From the discovery of competitive hexapeptide inhibitors, potent and selective HCV NS3 protease macrocyclic inhibitors have been identified. Structure-activity relationship studies were performed focusing on optimizing the N-terminal carbamate and the aromatic substituent on the (4R)-hydroxyproline moiety. Inhibitors meeting the potency criteria in the cell-based assay and with improved oral bioavailability in rats were identified. BILN 2061 was selected as the best compound, the first NS3 protease inhibitor reported with antiviral activity in man.
Nucleoside analogs such as acyclovir (ACV) and penciclovir and prodrugs thereof have been approved as drugs of choice for the treatment of herpes simplex virus (HSV) infections (1, 11). While nucleoside-based therapeutics are quite effective for the treatment of primary and recurrent mucocutaneous infections, present medications are not effective for the treatment of nucleoside-resistant herpesvirus infections in immunocompromised individuals (4,5,29). Moreover, nucleoside-based antiviral therapeutics have limited effects on the establishment of latent HSV infections (6,17,31). Therefore, significant improvements in therapy may be achievable with distinct inhibitors with improved efficacy and pharmacokinetic (PK) properties and a new mechanism of action. We have reported previously on the discovery of specific non-nucleoside-based inhibitors of the HSV type 1 (HSV-1) helicase-primase (10). This enzyme is composed of the virus-encoded UL5, UL8, and UL52 gene products, which are all essential for HSV DNA replication and growth (3,7,9,19,23). The most optimized aminothiazolyl-phenyl compounds exhibited potent antiviral activity against a series of HSV strains analyzed in vitro, including HSV-1 and HSV-2 strains that are ACV r . One of the inhibitors, BILS 179 BS, showed antiviral activity in murine models of wild-type HSV disease after oral administration (10), but its activity against ACV r HSV disease had not been evaluated. In the present report, we provide a detailed examination of the PK and pharmacodynamic properties of a helicase-primase inhibitor, BILS 45 BS, in a mouse model of ACV r HSV infection. PK studies were done with hairless mice, where BILS 45 BS demonstrated in vivo efficacy against wild-type HSV-1 infection with a 50% effective dose (ED 50 ) of 56 mg/kg. The athymic nude mouse model was chosen for the present study because ACV r HSV-1 fails to induce significant disease in normal mice (2, 14-16). In addition, since ACV r HSV infections cause significant disease mainly in the immunocompromised patient population, information obtained with these immunodeficient animals may have clinical relevance (14-16). Our results show that BILS 45 BS, an analog structurally related to BILS 179 BS, exhibited excellent oral efficacy against ACV r HSV-1 infections in nude mice, highlighting the potential of this novel class of antiherpetic agents for the treatment of ACV r HSV disease in humans. MATERIALS AND METHODSCell culture and viruses. All of the cell culture reagents and media used in this study were obtained from Gibco BRL (Burlington, Ontario, Canada). Cells were from the American Type Culture Collection (Manassas, Va.). Vero (African green monkey kidney) cells were grown in Dulbecco's modified Eagle's medium supplemented with 8% fetal bovine serum, 100 U of penicillin per ml, 100 g of streptomycin sulfate per ml, and 100 g of kanamycin sulfate per ml. Baby hamster kidney (BHK) 21/C13 (ATCC CCL10) cells were grown in ␣-MEM instead of Dulbecco's modified Eagle's medium. All cells were grown at 37°C in an ...
A series of HIV protease inhibitors containing a novel (hydroxyethyl)amidosuccinoyl core has been synthesized. These peptidomimetic structures inhibit viral protease activity at low nanomolar concentrations (IC50 < 10 nM for HIV-1 protease). The inhibition constant (Ki) for inhibitor 19 was determined to be 7.5 pM against HIV-1 and 1.2 nM against HIV-2 proteases, respectively. Several compounds (19-24) inhibited HIV-1 replication in cell culture assays with 50% effective concentrations (EC50) = 3.7-35 nM. This series of inhibitors was found to exhibit poor bioavailability (< 10%) in the rat, following oral administration. The synthesis and biological properties of these compounds are discussed. In addition, an X-ray structure of one of these inhibitors (23) in complex with HIV-2 protease provides insight into the binding mode of this novel class of HIV protease inhibitors.
The role of endothelin (ET-1) in mediating the development of blood pressure was investigated in the spontaneously hypertensive (SHR) rat using the Wistar-Kyoto (WKY) rat as the normotensive control. The following were characterized in both rat strains: age-dependent changes in mean arterial blood pressure (MAP), tissue (blood, lung, heart, and kidney) levels of immunoreactive ET-1 like related peptides (ET-1RP), aortic ring responses to ET-1, and specific high-affinity tissue (lung, atrium, ventricle, aorta, and kidney) binding sites for 125I-labelled ET-1. Commencing at age 10 weeks through to 12 weeks, SHR rats but not WKY rats developed a significant increase in MAP (from 152 +/- 7 to 189 +/- 3 mmHg) (1 mmHg = 133.32 Pa). However, in both WKY and SHR rats immunoreactive levels of ET-1RP increased (100 and 80%, respectively) throughout the same measurement period. The potency of ET-1 to contract aortic rings from SHR rats was slightly but not significantly greater than that for aortic rings from WKY rats, although aortic rings from SHR rats contracted in the presence of 0.5 nM ET-1, while those from WKY rats did not. The levels of immunoreactive ET-1RP were significantly reduced (32%) in the kidney and unchanged in the heart and lung of SHR rats compared with WKY rats. Specific 125I-labelled ET-1 binding sites displayed an increase and a significant decrease (24%) of density in the atrium and ventricle, respectively, a significant increase (31%) of affinity in the lung, and were unchanged in the kidney and aorta of SHR rats compared with WKY rats following the development of hypertension. The lack of a correlation between circulating levels of immunoreactive ET-1RP and the development of hypertension coupled with a lack of significant differences in vascular reactivity suggest that ET-1 is not the sole mediator of hypertension in this animal model. However, the tissue-specific changes in immunoreactive ET-1RP and 125I-labelled ET-1 binding sites suggest that ET-1 may be a partial mediator of hypertension and is subject to compensatory changes in response to the increased total peripheral resistance in SHR rats.
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