Rudi Pauwels

Antiviral activity of phosphonylmethoxyalkyl derivatives of purine and pyrimidines

Various 3-hydroxy-2-phosphonylmethoxypropyl (HPMP) and 2-phosphonylmethoxyethyl (PME) derivatives of purine [adenine (A), guanine (G), 2,6-diaminopurine (DAP), 2-monoaminopurine (MAP), hypoxanthine (HX)] and pyrimidine [cytosine (C), uracil (U), thymine (T)] have been evaluated for their antiviral properties. PMEDAP, (S)-HPMPA [and the cyclic phosphonate thereof, (S)-cHPMPA)], (S)-HPMPC, PMEG, PMEA, HPMPG and HPMPDAP proved to be effective inhibitors of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). (S)-HPMPA and (S)-cHPMPA were the most effective inhibitors of varicella-zoster virus (VZV), and (S)-HPMPC was the most effective inhibitor of cytomegalovirus (CMV). Against adenovirus (types 2, 3 and 4) and vaccinia virus again (S)-HPMPA and (S)-cHPMPA showed the greatest inhibitory activity. As a rule, the PME derivates were much less inhibitory to VZV, CMV, vaccinia and adenovirus than the HPMP derivatives. However, PMEA, PMEDAP and PMEMAP showed marked and selective activity against the human immunodeficiency virus (HIV). (S)-HPMPA was selected for further evaluation in animal model infections. It proved efficacious in the topical treatment of HSV-1 keratitis in rabbits and cutaneous HSV-1 infection in hairless mice, and in the systemic treatment of both HSV-1 and vaccinia virus infections in mice.

Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus.

Several sulfated polysaccharides (dextran sulfate, pentosan polysulfate, fucoidan, and carrageenans) proved to be potent inhibitors for herpes simplex virus, human cytomegalovirus, vesicular stomatitis virus, Sindbis virus, and human immunodeficiency virus. They were moderately inhibitory to vaccinia virus but not inhibitory to adenovirus, coxsackievirus, poliovirus, parainfluenza virus, and reovirus. These results indicate that, with the exception of parainfluenza virus, enveloped viruses are specifically susceptible to the inhibitory activity of sulfated polysaccharides.

TMC125, a Novel Next-Generation Nonnucleoside Reverse Transcriptase Inhibitor Active against Nonnucleoside Reverse Transcriptase Inhibitor-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC50] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC50 = 3.5 μM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of α1-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC50 of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC50 < 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.

TMC114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitor-resistant viruses, including a broad range of clinical isolates

ABSTRACT The purpose of this study was to characterize the antiviral activity, cytotoxicity, and mechanism of action of TMC114, a novel human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI). TMC114 exhibited potent anti-HIV activity with a 50% effective concentration (EC50) of 1 to 5 nM and a 90% effective concentration of 2.7 to 13 nM. TMC114 exhibited no cytotoxicity at concentrations up to 100 μM (selectivity index, >20,000). All viruses in a panel of 19 recombinant clinical isolates carrying multiple protease mutations and demonstrating resistance to an average of five other PIs, were susceptible to TMC114, defined as a fold change in EC50 of <4. TMC114 was also effective against the majority of 1,501 PI-resistant recombinant viruses derived from recent clinical samples, with EC50s of <10 nM for 75% of the samples. In sequential passage experiments using HIV-1 LAI, two mutations (R41T and K70E) were selected. One selected virus showed a 10-fold reduction in susceptibility to TMC114, but <10-fold reductions in susceptibility to the current PIs (atazanavir was not assessed), except saquinavir. However, when the selected mutations were introduced into a laboratory strain by site-directed mutagenesis, they had no effect on susceptibility to TMC114 or other PIs. There was no evidence of antagonism between TMC114 and any currently available PIs or reverse transcriptase inhibitors. Combinations with ritonavir, nelfinavir, and amprenavir showed some evidence of synergy. These results suggest that TMC114 is a potential candidate for the treatment of both naïve and PI-experienced patients with HIV.

Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus (HIV) in vitro.

The polyanionic substances dextran sulfate and heparin were investigated for their antiviral effect on the human immunodeficiency virus (HIV) in vitro. Dextran sulfate and heparin effected a 50% reduction in the cytopathogenicity of HIV for MT-4 cells at a concentration of 4.7 and 7.5 micrograms/ml, respectively. In Molt-4 (clone 8) cells, these values were slightly higher (14.1 and 15.6 micrograms/ml, respectively). No toxicity for the host cells was noted with these compounds at a concentration up to 400 micrograms/ml, so that the selectivity indexes, as based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, were well in excess of 100. These findings may have far reaching implications both diagnostically, when attempts are made to isolate HIV from heparinized blood samples, as therapeutically, to the extent that dextran sulfate or heparin may be useful in blocking HIV replication in vivo.

