Geoffrey W. Henson

AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-gamma receptor-deficient mice.

Autoimmune collagen-induced arthritis (CIA) in IFN-γR-deficient DBA/1 mice was shown to be reduced in severity by treatment with the bicyclam derivative AMD3100, a specific antagonist of the interaction between the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4. The beneficial effect of the CXCR4 antagonist was demonstrable when treatment was initiated between the time of immunization and appearance of the first symptoms. Treatment also reduced the delayed-type hypersensitivity response to the autoantigen, collagen type II. These observations are indicative of an action on a late event in the pathogenesis, such as chemokine-mediated attraction of leukocytes toward joint tissues. The notion of SDF-1 involvement was further supported by the observation that exogenous SDF-1 injected in periarthritic tissue elicited an inflammatory response that could be inhibited by AMD3100. The majority of leukocytes harvested from inflamed joints of mice with CIA were found to be Mac-1+ and CXCR4+, and AMD3100 was demonstrated to interfere specifically with chemotaxis and Ca2+ mobilization induced in vitro by SDF-1 on Mac-1+/CXCR4+ splenocytes. We conclude that SDF-1 plays a central role in the pathogenesis of murine CIA, by attracting Mac-1+/CXCR4+ cells to the inflamed joints.

Bicyclams, a class of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/CXCR-4.

Bicyclams are a novel class of antiviral compounds which are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. The prototype compound, AMD3100, has an IC50 of 1-10 ng/ml, which is a least 100,000 fold lower than the cytotoxic concentration. AMD3100 does not inhibit virus binding to the CD4 receptor and based on time-of-addition experiments, has been assumed to interact with the HIV fusion-uncoating process. Resistance of HIV-1 strains to AMD3100 is associated with the accumulation of several mutations in the viral envelope glycoprotein gp120. Here, we demonstrate that AMD3100 interacts with fusin (CXCR-4), the coreceptor used by T-tropic viruses to infect the target cells. The replication of NL4-3 wild type virus and NL4-3 dextran sulfate-resistant virus was inhibited by the CXC-chemokine, stromal cell-derived factor 1 (SDF-1), the natural ligand for CXCR-4. In contrast, the replication of the HIV-1 NL4-3 AMD3100-resistant virus was no longer inhibited by SDF-1. The bicyclams are the first low-molecular-weight anti-HIV agents shown to interact with the coreceptor for T-tropic viruses.

Leukocytosis and Mobilization of CD34+ Hematopoietic Progenitor Cells by AMD3100, a CXCR4 Antagonist

Stromal cell-derived factor-1 (SDF-1/CXCL12) plays a key regulatory role in the trafficking of hematopoietic cells. AMD3100 is a specific antagonist of the binding of SDF-1 to its receptor, CXCR4. This phase I study assessed the hematological effects, pharmacokinetics, and safety of administration of AMD3100 to 32 healthy volunteers, including its ability to mobilize CD34+ hematopoietic progenitor cells. A generalized leukocytosis occurred after a single subcutaneous injection of AMD3100 (80 microg/kg) resulting in a maximum white blood cell count of 19.49 +/- 1.27 x 103/microL (mean +/- SEM) at 6 hours. No changes were observed in erythrocyte or platelet counts. Circulating CD34+ cells increased 5-fold after administration of AMD3100 at 80 mug/kg and 15.5-fold in response to AMD3100 at 240 mug/kg, both at 9 hours after injection. Myeloid progenitor cells-colony forming unit granulocytemacrophage (CFU-GM); CFU-granulocyte, eosinophil, monocyte, megakaryocyte (CFU-GEMM); and burst forming units-erythroid showed similar increases in mobilization to the blood with increasing doses of AMD3100. The mobilized cells were in a slow or noncycling state as determined by in vitro high specific activity of 3H-thymidine. Pharmacokinetic studies showed a near linear increase in peak drug levels with increasing doses and nearly complete elimination of the drug by 24 hours. AMD3100 was well tolerated with only mild and transient toxicities (injection site erythema, headache, paresthesia, nausea, and abdominal distension) observed. These observations suggest that AMD3100 may be a clinically useful agent for hematopoietic progenitor cell mobilization.

INX-08189, a Phosphoramidate Prodrug of 6-O-Methyl-2′-C-Methyl Guanosine, is a Potent Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic and Pharmacodynamic Properties

ABSTRACT INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2′-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10 ± 6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35 ± 8 nM at 24 h. An intracellular 2′-C-methyl guanosine triphosphate (2′-C-MeGTP) concentration of 2.43 ± 0.42 pmol/106 cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344 ± 170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2′-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2′-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2′-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

The bicyclams, a new class of potent human immunodeficiency virus inhibitors, block viral entry after binding

