PEGylated Cholecystokinin: I. Preparation and characterization II. Pharmacological aspects of food intake
GePEGyleerd Cholecystokinine: I. Synthese en identificatie II. Farmacologische aspecten van voedselinname
Leon Tamariz, Fabian; M0330417
Cholecystokinin (CCK) is a neuro-gastrointestinal peptide playing an important role in the energy intake homeostasis. In spite of the amount ofpublished literature resulting from many years of research on its working mechanism, a lot of controversy on this topic still exists. Two analytical problems resulting from some biological properties of CCK have been mentioned as reasons for this disagreement. The first is the extremelyrapid degradation of exogenously injected CCK (T½ = 1-2 min); and the second is the lack of a reliable analytical method that allows the accurate measurement of CCK in biological samples.The aim of this work was to synthesize a long-acting CCK that allows aneasier analysis of the peptides working mechanism and in a later stadium its relationship with other gastrointestinal hormones and molecules with possible anorexigenic activity as for example the cyanoglucoside simmondsin. In order to achieve our goal, the CCK analog CCK-9 was covalently coupled to one molecule of the synthetic polymer poly (ethylene glycol) (PEG). This conjugation reaction receives the name of PEGylation and has beenrecently proposed as an alternative to solve some typical problems in the therapeutics with small drug molecules, peptides and proteins such asthe short circulation half-life, toxicity, immunogenicity, proteolytic degradation and others. The rationale for the chemical modification of peptides or proteins is based on the assumption that properties of the macromolecular conjugate are dominated by the properties of the polymeric carrier determining the alteration on pharmacological and pharmacokinetic profiles.In the first part of this work the synthesis and characterization of a series of PEG-CCK-9 conjugates using different molecular weight polymersis described. The PEGylation of CCK-9 implies the nucleophilic attack of the amino terminal group of the peptide on the NHS activated carboxyl group of the polymer to form a stable amide group. This reaction resulted in high yields of products that were purified and quantified by means of C-18 analytical HPLC using photodiode array detector (DAD) and Corona charged aerosol detector (CAD) alone or in series. The characterization of the obtained products was done by means of analytical techniques asMALDI-TOF MS and 1H NMR. The synthesized PEG-CCK-9 conjugates showed a significant longer resistance to enzymatic degradation in comparison to non-modified CCK-9 when they were incubated in fresh rat plasma.The FI reducing activity of the synthesized products was measured in vivo after i.p. injection in rats. The PEG-CCK-9 conjugates resulted in a significant prolongation of the anorectic capacity in comparison to the non-modified CCK-9 resulting in a reduction of the cumulative food intake, related to the molecular weight of PEG, during periods between 4 to 23 h. The best results were obtained with PEG10kDa-CCK-9 showing a cumulative FI reduction of 13 % over a 23 h period.The second part of this work deals with pharmacokinetic, biodistribution and mechanistic aspects of the PEG10kDa conjugate.Considering the lack of a reliable method for CCK detection in biological samples the radiolabeled conjugate, PEG10kDa-[123I]CCK-10 was synthesized for an easy detection. CCK-10 was used to couple with PEG10kDa instead of CCK-9; this CCK analogue presents the same amino acid sequence asthe used nonapeptide plus one tyrosine moiety, which is necessary for electrophilic substitution with iodine and for PEGylation through its amino terminal group. No pharmacological differences expressed as food intake values could be observed, first, between PEG10kDa-CCK-9 and PEG10kDa-CCK-10 and second, between the PEG10kDa-CCK-9 conjugate and the labeled PEG10kDa-CCK-10 after injections to rats. These results confirmed the suitability of the PEG10kDa-[123I]CCK-10 for the planned experiments.The AUC values and the Cmax values detected in the blood for PEG10kDa-[123I]CCK-10 after injection by different routes (i.v.; i.p.; i.m.; and i.n.) were in harmony with the previously obtained pharmacological results, confirming the prolongation of CCK half-life after PEGylation due to the resistance to enzymatic degradation. This capacity allowed the conjugate to exert its anorexigenic activity when administered through routeswhere enzymatic degradation was reported to have been the limiting factor (e.g. i.m.).The biodistribution of the labeled conjugate showed higher accumulationin organs expressing CCK1 receptors such as pancreas, duodenum, small intestine and stomach wall, indicating that the working mechanism of the conjugate is indeed related to those CCK1 receptors.No indication of crossing the blood brain barrier could be observed forPEG10kDa-[123I]CCK-10 independently of the injection route used.High amounts of radioactivity could be detected in the urine after PEG10kDa-[123I]CCK-10 administration by different routes suggesting this pathway as the more important for product elimination. Those different results indicate that the prolongation of the anorexigenic effect observed after CCK PEGylation seems to be the sum of biological modifications exerted by the presence of the polymer.