Fabian Léon-Tamariz

PEGylated cholecystokinin prolongs satiation in rats: dose dependency and receptor involvement

Acute intraperitoneal (i.p.) administration of cholecystokinin (CCK) is known to induce a significant, but short‐lasting, reduction in food intake, followed by recovery within hours. Therefore, we had covalently coupled CCK to a 10 kDa polyethylene glycol and showed that this conjugate, PEG‐CCK9, produced a significantly longer anorectic effect than unmodified CCK9. The present study assessed the dose–dependency of this response and the effect of two selective CCK1 receptor antagonists, with different abilities to cross the blood‐brain barrier (BBB), on PEG‐CCK9‐induced anorexia.

PEGylation of cholecystokinin prolongs its anorectic effect in rats

The anorectic compound CCK-9 was coupled to polyethylene glycol 5 kDa, 10 kDa, 20 kDa and 30 kDa, under different reaction conditions. Conjugates were purified by HPLC and characterized by MALDI-TOF MS. A 96% PEGylation yield was obtained in buffer pH 7.5 after 6h reaction at 20 degrees C. The anorectic activity was tested in vivo in rats. A single bolus intra-peritoneal injection of non-modified CCK-9 resulted in a significant initial food intake reduction 30 min after food presentation (87% compared to paired control group). When PEG-CCK-9 conjugates modified with polymers of molecular weight up to 20 kDa were injected, lower but statistically significant initial food intake reductions were obtained (76% for PEG 10 kDa-CCK-9 conjugate compared to control group). The cumulative food intake reduction of non-modified CCK-9 is normalized within 1-2h, whereas the PEG-CCK-9 molecules showed a prolonged anorectic activity lasting for 6h for PEG 5 kDa-CCK-9; 23 h for PEG 10 kDa-CCK-9 and between 8h and 23 h for PEG 20 kDa-CCK-9. For PEG 30 kDa-CCK-9 conjugate, neither an initial nor a cumulative FI reduction was observed. PEG-CCK-9 conjugates show a significantly prolonged anorectic activity in comparison to the non-modified peptide. This effect is most evident for the PEG 10 kDa-CCK-9 conjugate.

PEGylated cholecystokinin is more potent in inducing anorexia than conditioned taste aversion in rats

The physiological involvement of endogenous cholecystokinin (CCK) in the termination of feeding has been challenged by evidence of aversive effects of exogenous CCK8. We previously prolonged the anorectic effect of CCK by conjugation to polyethylene glycol (PEGylation) to produce PEG‐CCK9. In this study, we investigated the ability of different doses of PEG‐CCK9 to induce conditioned taste aversion (CTA) and satiety and identified the receptors involved in CTA induction.

Lack of tolerance development with long-term administration of PEGylated cholecystokinin

Cholecystokinin (CCK) is a short acting satiating peptide hormone produced in the proximal small intestine. Daily CCK injection in rats initially inhibits food intake, but after several days, food intake is no longer affected, suggesting development of tolerance. Previously, we covalently coupled CCK to a 10kDa polyethylene glycol (mPEG-OH) and showed that this conjugate, PEG-CCK(9), produced a significantly longer anorectic effect than unmodified CCK(9). The present study examined whether tolerance to the anorectic effect develops during long-term administration of PEG-CCK(9). For 14 consecutive days, male Wistar rats (n=12) received a daily i.p injection of 8microgkg(-1) of PEG-CCK(9) and a control group received a daily control injection of mPEG-OH. Body weight and food intake were monitored daily during the experiment. Effects on the pancreas were investigated. On each day, injection of PEG-CCK(9) induced an anorectic effect lasting 3-6h, but failed to significantly reduce daily total food intake compared to controls. The body weight gain of the PEG-CCK(9)-treated animals was not different from controls. The PEG-CCK(9)-treated group had a significantly higher pancreas weight, mainly due to hyperplasia. In conclusion, PEG-CCK(9) continued to have a daily suppressive effect on food intake when administered for 14 consecutive days, showing there was no development of tolerance.

Biodistribution and pharmacokinetics of PEG-10kDa-cholecystokinin-10 in rats after different routes of administration.