The bicyclams represent a new class of highly potent and selective HIV inhibitors. Time-of-addition experiments have previously shown that these compounds interfere with an early event in the viral replicative cycle. Additional experiments have now been carried out in order to investigate in more detail the mechanism of action of these promising compounds. As described in this paper, PCR experiments revealed that no viral DNA was formed following viral infection, thus confining the target(s) of action of the bicyclams to an early stage of HIV infection. An assay, using pseudotype virions containing the envelope of HIV-1 and the genome of a plaque-forming virus (Cocal Virus), pointed to viral entry as the main target of the bicyclams. HIV-1 strains resistant to two prototype bicyclams, JM2763 and SID791 (JM3100), were raised. Results obtained with SID791 with respect to syncytium formation induced by SID791-sensitive and -resistant HIV-1 strains and the cross-resistance observed for dextran sulfate, suggest inhibition of binding and/or fusion as a plausible target of SID791. Additional experiments enabled us to exclude SID791 and JM2763 as binding inhibitors and to conclude that bicyclams block the entry of cell-bound virus. Furthermore, a monoclonal antibody recognising the V3 loop of wild-type gp120 did not bind to this region in the two bicyclam-resistant strains. Our results point to gp120 as a possible target for the HIV-inhibitory effects of the bicyclams.

The CXCR4 Antagonist AMD3100 Efficiently Inhibits Cell-Surface-Expressed Human Immunodeficiency Virus Type 1 Envelope-Induced Apoptosis

ABSTRACT Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death by apoptosis in infected and uninfected cells. The envelope glycoprotein complex ([gp120/gp41]n) of X4 HIV-1 isolates is involved in both infected and uninfected cell death via its interaction with cellular receptors CD4 and CXCR4. We studied the effect of the blockade of CXCR4 receptors by the agonist stromal derived factor (SDF-1α) and the antagonist bicyclam AMD3100 on apoptotic cell death of CD4+cells in different models of HIV infection. In HIV-infected CEM or SUP-T1 cultures, AMD3100 showed antiapoptotic activity even when added 24 h after infection. In contrast, other antiviral agents, such as zidovudine, failed to block apoptosis under these conditions. The antiapoptotic activity of AMD3100 was also studied in coculture of peripheral blood mononuclear cells or CD4+ cell lines with chronically infected H9/IIIB cells. AMD3100 was found to inhibit both syncytium formation and apoptosis induction with 50% inhibitory concentrations ranging from 0.009 to 0.24 μg/ml, depending on the cell type. When compared to SDF-1α, AMD3100 showed higher inhibitory potency in all cell lines tested. Our data indicate that the bicyclam AMD3100 not only inhibits HIV replication but also efficiently blocks cell-surface-expressed HIV-1 envelope-induced apoptosis in uninfected cells.

Pharmacokinetics and Safety of FV-100, a Novel Oral Anti-Herpes Zoster Nucleoside Analogue, Administered in Single and Multiple Doses to Healthy Young Adult and Elderly Adult Volunteers

ABSTRACT FV-100 is the prodrug of the highly potent anti-varicella zoster virus bicyclic nucleoside analogue CF-1743. To characterize the pharmacokinetics and safety of oral FV-100, 3 randomized, double-blind, placebo-controlled clinical trials were conducted: (i) a single-ascending-dose study in 32 healthy subjects aged 18 to 55 years (100-, 200-, 400-, and 800-mg doses) with an evaluation of the food effect in the 400-mg group; (ii) a multiple-ascending-dose study in 48 subjects aged 18 to 55 years (100 mg once daily [QD], 200 mg QD, 400 mg QD, 400 mg twice a day, and 800 mg QD for 7 days); and (iii) a 2-part study in subjects aged 65 years and older with a single 400-mg dose in 15 subjects and a 400-mg QD dosing regimen for 7 days in 12 subjects. FV-100 was rapidly and extensively converted to CF-1743, the concentration of which remained above that required to reduce viral activity by 50% for the 24-hour dosing period. Renal excretion of CF-1743 was very low. A high-fat meal reduced exposure to CF-1743; a low-fat meal did not. Pharmacokinetic parameters for the elderly subjects were comparable to those for the younger subjects. FV-100 was well tolerated by all subjects. The pharmacokinetic and safety profiles of FV-100 support its continued investigation for the treatment of herpes zoster and prevention of postherpetic neuralgia with once-daily dosing and without dose modifications for elderly or renally impaired patients.

Pharmacokinetics of antiviral polyoxometalates in rats.

