagellates (9). We previously reported antischistosomal activity for (S)-HPMPA (4), as it caused significant reductions in vivo in worm loads, tissue egg loads, and the frequency of egg developmental stages. Most prominently, (S)-HPMPA treatment resulted in the nearly complete disappearance of mature and immature eggs (4). In vitro, (S)-HPMPA did not significantly affect the muscle tension of S. mansoni worms regardless of the concentration tested (4). In this report we have evaluated the antischistosomal activity of various alkoxyalkyl esters of (S)-HPMPA, CDV, and other acyclic nucleoside phosphonate compounds to assess their potential antischistosomal activities.     

Disposition of the Acyclic Nucleoside Phosphonate (S)-9(3-Hydroxy-2-Phosphonylmethoxypropyl)Adenine

The acyclic nucleoside phosphonate (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] has been shown to be active against pathogens, like hepatitis B viruses and Plasmodium parasites, that infect parenchymal liver cells. (S)-HPMPA is therefore an interesting candidate drug for the treatment of these infections. To establish effective therapeutic protocols for (S)-HPMPA, it is essential that the kinetics of its hepatic uptake be evaluated and that the role of the various liver cell types be examined. In the present study, we investigated the disposition of (S)-HPMPA and assessed its hepatic uptake. Rats were intravenously injected with [³H](S)-HPMPA, and after an initial rapid distribution phase (360 ± 53 ml/kg of body weight), the radioactivity was cleared from the circulation with a half-life of 11.7 ± 1.4 min. The tissue distribution of [³H](S)-HPMPA was determined at 90 min after injection (when >99% of the dose cleared). Most (57.0% ± 1.1%) of the injected [³H](S)-HPMPA was excreted unchanged in the urine. The radioactivity that was retained in the body was almost completely recovered in the kidneys and the liver (68.4% ± 2.5% and 16.1% ± 0.4% of the radioactivity in the body, respectively). The uptake of [³H](S)-HPMPA by the liver occurred mainly by parenchymal cells (92.1% ± 3.4% of total uptake by the liver). Kupffer cells and endothelial cells accounted for only 6.1% ± 3.5% and 1.8% ± 0.8% of the total uptake by the liver, respectively. Preinjection with probenecid reduced the hepatic and renal uptake of [³H](S)-HPMPA by approximately 75%, which points to a major role of a probenecid-sensitive transporter in the uptake of (S)-HPMPA by both tissues. In conclusion, we show that inside the liver, (S)-HPMPA is mainly taken up by parenchymal liver cells. However, the level of uptake by the kidneys is much higher, which leads to nephrotoxicity. An approach in which (S)-HPMPA is coupled to carriers that are specifically taken up by parenchymal cells may increase the effectiveness of the drug in the liver and reduce its renal toxicity.     

Plasma (1→3)-β-D-glucan assay and immunohistochemical staining of (1→3)-β-D-glucan in the fungal cell walls using a novel horseshoe crab protein (T-GBP) that specifically binds to (1→3)-β-D-glucan

A highly sensitive enzyme-linked immunosorbent assay specific to (1→3)-β-D-glucans (GBP-ELISA) has been developed using a novel (1→3)-β-D-glucan-binding protein (T-GBP), which was purified from the amebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. This method allowed quantitation of the glucans in a concentration range of 0.1–1,000 ng/ml, regardless of linear and branched structures, and was applied to determine the amounts of (1→3)-β-D-glucan in human and animal plasmas for diagnosis of fungemia. High levels of plasma glucan contents in clinical samples were found to be correlated closely with the severity of fungal infection. T-GBP was successfully utilized for indirect immunofluorescence staining of (1→3)-β-D-glucan in Candida albicans cell walls. J. Clin. Lab. Anal. 11:104–109. © 1997 Wiley-Liss, Inc.