Prediction of time-dependent CYP3A4 drug-drug interactions by physiologically based pharmacokinetic modelling: impact of inactivation parameters and enzyme turnover.

Predicting the magnitude of time-dependent metabolic drug-drug (mDDIs) interactions involving cytochrome P-450 3A4 (CYP3A4) from in vitro data requires accurate knowledge of the inactivation parameters of the inhibitor (K(I), k(inact)) and of the turnover of the enzyme (k(deg)) in both the gut and the liver. We have predicted the magnitude of mDDIs observed in 29 in vivo studies involving six CYP3A4 probe substrates and five mechanism based inhibitors of CYP3A4 of variable potency (azithromycin, clarithromycin, diltiazem, erythromycin and verapamil). Inactivation parameters determined anew in a single laboratory under standardised conditions together with data from substrate and inhibitor files within the Simcyp Simulator (Version 9.3) were used to determine a value of the hepatic k(deg) (0.0193 or 0.0077h(-1)) most appropriate for the prediction of mDDIs involving time-dependent inhibition of CYP3A4. The higher value resulted in decreased bias (geometric mean fold error - 1.05 versus 1.30) and increased precision (root mean squared error - 1.29 versus 2.30) of predictions of mean ratios of AUC in the absence and presence of inhibitor. Depending on the k(deg) value used (0.0193 versus 0.0077h(-1)), predicted mean ratios of AUC were within 2-fold of the observed values for all (100%) and 27 (93%) of the 29 studies, respectively and within 1.5-fold for 24 (83%) and 17 (59%) of the 29 studies, respectively. Comprehensive PBPK models were applied for accurate assessment of the potential for mDDIs involving time-dependent inhibition of CYP3A4 using a hepatic k(deg) value of 0.0193h(-1) in conjunction with inactivation parameters determined by the conventional experimental approach.

Characterizing the mu-conotoxin binding site on voltage-sensitive sodium channels with toxin analogs and channel mutations.

The three-dimensional organization of the domains of the rat skeletal muscle sodium channel subtype 1 (rSkM1) and the toxin-channel interaction surface have been explored by a complementary mutagenesis approach. This method involves probing mutant channels with analogs of the peptide toxin, mu-conotoxin (mu-CTX), for which the tertiary structure has been determined. mu-CTX has an overall net charge of +5. The blocking of Na+ currents of rSkM1 expressed in Xenopus oocytes by mu-CTX analogs in which negative charge had been removed by Asn substitution for Asp or positive charge had been decreased by Gln substitution for Arg or Lys was studied; the mu-CTX analogs exhibited decreased blocking potencies of up to 228-fold compared with an IC50 = 51.4 +/- 2.2 nM for native mu-CTX on wild-type rSkM1. Mutations at Arg 13 of mu-CTX were the most critical in decreasing potency and at Lys9 were the least critical. Charge alone, however, was not the essential factor in some toxin substitutions: the IC50 value for Asp12Asn showed little change while that for Asp12Glu was increased approximately 100-fold due to a change in conformation (revealed by NMR measurements of the toxin in solution). Focusing on the sites in the channel which might be involved in toxin binding, mutations were introduced involving substitutions at more than a dozen mostly anionic sites in putative extracellular residues of rSkM1. The toxin binding results indicate: firstly, many channel mutations at anionic sidechains on the putative extracellular surface of mu-CTX-sensitive channels, thought to be possible sites of interaction with toxin, have been shown to have no effect on toxin binding. Secondly, one channel mutation, rSkM1/Tyr401Cys, (in the loop between S5 and S6 of Domain 1), affected mu-CTX potency causing a 3.7-fold increase in IC50 value. The ratio of toxin blocking potencies was not significantly different when wild-type and the mutant (Tyr401Cys) rSkM1 channels were studied with two toxin analogs, Arg19Gln and Arg13Gln, in contrast to all other toxin derivatives examined. Since Tyr401 is known to be in the channel pore, these results suggest that either or both of the Arg residues at positions 13 and 19 of mu-CTX interact(s) with residue Tyr401 of rSkM1 and, therefore, indicate that mu-CTX extends into the pore region of the channel.

Potent peptide inhibitors of stromelysin based on the prodomain region of matrix metalloproteinases.

Stromelysin is secreted as an inactive zymogen that is activated in the extracellular space by cleavage of the His81-Phe82 bond with the release of the 81-amino acid propeptide domain. This segment contains a 12-amino acid sequence (MRKPRC75GVPDVG) that is highly conserved in all matrix metalloproteinases. Previous studies have shown that the hexapeptide, Ac-RCGVPD-NH2, and the pentapeptide, Ac-RCGVP-NH2, based on this region retain significant inhibitory activity. This new structure-activity relationship study of both peptides has shown that only Cys75 and Val77 are essential for inhibitory activity. Peptides based on this series inhibited stromelysin and collagenase with equal potency. Additional peptides spanning this region were synthesized in order to focus on these two sites. Significantly, isocysteine was substituted for Cys75 without significant loss of inhibitory activity. Tyr-(2,6-dichlorobenzyl) was substituted for Val77. The introduction of these 2 new residues into Ac-CGVP-NH2 produced a very potent inhibitor, Ac-isoCGY-(2,6 dichlorobenzyl)-P-NH2 with an IC50 of 3 microM. The following factors, acting in combination, determine the inhibitory activity of peptides in this series: distance between the sulfur atom and the peptide backbone, coordination geometry of the thiol side chain with the active-site zinc, and conformational flexibility of the side-chain.

Inhibition of human stromelysin by peptides based on the N-terminal domain of tissue inhibitor of metalloproteinases-1.

