Anti-HIV-1 activity in vitro of ceftazidime degradation products.

Cephalosporins in aqueous solutions generate degradation products that inhibit in vitro HIV-1 replication in cell lines, as well as in primary cells (lymphocytes and macrophages). This effect is observed at concentrations that do not interfere with the normal functions of these cells. Upon chromatographic fractionation of an aqueous solution of hydrolysed ceftazidime, a high molecular weight fraction (MW 8000) with antiviral activity was isolated. The exact chemical nature of the active component responsible for the anti-HIV activity in vitro appears to be complex and is currently unknown. Inhibition of HIV-1 reverse transcriptase and RNase H activity was observed, however, higher concentrations than those needed to inhibit HIV replication were required. The inhibitory action of the hydrolysed ceftazidime was manifested during the early phase of the HIV-1 life-cycle. Despite a lack of a direct effect of the CD4/gp120 interaction, HIV-1 mediated cell fusion was inhibited by the hydrolysed ceftazidime, suggesting that the active principle acts in a very early stage of the viral life-cycle.

New aza-dipeptide analogues as potent and orally absorbed HIV-1 protease inhibitors: candidates for clinical development.

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

Profile of CGP 61755: a novel and potent HIV-1 protease inhibitor that shows enhanced anti-HIV activity when combined with other antiretroviral agents in vitro.

CGP 61755 is a novel hydroxyethylene derivative produced by a high yield 10 step chemical synthesis. It is highly specific for HIV-1 protease with an IC50 of 1 nM. The ED90 in MT-2, PBLs and macrophages is infected with laboratory strains of HIV-1 or clinical isolates is 30-100 nM. In chronically infected macrophages the ED90 is 1000 nM (1000 nM for saquinavir and 10 microM for indinavir). When the antiviral activity of CGP 61755 on HIV-1 infected lymphocytes was examined using serum free medium an ED99 of 60 nM was determined, while in the presence of 10% human serum the same activity was achieved with 120 nM. When examined in combination with RT inhibitors or protease inhibitors, either in a co-culture of CEM-SS and chronically infected H9IIIB cells or in a free virus lymphocyte infection, cooperativity of the antiviral activities was observed. Dog pharmacokinetic studies comparing p.o. and i.v. data indicate that CGP 61755 has a bioavailability between 50 and 80%. Following oral administration the area under the concentration curve (AUC) values increased in a dose proportional manner. The plasma levels of the drug at 6 hours after oral administration were above the ED90. Based on these properties we believe that CGP 61755 has an attractive profile that justifies further preclinical evaluation of the drug.

Aza-peptide analogs as potent human immunodeficiency virus type-1 protease inhibitors with oral bioavailability.

A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with antiviral activity in the low-nanomolar range. The S-configuration of the transition-state hydroxyl group was preferred in this series. Small modifications of the P2P3 and P2'P3' substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic profile. As a result of these studies, the symmetrically acylated compound 8a and its close analog 24a bearing a methyl carbamate in P3 and an ethyl carbamate in P3' position were identified as potent inhibitors with plasma concentrations exceeding antiviral ED50 values 150-fold following oral application in mice.

Synthesis of potent and orally active HIV-protease inhibitors.

A series of potent HIV-protease inhibitors has been prepared. Several of the newly synthesized compounds showed high plasma even after oral administration to animals. Based on the overall biological profile, CGP 61755 was chosen for further preclinical evaluation. For this compound, a 10 step synthesis potentially suitable for large scale production was developed.

CGP 53437, an orally bioavailable inhibitor of human immunodeficiency virus type 1 protease with potent antiviral activity.