Cholecystokinin, produced in the proximal small intestine, is a short acting satiating peptide hormone. CCK-10, before and after mono-iodination, was previously coupled to 10kDa polyethylene glycol (PEG). The formed conjugates PEG10kDa-CCK-10 and PEG10kDa-[(127)I]-CCK-10 show after i.p. administration to rats a sustained food intake reduction during 8h in comparison to 2h for free CCK-10. The present study examined the blood pharmacokinetics of this pharmacological interesting molecule by means of PEG10kDa-[(123)I]-CCK-10 following intravenous, intraperitoneal, intramuscular and nasal administration and the biodistribution after i.p. administration. HPLC analysis with radiometric detection allowed the differentiation between inorganic iodide and the intact tracer in blood. Blood kinetics after i.v. injection was fitted to a bi-exponential with a distribution half-life of 15 min and with an elimination half-life of 8 hours for intact PEG10kDa-[(123)I]-CCK-10. The biodistribution studies showed a higher accumulation of the tracer for all administration routes in organs expressing CCK receptors localized in the gastrointestinal tract such as pancreas, duodenum and small intestine. No indication of blood brain barrier crossing for the conjugate could be observed independently of the administration route. Main clearance was via the urinary pathway.

Dose―response effects of PEGylated cholecystokinin on the behavioral satiety sequence

Cholecystokinin (CCK) is known to have a short biological half-life. In order to prolong the half-life and create a new investigative tool, we previously PEGylated the peptide, yielding PEG-CCK(9), and demonstrated that it had a dose-dependent prolonged anorectic effect. The aim of this study was to investigate whether PEG-CCK(9) reduces food intake by inducing satiation or by abnormal physiological effects, such as pain, malaise, or nausea. An observational study was performed to examine the effects of different doses of PEG-CCK(9) (1, 2, 4, 8, or 16 microg kg(-1)) on feeding and other behaviors. The behavioral sequence associated with satiety (BSS), i.e. the orderly progression from eating, through grooming and activity, to resting, was analyzed. From the lowest dose tested (1 microg kg(-1)), PEG-CCK(9) caused a dose-dependent reduction in food intake due to a dose-related reduction in both the duration and frequency of eating and a dose-dependent increase in duration of rest. A dose-dependent acceleration in the temporal profile of the BSS was observed, while the normal structure of feeding behaviors was well preserved, except at the dose of 16 microg kg(-1) of PEG-CCK(9), at which a decrease in eating rate and grooming behavior was observed, together with the occurrence of a significant number of abdominal cramps. These findings suggest that the hypophagic response to PEG-CCK(9) is mainly induced by natural mechanisms of satiety, although abnormal physiological effects, such as abdominal cramps, might reinforce the food inhibitory effect, especially at high doses of PEG-CCK(9) (>8 microg kg(-1)).

Synthesis, Characterization and Satiety in Rats of PEG10kDa-CCK-10 and (Radio)Iodinated PEG10kDa-CCK-10

The iodination of CCK-10 and coupling with PEG 10kDa is described along with evaluation of their food intake reducing effects and in vitro stability. Labeling was performed by electrophilic substitution with both [127I] and [123I] iodide, using iodo-beads® as the oxidant. The reaction conditions were optimised. HPLC was used to follow reaction progression and for purification of the labeled compounds. The synthesized compounds were characterized by MALDI-TOF MS and 1H NMR spectroscopy. Radiochemical yields were 40 ± 3% for [123I]CCK-10; the conjugation with PEG 10kDa was quantitative. A specific activity of 2438 GBq/mmol was obtained. Plasma stability studies with PEG10kDa-[123I]- CCK-10 showed a de-iodination half-life of 27 hours. Food intake experiments in rats with PEG10kDa-[127I]-CCK-10 showed after a single bolus intra-peritoneal injection a food intake reduction during 8 hours equivalent with the results obtained for PEG10kDa-CCK-10 and with PEG10kDa-CCK-9. The pharmacological results obtained indicate that neither the introduction of an extra tyrosine in CCK-9 nor the introduction of a iodine label in tyrosine affects the prolonged food reduction activity of the CCK conjugate. The suitability of PEG10kDa [123I]-CCK-10 as an agent for mapping CCK receptors and pharmacokinetic studies has been shown.