Safety, tolerance, and pharmacokinetic studies of OPT-80 in healthy volunteers following single and multiple oral doses.

Current therapies for Clostridium difficile infection (CDI) are encumbered by treatment failures and recurrences. Due to its high in vitro activity against C. difficile but low activity against the typical intestinal flora, minimal absorption, and durable cure in the hamster model of C. difficile infection, OPT-80 was considered for clinical development as a therapy for CDI. This trial consisted of two phases. Four single oral doses of OPT-80 (100, 200, 300, and 450 mg) were administered in a crossover manner to 16 healthy volunteers in a double-blind, placebo-controlled phase 1A study; a 1- to 2-week washout interval separated the treatments. In the double-blind phase 1B study, 24 healthy subjects were randomized to receive OPT-80 (150, 300, or 450 mg) or placebo for 10 days. In both studies, OPT-80's safety and tolerability were evaluated and the concentrations of OPT-80 and its primary metabolite (OP-1118) in plasma and feces were determined. OPT-80 levels in the urine were also analyzed for the phase 1A study. In both the single-dose and the multiple-dose studies, OPT-80 was well tolerated by all subjects in all dose groups. Maximal plasma concentrations were near or below the limit of quantification (5 ng/ml) across the dose range; urine concentrations were below the detection limit. The fecal total recovery of OPT-80 plus its major metabolite, OP-1118, approximated 100%. The tolerability, high fecal concentration, and low systemic exposure data from these studies support the further clinical development of OPT-80 as an oral therapy for CDI.

Synthesis of solid-supported mirror-image sugars: a novel method for selecting receptors for cellular-surface carbohydrates.

We introduced a novel method, through mirror-image phage display, for the identification of high-affinity D-peptides to target specific cell-surface carbohydrates. Both 3-deoxy-alpha-L-manno-2-octulosonic acid (L-KDO) and L-sialic acid and an L-sialo-disaccharide have been synthesized and attached to a solid support for selection of high-affinity peptide binders displayed on phages. Our initial studies in this effort produce single-chain Fab sequences and dodecapeptides that bind to sialic acid and KDO with nanomolar and high micromolar affinity.

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis.

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis.

Binding studies with intact rat prolactin and a 16K fragment of the hormone.

A cleaved form of PRL has been found in the pituitary of rodents and humans and in human blood. A 16K fragment derived from cleaved rat (r) PRL has mitogenic and lactogenic activities in PRL bioassays. In this report we analyzed whether 16K rPRL acts through PRL and/or specific 16K receptors. The 16K PRL displaced 23K PRL label with a potency that varied among different tissues. With liver membranes the fragment had only 5-6% the potency of intact PRL, but in whole brain its potency was similar to that of the 23K form. By contrast, 16K PRL was 30 times more effective at displacing [125I]23K rPRL from the kidney membranes than was intact PRL. [125I]16K PRL bound specifically to membranes of kidney, liver, brain, and skeletal muscle, and it was not displaced by intact or cleaved 23K rPRL, intact ovine PRL, or rat or bovine GH. The specific binding of 16K PRL to the kidney changed with incubation time, temperature, and pH and was significantly higher than that of 23K PRL to brain and kidney membranes. However, the specific binding of the 16K was lower in liver and skeletal muscle. The binding sites for 16K PRL in the kidney had a higher affinity than did those for 23K PRL (Kd = 6.9 x 10(-9) vs. 0.8 x 10(-8), respectively), but the opposite was found in the other tissues. The specific 16K PRL-binding sites in different tissues may represent receptors that mediate actions of the fragment that are unshared by intact PRL. Furthermore, 16K PRL binds to 23K receptors with an activity that varies among the target tissues. These results support the notion of distinct PRL isoreceptors in different tissues.

Biological and immunological characterization of cleaved and 16K forms of rat prolactin.

A cleaved form of rat PRL (rPRL) has been reported to exist in the pituitary gland and to be the main product of PRL proteolysis by its target tissues. A 16K fragment derived from cleaved PRL (cPRL) has mammary mitogenic activity in rats in vivo, and we found that both cleaved and 16K PRLs are capable of binding to hepatic PRL receptors. To analyze the functional significance of cleaved and 16K PRLs, ample amounts of cPRL were generated by incubation of 24K rPRL in vitro with a 25,000 X g rat mammary gland pellet. cPRL was reduced (2-mercaptoethanol), and the 16K and 8K fragments were separated by gel filtration. The purity of the cleaved and 16K samples was established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their mitogenic [pigeon crop-sac, Nb2 lymphoma cell, and mammary cell bioassays (BAs)], lactogenic (casein BA), and immunologic (PRL RIA) activities were determined. cPRL had the same mitogenic activity, but half the immunoactivity, as intact PRL. The 16K fragment was biologically active in all assays. Its estimated mitogenic and lactogenic potencies were 65% and 10% those of the 24K PRL, respectively, and it had only 2% the immunoactivity of the intact hormone. Thus, the 16K fragment had higher BA to RIA ratios than cleaved or intact 24K PRLs. The functional significance of 16K PRL may have been underestimated owing to its low RIA activity.