CGP 53437 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease containing a hydroxyethylene isostere. The compound inhibited recombinant HIV-1 protease with a Ki of 0.2 nM. The inhibition constant versus human cathepsin D and human cathepsin E was 4 nM. Human pepsin and gastricsin were inhibited with Kis of 8 and 500 nM, respectively, and human renin was inhibited with a Ki of 190 microM. The replication of HIV-1/LAV, HIV-1/Z-84, and HIV-1/pLAI was inhibited with a 90% effective dose of 0.1 microM in acutely infected MT-2 cells. The 50% cytotoxic dose was 100 microM. Similar antiviral activity was observed when the compound was added up to 10 h after infection. At the effective concentration, processing of Gag precursor protein p55 was greatly reduced, confirming an action on the late stage of the virus life cycle, as expected. The efficacy of the inhibitor was also demonstrated by using primary human peripheral blood lymphocytes infected with the HIV-1/LAV strain, low-passage clinical isolates obtained from HIV-1-seropositive individuals (including a zidovudine-resistant strain), and HIV-2/ROD. In these cells, CGP 53437 delayed the onset of HIV replication in a dose-dependent fashion (substantial effects with concentrations of > or = 0.1 microM) as long as the inhibitor was maintained in the culture. CGP 53437 was orally bioavailable in mice. Concentrations in plasma 10-fold in excess of the in vitro antiviral 90% effective dose could be sustained for several hours after oral application of 120 mg/kg. Therefore, CGP 53437 has the potential to be a therapeutically useful anti-HIV agent for the treatment of AIDS.

Significant differences in therapeutic responses to a human interferon-alpha B/D hybrid in Rauscher or Friend murine leukemia virus infections.

Treatment of mice infected with Rauscher (RMLV) or Friend (FMLV) murine leukemia viruses at an early stage of disease (beginning at day 0 and continuing every other day for 21 days) with 5 x 10(7) units/kg body weight of a cross-species-active recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D) was more effective in FMLV than in RMLV infections. In contrast, treatment with 5 x 10(7) units/kg body weight of IFN beginning as late as 15 days postinfection and continuing every other day for 21 days was more effective in RMLV than in FMLV infections. These differences were consistent with observed changes in circulating white blood cells, spleen weight, and reverse transcriptase levels. Additionally, biweekly long-term administration (beginning at day 0) of 5 x 10(6) units/kg of rHuIFN-alpha B/D (an ineffective treatment in short-term therapy) significantly prolonged the mean survival time of RMLV-infected mice, but only weakly prolonged that of FMLV-infected mice.

Antiviral activity of a novel recombinant human interferon-alpha B/D hybrid.

The antiviral potential of a novel cross-species active, recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D), was evaluated for its efficiacy in cultured human monocytes and in several murine models of viral disease. When examined in 14-day-old human monocyte cultures, rHuIFN-alpha B/D was highly effective in preventing viral replication and cell destruction caused by herpes simplex virus type 1 (HSV-1/VR3). The effect observed with 100 units of this hybrid IFN was as good or higher than that observed with equivalent amounts of rHuIFN-alpha A or IFN-gamma. In addition, a single dose (5 X 10(7) U/kg) of rHuIFN-alpha B/D administered several hours after intranasal infection with HSV-1/VR3 suppressed pulmonary virus replication and prevented death due to interstitial pneumonia. Similarly, mice infected with a more aggressive strain of HSV-1 (McIntyre) were protected when this IFN preparation was administered at the time of virus infection and 1 day later. The anti-retroviral activity of rHuIFN-alpha B/D was examined in two murine leukemia retroviral models, Rauscher (RMLV) and Friend (FMLV), and a murine model of acquired immunodeficiency (LP-BM5). Treatment of RMLV or FMLV infected mice significantly prolonged mean survival times and the number of long-term FMLV survivors. These therapeutic effects were demonstrated when IFN was administered on the day of virus infection or as late as 3 days following infection. Transient reversal of the immunosuppressive effects induced by LP-BM5 infection was observed when rHuIFN-alpha B/D treatment was initiated at the time of virus infection. Moreover, when rHuIFN-alpha B/D was used together with azidothymidine (AZT), the effect of the combination was better than either drug alone.