Treatment with insulin-like growth factor-1 increases chondrogenesis by periosteum in vitro.

The repair of defects in articular cartilage with hyaline tissue that is resilient to wear is a challenging problem. Fibrocartilaginous tissue forms in response to injury through the articular surface and degenerates under mechanical load. Because periosteum contains cells, which are capable of synthesizing cartilage matrix proteins, it has been used to repair defects in articular surfaces. Treatment of periosteal grafts with growth factors, particularly those that elicit chondrocyte gene expression, may improve tissue regeneration. Gene expression by periosteal explants in vitro was measured. Expression of type II collagen and aggrecan mRNA was increased in response to treatment with IGF-I. Furthermore, IGF-I treatment caused an increase in type II collagen and aggrecan mRNA that was time and concentration dependent. The effect of short and long-term (continuous) incubations was compared to determine if a pretreatment could be used to condition a graft for subsequent surgical use. Short-term incubation in vitro with IGF-I followed by incubation without IGF-I was nearly as effective at increasing expression of type II collagen and aggrecan mRNA as incubation for the same length of time with IGF-I present continuously in the culture media. Treatment with IGF-I also produced cell clustering and nodule formation which are indicative of chondrogenesis. These results suggest that pretreatment with IGF-I in vitro may enhance the effectiveness of a graft to produce hyaline cartilage in vivo. Whether the cellular and molecular changes we have observed can lead to the formation of tissue that withstands the mechanical forces exerted by weight bearing remains to be determined.

2',6'-Dimethylphenoxyacetyl: a new achiral high affinity P(3)-P(2) ligand for peptidomimetic-based HIV protease inhibitors.

Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1-2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors.

Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy.

A new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P2 and P3 positions has been identified. The optimized inhibitors have IC50 values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and 7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development.

Novel small renin inhibitors containing 4,5- or 3,5-dihydroxy-2-substituted-6-phenylhexanamide replacements at the P2-P3 sites.

Renin inhibitors containing a 4,5- or a 3,5-dihydroxy-2-substituted-6-phenylhexanamide fragment at the P2-P3 sites have been prepared and evaluated. The four possible diastereomeric diols of the two series of inhibitors were synthesized to determine the optimal configuration of the carbinol centers for these replacements. The most potent inhibitors of each series, la and 2c have a molecular weight of only 503 and IC50 values of 23 and 20 nM in a human plasma renin assay at pH 6.0. Their very low aqueous solubility limited their further evaluation. The efficacy of these P2-P3 replacements is a result of their ability to maintain the important hydrogen-bonds with the enzyme. Due to conformational differences with the dipeptide, adjustment at the P2 side chain was required. These 4,5- and 3,5-dihydroxyhexanamide segments could be seen as novel N-terminal dipeptide replacements.

Potent HIV protease inhibitors containing a novel (hydroxyethyl)amide isostere.

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.

Antiviral properties of palinavir, a potent inhibitor of the human immunodeficiency virus type 1 protease.

Palinavir is a potent inhibitor of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases. Replication of laboratory strains (HIV-1, HIV-2, and simian immunodeficiency virus) and HIV-1 clinical isolates is inhibited by palinavir with 50% effective concentrations ranging from 0.5 to 30 nM. The average cytotoxic concentration of palinavir (35 microM) in the various target cells indicates a favorable therapeutic index. Potent antiviral activity is retained with increased doses of virus and with clinical isolates resistant to zidovudine (AZT), didanosine (ddI), or nevirapine. Combinations of palinavir with either AZT, ddI, or nevirapine demonstrate synergy or additivity in the inhibition of HIV-1 replication. Palinavir retains anti-HIV-1 activity when administered postinfection until times subsequent to the reverse transcription step. In chronically infected CR-10 cells, palinavir blocks Gag precursor polyprotein processing completely, reducing greater than 99% of infectious particle production. The results indicate that the antiviral activity of palinavir is specific to inhibition of the viral protease and occurs at a late stage in the replicative cycle of HIV-1. On the basis of the potent in vitro activity, low-level cytotoxicity, and other data, palinavir was selected for in-depth preclinical evaluation.

Spider bites in the United States.

The bite of the brown recluse spider rapidly causes an infarction in skin that is painful and can be slow to heal. This is the only North American spider to cause such severe necrosis. The bite sometimes can produce a sudden, severe hemolysis. Conservative care without drugs is the safest proven treatment.

Several polyhydroxymonoamide renin inhibitors assume similar conformations in the unbound and renin-bound states.

The solution conformations of three polyhydroxymonoamide renin inhibitors which differ in the relative configuration and position of the hydroxyl groups at the P3 position were investigated by NMR spectroscopy. The NMR data are consistent with a predominant conformation in DMSO with the exception that two inhibitors exhibit conformational averaging about a torsion angle along P3. Comparisons with the renin-bound structures determined by X-ray crystallography [Tong et al., (1995) J. Mol. Biol. 250, 211] show that the unbound and renin-bound conformations are similar (with exceptions in the P3 position). This similarity suggests that gross conformational changes of the inhibitor are not a prerequisite for binding to renin. Apart from being able to tolerate different dihydroxylated structures at P3, renin can also accommodate different conformations at P3. Differences were observed at the P3 position between the inhibitors in the unbound state, between the unbound and renin-bound states, and between the renin-bound states.

High resolution crystal structures of recombinant human renin in complex with polyhydroxymonoamide inhibitors.

The crystal structures of recombinant glycosylated human renin in complex with several polyhydroxymonoamide inhibitors have been determined at up to 1.8 A resolution. The high resolution structures permit a detailed analysis of the conformation of renin, the interactions between the inhibitors and renin, and the network of ordered water molecules. The polyhydroxymonoamide inhibitors are bound with their backbones in an extended conformation, and with their side-chains occupying the S3 to S1 pockets. The inhibited renin molecules are shown to exist in both the closed and the open conformations. Inhibitors bound to the two distinct forms of renin can assume different conformations at the P3 position.