Valaminols, probably the most simplified peptide-analogs acting as pepsin inhibitors.

Recently, we have introduced simplified analogs of pepstatin A, representing 'tripeptides' with two valine residues which are C-terminated by an amino alcohol moiety. In the present study, we have deleted one valine unit, yielding simple molecules-called 'valaminols'-which can be prepared in big scale under really low costs. First investigations on structure-activity relationships revealed that the most active compound is a valaminol bearing both natural substituents either at the N-terminus or at the C-terminal side chain. Its inhibitory activity fully corresponds to the activity of tripeptides, hitherto reported by us, although one valine residue has been omitted.

The dermal delivery of lignocaine: influence of ion pairing.

The purpose of the present study was to determine the significance of ion pairing on the permeation of lignocaine. Results of diffusion studies through polydimethylsiloxane (PDMS) at different pH values 4. 0, 6.0, 7.0, 8.0 indicated that lignocaine hydrochloride (L-HCl) flux significantly increased with the amount of unionized base. In order to see if similar results could be obtained using human skin, permeation runs were performed with human skin at pH of 4.0, 5.5 and 7.0. These values were chosen to simulate an appropriate range of physiological conditions. Results of the experiments with human epidermis showed increasing L-HCl flux with increasing pH, confirming the trends seen with PDMS membranes. A linear relationship was found between the apparent partition coefficient and the steady state flux. Further experiments were conducted at donor pH 4.0 to minimise the contribution of the unionized species. Although an excess of different ions such as nitrate, mesylate and bromide increased the apparent partition coefficient, the steady state flux was not significantly increased. The steady state lignocaine flux was increased up to 2.45-fold using different counter ions. The highest flux was measured from lignocaine morpholinopropane sulfonate (L-mps). It is possible to enhance the flux of salts across lipophilic membranes by using an ion pair approach. The degree to which this is possible depends on the lipophilicity of the counter ion, the medium in which the ion pair forms, and the ionic strength.

Synthesis, development and in vitro evaluation of drug delivery systems with protective effect against degradation by pepsin.

A (poly)peptide drug delivery system providing a protective effect against degradation by pepsin has been generated. A simplified pepstatin analogue was thereby synthesised displaying a terminally located primary amino group allowing an easy covalent attachment to anionogenic mucoadhesive polymers by the formation of amide bonds. The IC50 of this novel inhibitor was determined to be 6.65+/-1.05x10(-6) M. Mediated by a carbodiimide it was covalently bound to polycarbophil and sodium carboxy-methyl cellulose (NaCMC). In contrast to polycarbophil-inhibitor conjugates, NaCMC-inhibitor conjugates displayed a high inhibitory effect towards pepsin. The protective effect of tablets containing a NaCMC-pepsin inhibitor conjugate (10%), NaCMC (56.7%), insulin (3.3%), and mannitol (30%) was evaluated in vitro. Tablets were therefore incubated for 2 h at 37 degrees C with simulated gastric fluid according to the Pharmacopoeia Europea. Following analysis demonstrated that 50.8+/-8.6% (mean +/- SD; n = 3) of insulin were degraded within the swollen carrier matrix, whereas insulin was completely metabolised in tablets without the NaCMC-pepsin inhibitor conjugate. This protective effect against degradation by pepsin might make such dosage forms useful tools for a targeted (poly)peptide delivery to the stomach.

Synthesis and in vitro multidrug resistance modulating activity of a series of dihydrobenzopyrans and tetrahydroquinolines.

A series of dihydrobenzopyrans and tetrahydroquinolines was synthesized and pharmacologically tested for their ability to inhibit P-glycoprotein mediated daunomycin efflux in multidrug resistant CCRF-CEM vcr1000 cells. Several compounds exhibit activities in the range of the reference compounds verapamil and propafenone. Preliminary structure-activity relationship studies propose the importance of high molar refractivity values of the compounds and the presence of an additional basic nitrogen atom.

Simplified pepstatins: synthesis and evaluation of N-terminally modified analogues.

The promising strategy of gastric ulcer healing with perorally administered epidermal growth factor (EGF) is so far strongly limited by the pepsinic degradation of this therapeutic polypeptide in the stomach. The incorporation of EGF in a bioadhesive polymer-pepsin inhibitor conjugate used as drug carrier matrix, however, might provide sufficient protection toward pepsinic degradation. The synthesis of appropriate pepsin inhibitors represents a prerequisite for the development of such polymer-inhibitor conjugates. The presented study demonstrates that modifications at the N-terminus of simplified analogues of pepstatin which can be synthesized in a simple and straight way result only in slight variations of the inhibitory activity. These analogues display only 10-fold reduced inhibitory activity, compared to pepstatin A, when bearing a greater N-terminal group like isovaleryl, Boc, or Cbz. Compounds which are substituted at the N-terminus by a shorter N-acyl group like propionyl or cyclopropylcarbonyl show further reduced activity (0.01, compared to pepstatin A). The presence of an amide or a urethane moiety at the N-terminus has no considerable effect on enzyme inhibition. Therefore, the N-terminus of these analogues is able to be modified forming a covalent bond to various bioadhesive polymers via a suitable functionality.

Auxiliary agents for the peroral administration of peptide and protein drugs: synthesis and evaluation of novel pepstatin analogues.

The peroral administration of (poly)peptide drugs requires the development of delivery systems, which provide a protective effect toward a gastrointestinal enzymatic attack. A promising strategy for such systems represents polymer-enzyme inhibitor conjugates in which the embedded therapeutic agent is protected. However, the practical use of polymer-inhibitor conjugates has so far been limited by high production costs of these auxiliary agents. To solve this problem for delivery systems shielding from pepsinic degradation, structurally simplified analogues of the pepsin inhibitor pepstatin A have been synthesized. The synthesis of tripeptide analogues, described by McConnell et al., led us to pursue further modifications varying the C-terminus. Our target to attach a spacer moiety-enabling the free access of pepsin to the inhibitor-should be combined with an attractive synthetic approach providing low production costs in large-scale preparation. Structure modifications comprised either the side chain of the third amino acid which served as starting compound designing the C-terminus (L-leucine, L-isoleucine, L-norvaline) as the length of the spacer link, simulated by a linear alkyl group (n-butyl, n-hexyl, and n-octyl). The inhibitory activities which have been evaluated by an enzyme assay were significantly dependent on the nature of the side chain, whereas the length of the spacer had no influence on the inhibitory effect. Analogues bearing the isobutyl or n-propyl moiety as side chain displayed a strong inhibitory effect which was comparable to that pepstatin A. These congeners represent promising auxiliary agents for the peroral administration of (poly)peptide drugs.

Binding and structure-activity-relation of benzo[f]isoquinoline- and norcodeinone-derivatives at mu-opioid receptors in the rat cerebral cortex.

1. We have probed the ligand binding site of the mu-opioid receptor using a series of isoquinoline- and norcodeinone-derivatives; in these morphine- and codeine-analogues, the position of the piperidine-nitrogen as well as its mobility is altered relative to that found in morphine. 2. The mu-receptor in rat cortical membranes was labelled with [3H]-naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine-nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity. 3. In contrast, we find that the piperidine-nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl-group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)-10, 11-epoxy-1, 2, 3, 4, 5, 6-hexahydro-9-hydroxy-3-methyl-4a,10b-butano- benzo[f]isochinolin-12-on(10). This compound is an agonist with an affinity comparable to that of morphine. 4. We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the mu-opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2A distance that exists between the position of the nitrogen in morphine and WB4/PH.