TMC125 Displays a High Genetic Barrier to the Development of Resistance: Evidence from In Vitro Selection Experiments

ABSTRACT TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to “classic” selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs. Both low- and high-MOI experiments demonstrated that the development of resistance to TMC125 required multiple mutations which frequently conferred cross-resistance to efavirenz and nevirapine. In high-MOI experiments, 1 μM TMC125 completely inhibited the breakthrough of resistant virus from wild-type and NNRTI-resistant HIV-1, in contrast to efavirenz and nevirapine. Furthermore, breakthrough of virus from site-directed mutant (SDM) SDM-K103N/Y181C occurred at the same time or later with TMC125 as breakthrough from wild-type HIV-1 with efavirenz or nevirapine. The selection experiments identified mutations selected by TMC125 that included known NNRTI-associated mutations L100I, Y181C, G190E, M230L, and Y318F and the novel mutations V179I and V179F. Testing the antiviral activity of TMC125 against a panel of SDMs indicated that the impact of these individual mutations on resistance was highly dependent upon the presence and identity of coexisting mutations. These results demonstrate that TMC125 has a unique profile of activity against NNRTI-resistant virus and possesses a high genetic barrier to the development of resistance in vitro.

Encoding microcarriers: present and future technologies

In answer to the ever-increasing need to carry out many assays simultaneously in drug screening and drug discovery, several microcarrier-based multiplex technologies have arisen in the past few years. The compounds to be screened are attached to the surface of microcarriers, which can be mixed together in a vessel that contains the target analyte. Each microcarrier has to be encoded to know which compound is attached to its surface. In this article, the methods that have been developed for the encoding of microcarriers are reviewed and discussed.

Effect of inhaled substance P and neurokinin A on the airways of normal and asthmatic subjects.

The neuropeptides substance P and neurokinin A are present in sensory airway nerves. Their effect on airway calibre was compared in six healthy non-smoking subjects and six asthmatic subjects. On separate days increasing concentrations (from 10(-9) to 10(-6) mol/ml) of each neuropeptide were administered by nebuliser and the airway response measured as change in specific airway conductance (sGaw). Substance P and neurokinin A caused no change in sGaw in the healthy subjects. Inhalation of substance P up to the highest concentration of 10(-6) mol/ml caused no change in sGaw in the asthmatic subjects. Neurokinin A, however, caused bronchoconstriction with a mean fall in sGaw of 48% (SEM 12%) after 5 x 10(-7) mol/ml. The onset of bronchoconstriction was rapid, but sGaw had returned to baseline values within one hour in all but one patient.

The effect of theophylline and enprofylline on allergen-induced bronchoconstriction.

The effect on the allergen-induced immediate and late bronchoconstriction of theophylline and enprofylline (3-propylxanthine), a new xanthine derivative with negligible ability to antagonize adenosine, was studied in nine patients with asthma. The patients were challenged three times at weekly intervals with the same dose of allergen. FEV1 and SGaw were followed up to 6 hours after challenge. The drugs were administered intravenously. Placebo was always administered on the first occasion. Theophylline and enprofylline were administered on test days 2 and 3 with a double-blind, randomized crossover technique. One hour before the allergen challenge, a loading dose was administered during 60 minutes followed by a constant infusion during 6 hours. The loading infusion was 7.2 mg/kg of theophylline and 2.7 mg/kg of enprofylline. The maintenance dose was 74 mg/hr and 71 mg/hr, respectively. Both theophylline and enprofylline caused a minor initial bronchodilatation. Theophylline and enprofylline slightly but significantly attenuated the immediate bronchoconstricting reaction after allergen inhalation. Theophylline and enprofylline had a significant attenuating effect on the late bronchial reaction. The mean plasma level of theophylline was 0, 10.8, 10.5, and 10.5 mg/L at 0, 1, 4, and 7 hours after the start of the loading infusion, respectively. The corresponding mean plasma levels of enprofylline were 0, 2.6, 2.7, and 2.7 mg/L. Theophylline and enprofylline caused headache in one patient. Two patients developed nausea and vomiting during the enprofylline infusion. The present data suggest that adenosine receptor antagonism may not be the main mode of action of xanthines in inhibiting bronchoconstriction after single dose antigen challenge.

Evolution of anti-HIV drug candidates. Part 3: diarylpyrimidine (DAPY) analogues

The synthesis and anti-HIV-1 activity of a series of diarylpyrimidines (DAPYs) are described. Several members of this novel class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) are extremely potent against both wild-type and a panel of clinically significant single- and double-mutant strains of HIV-1.