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.

Disruption of the behavioral satiety sequence by simmondsin.

Simmondsin, a cyanoglycoside from jojoba meal, reduces food intake after oral administration. To diagnose if it acts by inducing satiation or by creating abnormal physiological effects, an observational study was undertaken to investigate the effects of simmondsin on feeding and other behaviors. Particular attention was paid to the behavioral sequence associated with satiety (BSS). At first contact, simmondsin non-significantly reduced food intake by 17% and had little effect on feeding and associated behaviors. The behavioral structure was preserved and a small shift of the onset of resting to the left was observed, suggesting a small satiative action of simmondsin at first contact. Simmondsin given for the second time caused a more pronounced food intake reduction of 52% due to a reduction in eating duration, mean bout intake and mean bout length, and to an increase in latency to eat. At second contact, simmondsin caused a strong switching in active behaviors, disrupting the BSS. The simmondsin-induced hyperactivity suggests that simmondsin produces aversiveness with second contact. Our results indicate that simmondsin exerts multiple effects. It probably facilitates a small natural process of satiation/satiety at first contact, but creates abnormal physiological effects resulting in aversive reactions from second contact on.

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.

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.

Receptor-mediated biological responses are prolonged using hydrophobized ligands.

Hormone-receptor interactions occur following three-dimensional diffusion of the ligand to the membrane-embedded receptor. However, prior hydrophobization of the ligand might restrict its movement to two dimensions along the membrane surface, and the biological response might therefore be modulated. This idea was tested using the C-terminal nonapeptide, CCK9, of the satiating hormone, cholecystokinin (CCK). The hormone was lipidated by linking it covalently to distearoylphosphatidylethanolamine via a poly(ethylene glycol) (PEG) spacer. The desired conjugate was isolated by thin-layer chromatography and incorporated into preformed small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles. The hormone-bearing vesicles were injected intraperitoneally into Wistar rats and food intake monitored. Compared to the biological effect elicited by the same amount of soluble non-derivatized CCK9, food intake reduction showed a delayed onset, but lasted for a significantly longer time. We believe this prolonged effect was due to the transfer of the derivatized CCK9 from the vesicles to the natural membrane containing the hormone receptor. Ultimately, this event may result in sustained receptor occupation and, thus, food intake reduction. The underlying mechanism for the physiological effects observed may be of relevance in interpreting results obtained using artificial measuring devices; for example, the signal produced by biosensors may be drastically affected by the hydrophobicity of the ligand.

Isolation and identification of molecular species of phosphatidylcholine and lysophosphatidylcholine from jojoba seed meal (Simmondsia chinensis).

A mixture of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) has been isolated by column chromatography from a jojoba meal (Simmondsia chinensis) extract. The molecular species of both classes could be separated and isolated by C18 reversed phase HPLC. The two major compounds were identified by 1D and 2D (1)H and (13)C NMR, by MS, and by GC-MS as 1-oleoyl-3-lysophosphatidylcholine and 1,2-dioleoyl-3-phosphatidylcholine. Eight other molecular species of LPC and four other molecular species of PC could be assigned by comparison of the mass spectra of the isolated compounds with the spectra of the two major compounds. Complete characterization of the individual molecular species was achieved by GC and GC-MS analysis of the fatty acyl composition from the isolated compounds. The PC/LPC proportion in the phospholipid mixture from three different samples is 1.6 +/- 0.1. LPC is considered to be an important bioactive compound; the results of this study suggest further research for the evaluation of potential health benefits of jojoba meal phospholipids.

Simmondsin: effects on meal patterns and choice behavior in rats.

Simmondsin, a glycoside from jojoba meal, decreases food intake after oral administration. The present experiments are designed to clarify the mechanism of simmondsin's anorectic activity. The meal pattern analysis shows that simmondsin supplementation at different doses results in a dose-dependent food intake reduction, which is more pronounced after prior simmondsin experience. The effect of simmondsin on meal patterns (decreased meal size, meal duration and eating rate, increased latency to eat) is most severe at the highest concentration. Rats familiar with simmondsin more seriously postpone their first meal than with first contact, resulting in a decrease of the meal frequency and the day/night feeding ratio. Rats given the choice between a control diet and a simmondsin-supplemented (0.5%) diet, after half an hour, have a significant preference for the control diet. Simmondsin seems to have a specific flavor when mixed in the food since rats recognise the feeder containing simmondsin. The ability of simmondsin to induce conditioned taste aversion (CTA) was also investigated. Rats receiving simmondsin at concentrations of 0.15%, 0.25% or 0.5% during their conditioning develop significant taste aversions to the saccharin solutions. The performed experiments indicate that the simmondsin activity shows some analogy with the satiating molecule cholecystokinin (CCK) at first contact, but shows more analogy with the illness-inducing agent lithium chloride (LiCl) after prior experience with simmondsin. Rats familiar with simmondsin avoid simmondsin-supplemented food by directly monitoring its presence, and by learning to relate it to the postingestive consequences of consumption.

Simultaneous determination of carbohydrates and simmondsins in jojoba seed meal (Simmondsia chinensis) by gas chromatography.

Separate methods for the analyses of soluble carbohydrates in different plants and simmondsins in jojoba seed meal are described. A reliable gas chromatographic procedure for the simultaneous quantification of D-pinitol, myo-inositoL sucrose, 5-alpha-D-galactopyranosyl-D-pinitol. 2-alpha-D-galactopyranosyl-D-pinitol, simmondsin, 4-demethylsimmondsin, 5-demethylsimmondsin and 4,5-didemethylsimmondsin as trimethylsilyl derivatives in jojoba seed meal has been developed. The study of different extraction mixtures allowed for the quantitative recovery of the 9 analytes by a mixture of methanol-water (80:20, v/v) in the concentration range between 0.1 and 4%. Comparison of the separation parameters on three different capillary stationary phases with MS detection allowed for the choice of the optimal gas chromatographic conditions for baseline separation of the analytes.