Polyoxometalates are soluble mineral compounds formed principally of oxide anions and early transition metal cations. The polyoxometalates K12H2[P2W12O48].24H2O (JM 1591), K10[P2W18Zn4(H2O)2O68].20H2O (JM 1596), and [(CH3)3NH]8[Si2W18Nb6O77] (JM 2820) demonstrate potent antiviral activity against human immunodeficiency virus types 1 and 2, herpes simplex virus, and cytomegalovirus in vitro. The preclinical pharmacokinetics of these three compounds were characterized after single-dose intravenous administration of 50 mg/kg to rats. Plasma, urine, and feces were collected for 168 h, and polyoxometalate concentrations were determined by atomic emission. Serum protein binding was measured by equilibrium dialysis. All three compounds were highly bound to serum proteins in a concentration-dependent manner. Total and unbound concentrations of the three compounds in plasma declined in a triexponential manner with terminal half-lives of 246.0 +/- 127.0, 438.4 +/- 129.4, and 32.2 +/- 5.37 h (mean +/- standard deviation) for JM 1591, JM 1596, and JM 2820, respectively. Systemic clearances based on total concentrations in plasma were low, averaging 0.016 +/- 0.002, 0.015 +/- 0.002, and 0.018 +/- 0.003 liter/h/kg for JM 1591, JM 1596, and JM 2820, respectively. The clearances of unbound compounds from plasma averaged 0.966 +/- 0.136, 0.050 +/- 0.005, and 0.901 +/- 0.165 liter/h/kg for JM 1591, JM 1596, and JM 2820, respectively. For JM 1596, the clearance of unbound compound from the kidneys was lower than the glomerular filtration rate (0.086 liter/h/kg), suggesting this polyoxometalate underwent renal tubular reabsorption. However, JM 1591 and JM 2820 appeared to undergo tubular secretion. The fraction of the dose recovered in urine was 11.5, 46.8, and 10.6% for JM 1591, JM 1596, and JM 2820, respectively. Approximately 5% of the dose of each polyoxometalate was recovered in feces. The steady-state volume of distribution based on total concentrations averaged 1.44 liters/kg for JM 1591, 2.39 liters/kg for JM 1596, and 0.59 liter/kg for JM 2820, indicating moderate to wide distribution throughout the body. All three compounds were detected in various tissues 1 week after single-dose administrations, with the highest levels found in the kidneys and liver. The results of this study indicate that the disposition of polyoxometalates is highly dependent on their molecular structure.

In vitro Activity of a Novel Series of Polyoxosilicotungstates against Human Myxo-, Herpes- and Retroviruses

A series of silicon-containing polyoxotungstates belonging to the ‘Keggin-type’ (‘Keggin’, ‘Keggin sandwich’) were evaluated for their antiviral activity against enveloped viruses (myxo-, herpes- and retroviruses). The compounds exhibited antiviral activity against influenza virus type A, respiratory syncytial virus (RSV), herpes simplex virus type-1 (HSV-1), type-2 (HSV-2), thymidine kinase-deficient (TIC) HSV-1, human cytomegalovirus (HCMV), human immunodeficiency virus type-1 (HIV-1) and type-2 (HIV-2) at concentrations that were well below their cytotoxic threshold. The ‘Keggin’ compound JM2815 (K5[Si-(TiCp)W11O39].12H2O) and the ‘Keggin sandwich’ compound JM1590 (K13[Ce(SiW11O39)2].26H2O) resulted in the highest selectivity indices against HIV-1 and HIV-2, and compound JM2820 ([Me3NH]8.[Si2Nb6W18O77]) was the most potent inhibitor of HSV and HCMV replication. These compounds proved active against HCMV and HSV when present during virus adsorption, and against influenza virus A and RSV when present after virus adsorption. Polyoxosilicotungstates inhibited the binding of radiolabeled HCMV particles to the cells at concentrations that were antivirally active, and the compounds were able to displace HCMV particles that were bound to a heparin-Sepharose matrix. Presumably, the polyoxosilicotungstates interact with positively charged domains on the viral envelope site(s) involved in the attachment of the (HCMV) virions to the cell surface receptor heparan sulphate.

Pharmacokinetics of the anti-HIV Bicyclam SID791 (JM3100) in Rabbits, as Determined by both Analytical and Bio-Assay Methods

The serum levels of the bicyclam derivative 1,1′-[1,4-phenylenebis(methylene)]-bis-1,4,8,11-etraazacyclotetradecane octahydrochloride dihydrate [SID791 (JM3100)], a potent inhibitor of HIV replication (De Clercq et al., 1994) were determined in rabbits using two different methods. A method based on the UV-absorption of the Cu-complex of SID791 was used to analyse by HPLC the serum drug concentrations, and an antiviral activity bio-assay was performed to investigate whether the drug in the rabbit serum was in an available active form. After subcutaneous (sc) administration of SID791 to rabbits at 25 mg kg−1 of body weight, the compound was cleared from the serum in a bi-exponential manner (β1-half-lives: 67 and 69 min; β2-half-lives: 320 and 245 min; distribution volumes: 0.40 and 0.37 I; total body clearance: 0.30 and 0.29 I h−1; and AUC: 83.3 and 86.2 h μg ml−1, as determined by HPLC and bio-assay, respectively). Thus, very similar kinetic parameters were noted if serum drug concentrations were determined by HPLC analysis or bio-assay, suggesting that in the rabbit serum the drug is present as an antiviral active agent.