The tissue inhibitors of metalloproteinases (TIMPs) represent a family of naturally occurring protein inhibitors of stromelysin and other members of the family of matrix metalloproteinases. A series of peptides based on the N-terminal sequence of natural TIMP-1 was synthesized and assessed for inhibitory activity against purified human stromelysin. Inhibitor peptides were identified in the loop (bounded by the disulfide bonds [C3-C99] and [C13-C124]), e.g., [C3(Acm)-C13], (IC50, 42 microM). It was established that inhibition was due to the free sulfhydryl group of either C13 or C124. However, peptides within [C70(Acm)-C98(Acm)] inhibited stromelysin independently of zinc co-ordination by cysteine. The binding epitope in TIMP-1 may be discontinuous and comprised of sequences from at least 2 loops.

High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations.

We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*1101 molecules, by screening of an M13 peptide display library (approximately 20 million independent nonapeptides) for DR-binding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect of anchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6- or 7-residue-long designer peptides is shown to result in a gradual increase of binding affinity to the level of 13-residue-long high-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elimination of other anchors results only in partial loss of binding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins.

Production of monoclonal antibodies to prostromelysin (ProMMP-3) and establishment of a quantitative prostromelysin ELISA assay.

Human prostromelysin (59 kDa) was purified from the conditioned medium of IL-1-stimulated human dermal fibroblasts and anti-prostromelysin monoclonal antibodies were produced and identified by ELISA assay. Using prostromelysin, a C-terminally truncated recombinant form of prostromelysin consisting of amino acids 1-255, and their respective activated enzymes, we have begun mapping the epitopes recognized by these monoclonal antibodies. Various patterns of reactivity against the proenzymes and activated enzymes were observed. In further attempts to map the epitopes, we employed synthetic peptides representing hydrophilic regions of the primary amino acid sequence of prostromelysin. Our monoclonal antibodies did not recognize these peptides, suggesting that the antibodies may be recognizing conformational epitopes composed of non-linear portions of prostromelysin. Using these monoclonal antibodies, we have developed a quantitative prostromelysin sandwich ELISA assay.

Peptides based on the conserved predomain sequence of matrix metalloproteinases inhibit human stromelysin and collagenase.

Prostromelysin, a member of the family of matrix metalloproteinases, is secreted as a zymogen which is activated after cleavage of the His81-Phe82 bond. The 82 amino acid propeptide that is removed during activation contains 12 amino acids, MRKPRC75GVPDVG, that are highly conserved in all MMPs. We evaluated a series of peptides that span this region for their ability to inhibit stromelysin. The hexapeptide, Ac-RCGVPD, and the pentapeptide, Ac-RCGVP had IC50 values of approx. 10 microM. The tetrapeptide, Ac-RCGV, was somewhat less potent with an IC50 of 60 microM. Smaller peptides, e.g. Ac-RCG, were significantly less potent as inhibitors. Substitutions of Cys75 with Ser resulted in a complete loss of inhibitory activity. The peptides in this series also inhibited human fibroblast collagenase.

Characterization of a tight-binding MMP-3 inhibitor using improved fluorescence spectroscopy techniques.

Accurate kinetic characterization of stromelysin (MMP-3) inhibitors is critical in the design of potent inhibitors of this enzyme. We have successfully modified a previously described assay [1] which used an internally quenched peptide substrate (Dnp-PYAYWMR) that, upon cleavage by MMP-3, produces the products, Dnp-PYA (quiet) and YWMR (a fluorophore at 360 nm). This improved assay uses purified human MMP-3 in the presence of either 5% methanol or 5% DMSO. Fluorescence intensities associated with total hydrolysis of substrate by enzyme have been successfully mimicked using a combination of the product peptides as a standard. We have determined a Km of 39.2 microM and Kcat/Km of 4.6 microM/h for MMP-3 (in 5% MeOH) using this peptide substrate. This assay was used successfully to characterize Ro 31-4724 ((N-[(N-[2-[(N-hydroxycarbamoyl)methyl]-4-methyl-valeryl]-L-leucyl ] - L-alanine ethyl ester) as a reversible, tightly binding, inhibitor with a Ki of 26 nm.

Thermal laser-assisted balloon angioplasty of the superficial femoral artery: a multicenter review of 602 cases.

A multicenter review of 602 procedures was performed to evaluate the effects of thermal laser-assisted balloon angioplasty on the superficial femoral artery. Four hundred thirty-nine (73%) patients underwent laser-assisted balloon angioplasty for claudication, and 163 (27%) underwent the procedure for limb salvage. Two hundred ninety-two laser-assisted balloon angioplasty procedures were performed for multifocal stenotic disease (greater than 80%) diameter reduction), 258 for total occlusion, and 52 procedures for both superficial femoral artery stenosis and occlusion. The initial recanalization rate was 89% (538/602) depending on the length of the lesion. Success of laser-assisted balloon angioplasty was verified by angioscopy or arteriography, and all patients underwent segmental Doppler studies before discharge and during midterm follow-up extending to 30 months (mean, 11.3 months). Complications occurred in 62 (10%) patients, but only one limb amputation occurred because of a complication related to a laser-assisted balloon angioplasty. Overall, 60% of initially successful procedures have remained patent, but long segment (greater than 7 cm) occlusions have fared poorly (25% patency at 30 months). This minimally invasive technology affords the opportunity to treat short segment (less than 7 cm) symptomatic superficial femoral artery occlusive disease with minimal risk. Initial success and midterm patency rates for appropriate lesions appear to make laser-assisted balloon angioplasty a viable adjunct in the treatment of superficial femoral artery occlusive disease.