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Protection of enkephalins against enzymatic degradation by the prodrug approach
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enzymatic stability as well as the lipophilicity of the derivative were investigated. The results obtained show that attachment of a phthalidyl group to the imidazole moiety of TRH makes its pyroglutamy1 peptide bond resistant towards cleavage by the TRHspecific pyroglutamyl aminopeptidase ( PAPase II) in serum. Since the N-phthalidyl group is readily removed from TRH by enzymatic hydrolysis effected by non-specific plasma esterases not attacking the pyroglutamyl peptide bond, Nphthalidylation of TRH may thus be a promising approach to obtain prodrug forms of TRH with the aim of protecting the peptide against rapid inactivation in the systemic circulation. Like previously studied N-alkoxycarbonyl derivatives the phthalidyl derivative did not protect TRH against cleavage by unspecific pyroglutamyl aminopeptidase (PAPase I) or intestinal prolyl endopeptidase. Regarding the feasibility of obtaining lipophilic prodrug forms capable of penetrating the blood-brain barrier, the lipophilicity of N-phthalidyl TRH is certainly too low (log P = - 1.25 ) . However, it should be recognized that it is readily possible to increase the lipophilicity by introducing e.g. alkyl groups in the phthalidyl phenyl moiety. Protection of enkephalins against enzymatic degradation by the prodrug approach. G.J. Rasmussen and H. Bundgaard, Dept. Pharm. Chem., Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark. A major obstacle to the application of peptides as clinically useful drugs is their poor biomembrane penetration and rapid enzymatic degradation. A promising approach to diminish these delivery problems of peptide drugs is derivatization of the peptides to produce prodrugs or transport forms which are more lipophilic than the parent peptides and capable of protecting these against degradation by enzymes present at the mucosal absorption barrier or in the blood. In the present work, various 4-imidazolidinone derivatives of Leu-enkephalin and Met-enkephalin were assessed as prodrug forms for these pentapeptides. Such derivatives were readily obtained by reacting the peptides with various aldehydes or ketones. The kinetics of hydrolysis of the derivatives were studied in aqueous buffer solutions and in the presence of various enzymes. Whereas the parent enkephalins were rapidly hydrolyzed by a purified aminopeptidase as well as in human plasma solutions, the 4-imidazolidinone derivatives were totally resistant to enzymatic cleavage in these media. The derivatives proved also to be much more stable than the parent peptides in rabbit intestinal homogenates. On the other hand, these derivatives are readily bioreversible, being converted to the parent enkephalin by spontaneous hydrolysis. The rate of hydrolysis was found to be dependent on the structure of the aldehyde or ketone used for the derivatization. For the nine 4-imidazolidinones studied the half-lives of the hydrolysis at pH 7.40 and 37 “C ranged from 3.1 to 149 h. The results suggest that 4-imidazolidinone formation can be a useful prodrug approach to protect the N-terminal amino
acid residue of enkephalins and other related peptides against cleavage by aminopeptidases. Furthermore, prodrug derivatives with increased lipophilicity can readily be obtained by appropriate selection of the carbonyl pro-moiety. Molecular modelling of asperlicine derived cholecystokinin-A receptor antagonists. A. van der Bent, A.M. ter Laak, B.J.M. van Vlijmen, A.P. IJzerman and W. Soudijn, Centerfor BioPharmaceutical Sciences, Division of Medicinal Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Cholecystokinin is a peptide involved in hormonal regulation and neurotransmission. Based on radioligand binding studies, two receptor subtypes for this peptide have been classified: the predominantly peripheral CCK-A receptor and the central CCK-B receptor. Devazepide (MK-329, L-364, 7 18 ) displays the highest affinity towards the CCK-A receptor observed so far. With this molecule as a template it was found that the absolute configuration at C3 is important for affinity, the S-isomer being far more potent. Furthermore, replacement of the 2-indolyl group by an ortho substituted phenyl ring generally decreases affinity considerably, whereas meta and para substituents are rather well tolerated (B.E. Evans et al., J. Med. Chem. (1988) 31, 2235). These data prompted us to investigate the conformational characteristics of these compounds in relation to affinity by means of molecular modelling. Quantum-chemical calculations were carried out with the semi-empirical molecular orbital package MOPAC using the AM1 Hamiltonian; visualization of data was achieved with CHEM-X, a molecular modelling package. The R and S isomers can be fitted in such a way that the aromatic side chains and benzoyl group overlap at the expense of matching the seven-membered ring and 5-phenyl group. Biological data seem to reflect this “upside down” mode of binding of the less potent R isomers. Also, benzolactam antagonists, highly potent compounds with a different seven-membered ring and alkyl chains replacing the 5-phenyl group, tit well into this model. With respect to the precise orientation of the C3 side chain it appeared that high affinity compounds prefer (near) coplanarity of the amide bond and indolyl or phenyl groups, whereas the low affinity ortho substituted compounds direct these groups in an out-of-plane orientation. Therefore, it seems likely that the C3-side chain binds to the receptor in a (near) planar conformation. Mechanistic evaluation of the intestinal absorption of GDOPA in humans, using a newly developed in vivo intestinal perfusion instrument, L.oc-I-Gut. H. Lennemis’, D. Nilsson’, SM. Aquilonius’, L.K. Paalzow’, 0. Ahrenstedt3, L. Knutssot? and ‘Dept. of Biopharmaceutics and PharmacokiR. Hlllgren’, netics, Uppsala University,S-751 23 Uppsala,Sweden ‘Dept. of Neurology, UniversityHospital, Uppsala 3Dept. of Surgery and Internal Medicine, UniversityHospital, Uppsala. Intubation techniques in humans have been used for several decades to investigate digestive processes. These multichannel tubes made it possible to perform simultaneous mea-
