Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Discovery of an orally active series of isoxazoline glycoprotein IIb/IIIa antagonists.

Using isoxazoline XR299 (1a) as a starting point for the design of highly potent, long-duration GPIIb/IIIa antagonists, the effect of placing lipophilic substituents at positions alpha and beta to the carboxylate moiety was evaluated. Of the compounds studied, it was found that the n-butyl carbamate 24u exhibited superior potency and duration of ex vivo antiplatelet effects in dogs. Replacement of the benzamidin-4-yl moiety with alternative basic groups, elimination of the isoxazoline stereocenter, and reversal of the orientation of the isoxazoline ring resulted in lowered potency and/or duration of action.

Discovery of potent isoxazoline glycoprotein IIb/IIIa receptor antagonists.

Using the isoxazoline as a common structural feature, three series of glycoprotein IIb/IIIa receptor antagonists were evaluated, culminating in the discovery of XR299 (30). In an in vitro assay of platelet inhibition, XR299 had an IC50 of 0.24 microM and was a potent antiplatelet agent when dosed intravenously in a canine model. It was shown through X-ray studies of the cinchonidine salt 49 that the receptor required the 5(R)-stereochemistry for high potency. The ethyl ester prodrug of XR299, XR300 (29), was orally active in the dog.

Cyclic HIV protease inhibitors: synthesis, conformational analysis, P2/P2' structure-activity relationship, and molecular recognition of cyclic ureas.

High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.

Epidemiologic variations in childhood sexual abuse.

We retrospectively reviewed records of 566 children ranging from 6 months to 16 years of age who presented to a sexual assault crisis center. They represented 33.2% of all alleged sexual assault victims seen over a 36 month period. There were 103 males (18.2%) and 463 females (81.8%). Significant differences in presentation were demonstrated with respect to the victim's age, sex, and race, but the major factor influencing the variation is the victim/assailant relationship. Younger children were more likely than older children to present with histories of multiple assaults (p less than .0005), by known assailants (p less than .0005), occurring in the child's or assailant's home (p less than .001) and to report less violence (p less than .05). More risk of violence (p less than .0005) or evidence of trauma (p less than .0005) and less reporting of home assaults (p less than .0005) or multiple assault episodes (p less than .0005) were found in assaults by strangers when compared with non-stranger assaults. A lower frequency of reporting was found in females between 7 and 11 years of age. The dynamics of childhood sexual abuse are discussed in relationship to these findings. A number of victim and assailant related factors determine reporting patterns of childhood sexual abuse.

Epidemiologic factors in sexual abuse of boys.

To increase understanding of sexual abuse in boys, we reviewed records of 142 victimized boys ranging in age from 6 months to 17 years. They represented 14% of victims under 18 years of age during a four-year period. Most had histories of single assaults (87%) that involved attempted anal intercourse (78%) by know assailants (59%). Half of the assaults were violent, with evidence of trauma found in 24%. Patterns of abuse were associated with the victim's age and the assailant-victim relationship. Younger victims reported more non-stranger assaults, less violence, and less anal intercourse. Assaults by strangers tended to involve older children and more violence and to occur away from home. Male children are at significant risk of sexual abuse, often at younger ages than girls. Consequences of sexual abuse remain unknown, but may relate to future child abuse.

Sexual abuse of children. Sex-, race-, and age-dependent variations.

In a retrospective review of 416 alleged cases of sexual assault on victims under the age of 16 years, we analyzed the data with respect to race, sex, and age. There were 72 boys (17.3%) and 344 girls (82.7%), with ages ranging from 6 months to 16 years. The male victims were significantly younger than the female victims (respective median ages, 7 and 10 years), reported more violence, and had more evidence of trauma when initially seen. White and Hispanic girls were older than the black female victims (respective median ages, 12 and 8 years), whereas racial origin had no effect on the age distribution among male victims. Younger victims, regardless of sex or racial origin, were more likely than older children to be assaulted repetitively by someone known to them and at home and to experience less violence.