Peter Raddatz

Substrate Analogue Renin Inhibitors Containing Replacements of Histidine in P2 or Isosteres of the Amide Bond Between P3 and P2 Sites

The search for orally active renin inhibitors as therapeutic agents continues to represent a challenging target for medicinal chemists.1 Analogues of the angiotensinogen region flanking the bond split by renin have turned out to be very potent and specific inhibitors of renin. However, the high affinity of these angiotensinogen analogues for human renin is often associated with fast hydrolysis between P3 and P2 sites by the intestinal serine protease chymotrypsin.2

Evaluation of absorption enhancement for a potent cyclopeptidic ανβ3-antagonist in a human intestinal cell line (Caco-2)

Abstract Different absorption enhancing principles for a potent cyclopeptidic ανβ3-antagonist (EMD 121974) were investigated in monolayers of a human intestinal cell line (Caco-2). Transepithelial transport was quantitated by reversed-phase high-performance liquid chromatography. Cytotoxic effects were characterized by determination of transepithelial electrical resistances (TEERs), propidium iodide (PI)-influx, FITC-phalloidin staining and the release of cytosolic lactate dehydrogenase (LDH). Medium chain fatty acids (MCFAs, NaC10, NaC12) and taurocholate (NaTC) were the most efficient enhancers of cyclopeptide and FITC-dextran 4400 permeability coefficients, displaying different time profiles of activity. Whereas NaTC (15 mM) showed almost a constant permeation enhancing effect from 20 min up to 120 min (ca. 12-fold), MCFA absorption enhancement was markedly dependent on incubation time (NaC10, 20 min: 1.2-fold, 120 min: 17-fold; NaC12, 20 min: 4.3-fold, 120 min: 13-fold). All cytotoxicity assays demonstrated that MCFAs were significantly more cytotoxic than NaTC. Ion pairing with hydrophobic amino acids and heptane sulfonate distinctly increased octanol–buffer partition coefficients of the cationic cyclopeptide but did not enhance its transepithelial permeability. Nanoparticles as well as β-cyclodextrin neither affected integrity of the cells nor transport properties of the cyclopeptide. In summary, significant absorption enhancement was only observed with NaTC or MCFAs. Increase in permeability coefficients using NaTC occurred rapidly with acceptable cytotoxicities and merits further investigations.

Prodrug Approach for αΙΙbβ3-Peptidomimetic Antagonists to Enhance Their Transport in Monolayers of a Human Intestinal Cell Line (Caco-2): Comparison of In Vitro and In Vivo Data

AbstractPurpose. Different lipophilic derivatives of a potent αΙΙbβ3- antagonist with benzamidino-oxazolidinone structure were investigated with respect to transport and metabolism properties to evaluate their potential as prodrugs with improved absorption behavior. Methods. Intestinal transport and metabolism of the compounds were studied in Caco-2 monolayers under in vitro conditions and quantitated by a reversed-phase HPLC- method. Peroral bioavailability in cynomolgus monkeys and inhibition of platelet aggregation (guinea pig) were compared to in vitro permeability coefficients. Results. N-alkoxycarbonyl- and N-benzoyl- derivatization of the benzamidine-parent drug increased the apparent permeabilities across Caco-2 monolayers by a factor of 25-100 fold. Most prodrugs were transported mainly by passive diffusion, whereas the methoxycarbonyl-derivative EMD 122347 displayed directional transport from basolateral (BL) to apical (AP). This polarized efflux was concentration dependent (saturable kinetics with Km = 207 μM, Vmax = 0.275 nmol cm-2 min-1) and could be reduced in the presence of verapamil (300 μM), an inhibitor of p-glycoprotein. Cell mediated cleavage of the prodrugs was low and showed only slight differences to hydrolysis in buffer solution, indicating a predominantly non enzymatic cleavage. Both peroral bioavailability (monkey) and the inhibition of ex-vivo platelet aggregation (guinea pig) gave the same rank order as the permeability coefficients obtained in Caco-2 monolayers. Conclusions. Alkoxycarbonylamidine- and benzoylamidine promoieties of a RGD mimetic αΙΙbβ3-antagonist considerably increased both effect bioavailabilities in animal experiments as well as in-vitro permeability in cell monolayers, demonstrating the potential of this approach to enhance transport of peptidomimetic drugs.