enzymatic stability as well as the lipophilicity of the derivative were investigated. The results obtained show that attachment of a phthalidyl group to the imidazole moiety of TRH makes its pyroglutamy1 peptide bond resistant towards cleavage by the TRHspecific pyroglutamyl aminopeptidase ( PAPase II) in serum. Since the N-phthalidyl group is readily removed from TRH by enzymatic hydrolysis effected by non-specific plasma esterases not attacking the pyroglutamyl peptide bond, Nphthalidylation of TRH may thus be a promising approach to obtain prodrug forms of TRH with the aim of protecting the peptide against rapid inactivation in the systemic circulation. Like previously studied N-alkoxycarbonyl derivatives the phthalidyl derivative did not protect TRH against cleavage by unspecific pyroglutamyl aminopeptidase (PAPase I) or intestinal prolyl endopeptidase. Regarding the feasibility of obtaining lipophilic prodrug forms capable of penetrating the blood-brain barrier, the lipophilicity of N-phthalidyl TRH is certainly too low (log P = - 1.25 ) . However, it should be recognized that it is readily possible to increase the lipophilicity by introducing e.g. alkyl groups in the phthalidyl phenyl moiety. Protection of enkephalins against enzymatic degradation by the prodrug approach. G.J. Rasmussen and H. Bundgaard, Dept. Pharm. Chem., Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark. A major obstacle to the application of peptides as clinically useful drugs is their poor biomembrane penetration and rapid enzymatic degradation. A promising approach to diminish these delivery problems of peptide drugs is derivatization of the peptides to produce prodrugs or transport forms which are more lipophilic than the parent peptides and capable of protecting these against degradation by enzymes present at the mucosal absorption barrier or in the blood. In the present work, various 4-imidazolidinone derivatives of Leu-enkephalin and Met-enkephalin were assessed as prodrug forms for these pentapeptides. Such derivatives were readily obtained by reacting the peptides with various aldehydes or ketones. The kinetics of hydrolysis of the derivatives were studied in aqueous buffer solutions and in the presence of various enzymes. Whereas the parent enkephalins were rapidly hydrolyzed by a purified aminopeptidase as well as in human plasma solutions, the 4-imidazolidinone derivatives were totally resistant to enzymatic cleavage in these media. The derivatives proved also to be much more stable than the parent peptides in rabbit intestinal homogenates. On the other hand, these derivatives are readily bioreversible, being converted to the parent enkephalin by spontaneous hydrolysis. The rate of hydrolysis was found to be dependent on the structure of the aldehyde or ketone used for the derivatization. For the nine 4-imidazolidinones studied the half-lives of the hydrolysis at pH 7.40 and 37 “C ranged from 3.1 to 149 h. The results suggest that 4-imidazolidinone formation can be a useful prodrug approach to protect the N-terminal amino
acid residue of enkephalins and other related peptides against cleavage by aminopeptidases. Furthermore, prodrug derivatives with increased lipophilicity can readily be obtained by appropriate selection of the carbonyl pro-moiety. Molecular modelling of asperlicine derived cholecystokinin-A receptor antagonists. A. van der Bent, A.M. ter Laak, B.J.M. van Vlijmen, A.P. IJzerman and W. Soudijn, Centerfor BioPharmaceutical Sciences, Division of Medicinal Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Cholecystokinin is a peptide involved in hormonal regulation and neurotransmission. Based on radioligand binding studies, two receptor subtypes for this peptide have been classified: the predominantly peripheral CCK-A receptor and the central CCK-B receptor. Devazepide (MK-329, L-364, 7 18 ) displays the highest affinity towards the CCK-A receptor observed so far. With this molecule as a template it was found that the absolute configuration at C3 is important for affinity, the S-isomer being far more potent. Furthermore, replacement of the 2-indolyl group by an ortho substituted phenyl ring generally decreases affinity considerably, whereas meta and para substituents are rather well tolerated (B.E. Evans et al., J. Med. Chem. (1988) 31, 2235). These data prompted us to investigate the conformational characteristics of these compounds in relation to affinity by means of molecular modelling. Quantum-chemical calculations were carried out with the semi-empirical molecular orbital package MOPAC using the AM1 Hamiltonian; visualization of data was achieved with CHEM-X, a molecular modelling package. The R and S isomers can be fitted in such a way that the aromatic side chains and benzoyl group overlap at the expense of matching the seven-membered ring and 5-phenyl group. Biological data seem to reflect this “upside down” mode of binding of the less potent R isomers. Also, benzolactam antagonists, highly potent compounds with a different seven-membered ring and alkyl chains replacing the 5-phenyl group, tit well into this model. With respect to the precise orientation of the C3 side chain it appeared that high affinity compounds prefer (near) coplanarity of the amide bond and indolyl or phenyl groups, whereas the low affinity ortho substituted compounds direct these groups in an out-of-plane orientation. Therefore, it seems likely that the C3-side chain binds to the receptor in a (near) planar conformation. Mechanistic evaluation of the intestinal absorption of GDOPA in humans, using a newly developed in vivo intestinal perfusion instrument, L.oc-I-Gut. H. Lennemis’, D. Nilsson’, SM. Aquilonius’, L.K. Paalzow’, 0. Ahrenstedt3, L. Knutssot? and ‘Dept. of Biopharmaceutics and PharmacokiR. Hlllgren’, netics, Uppsala University,S-751 23 Uppsala,Sweden ‘Dept. of Neurology, UniversityHospital, Uppsala 3Dept. of Surgery and Internal Medicine, UniversityHospital, Uppsala. Intubation techniques in humans have been used for several decades to investigate digestive processes. These multichannel tubes made it possible to perform simultaneous mea-