Solid-state stability studies of cholecystokinin (CCK-4) peptide under nonisothermal conditions using thermal analysis, chromatography and mass spectrometry.

The solid-state stability of cholecystokinin (CCK-4) peptide under nonisothermal conditions was studied by differential scanning calorimetry (DSC), chromatography and mass spectrometry, identifying and schematizing the degradation products. To model the degradation mechanism of the peptide using the combined Kissinger and direct-differential methods, the observed degradation process was characterized by decomposition temperature (T(m)), reacted fraction (alpha(m)), activation energy (E(a)), and pre-exponential factor (A). Results obtained by the two calculation methods were similar. The cleavage reaction on both N- and C-terminal sides of aspartic acid was the principal degradation pathway, although the reaction can occur consecutively and/or in parallel. Therefore to determine the relative importance of the different degradation pathways, a system of differential equations relevant to each degradation reaction was analysed using the R((R)) statistical program. The results obtained show that the consecutive reaction was the less plausible, whereas a slightly better fit was obtained for the reaction with both processes than for the in-parallel reaction. In this situation, the F-test was applied to discriminate between the models, indicating that the simpler model is the most probable. In conclusion, the results demonstrate for the first time that, in solid-state, n-1 cleavage occurs in parallel to n+1 cleavage at aspartic acid residues and not consecutively.

Data analysis of kinetic modelling used in drug stability studies: isothermal versus nonisothermal assays.

PURPOSE: Kinetic modelling was applied to predict the stability of cholecystokinin fragment CCK-4 in aqueous solution, which was analyzed by isothermal and nonisothermal methods using a validated stability indicating HPLC method. METHODS: The isothermal studies were performed in the temperature range 40 to 80 degrees C at pH 12 and ionic strength 0.01 M as constants, whereas nonisothermal stability studies were performed using a linear increasing temperature program, heating rate 0.25 degrees C/h and a temperature interval 40-82 degrees C. The isothermal studies require two-step linear regression to estimate the parameters, resulting in a well-defined confidence interval. Nonisothermal kinetic studies require nonlinear or linear regression by previous transformation of data to estimate the parameters. In this case, the two most popular approaches, derivative and integral, were used and compared. RESULTS: Under isothermal conditions, an apparent first-order degradation process was observed at all temperatures. The linear Arrhenius plot suggested that the CCK-4 degradation mechanism was the same within the studied temperature range, with quite large uncertainties due to the small number of degrees of freedom based only on the scatter in the plot, and giving an estimated shelf life at 25 degrees C of 35.2 days. The derivative approach yields high variability in the Arrhenius parameters, since they are dependent on the number of polynomial terms chosen, so several statistical criteria were applied to select the best model. The integral approach allows activation parameters to be calculated directly from experimental data, and provides results in good agreement with those of the traditional method, but have the advantage that the uncertainty in the final result directly reflects the goodness of fit of the experimental data to the chosen kinetic model. The application of the bootstrap technique to estimating confidence limits for the Arrhenius parameters and shelf life is also illustrated, and shows there is no difference between the asymptotic and bootstrap confidence intervals. CONCLUSIONS: Nonisothermal studies give us fast and valuable information about drug stability, although their potential for predicting isothermal behaviour is conditioned by the data analysis method applied.

The effect of 5-hydroxytryptophan on cholecystokinin-4-induced panic attacks in healthy volunteers.

Previous studies suggest a modulatory role of serotonin (5-HT) in experimentally-induced panic attacks. In the current study, we investigated the acute effects of 5-HT precursor l-5-hydroxytryptophan (5-HTP) on the response to panicogenic challenge with cholecystokinin-tetrapeptide (CCK-4) in healthy volunteers. Thirty-two subjects were randomized to receive either 200 mg of 5-HTP or placebo with the CCK-4 challenge following in 90 min in a double-blind, parallel-group design. The results showed a nonsignificant difference between the groups in panic rate (19% after 5-HTP and 44% after placebo, p = 0.13) with a trend for lower intensity of symptoms after 5-HTP (p = 0.08). Further analysis by gender revealed that females in the 5-HTP group had a significantly lower panic rate and intensity of cognitive symptoms whereas, in males, the effect of 5-HTP was limited to lowering the intensity of somatic panic symptoms. Thus, an increased availability of 5-HT may have a gender-dependent protective effect in CCK-4-induced panic.

Permeability characteristics of tetragastrins across intestinal membranes using the Caco-2 monolayer system: comparison between acylation and application of protease inhibitors.

PURPOSE: Three types of acyl tetragastrin (TG), acetyl-TG (C2-TG), butyryl-TG (C4-TG) and caproyl-TG (C6-TG) were synthesized and their in vitro intestinal permeability characteristics were examined using Caco-2 monolayers. METHODS: The disappearance of acyl-TGs from the apical side of Caco-2 monolayers was estimated by analyzing degradation and permeation processes in terms of clearance. RESULTS: The amount of native TG transported to the basolateral side was very low due to its large degradation clearance (CLd) on the apical side. Degradation of TG was reduced by chemical modification with fatty acids, which resulted in an increase in the transport of TG across Caco-2 monolayers. In addition, the permeation clearance (CLp) value of carboxyfluorescein (CF), a paracellular transport and undegradable marker, was increased in the presence of acyl-TGs. Furthermore, we investigated the effects of the protease inhibitors bacitracin and gabexate on the transport of TG across Caco-2 monolayers. In the presence of a low concentration (0.1 mM) of protease inhibitor, the CLd value of TG was reduced, but they did not affect its CLp value. However, a higher concentration (1.0 mM) of bacitracin significantly reduced TG degradation on the apical side, and further increased its CLp value. CONCLUSIONS: We demonstrated that acylation of TG made it resistant to intestinal proteases and caused it to enhance absorption of drugs, including itself, across Caco-2 monolayers. Further, bacitracin acted as both a protease inhibitor and an absorption enhancer.

[Improvement of intestinal absorption of peptide and protein drugs by chemical modification with fatty acids].

It is well known that the oral bioavailability of peptide and protein drugs is generally poor because they are extensively degraded by proteases in the gastrointestinal tract and impermeable through the intestinal mucosa. Therefore, various approaches have been examined to overcome the delivery problems of these peptides and to improve their absorption via the gastrointestinal tract. Of these approaches, a potentially useful approach to solve these delivery problems may be chemical modification of peptides and proteins to produce prodrugs and analogues. Thus, it is plausible that this approach may protect peptides against degradation by peptidases and other enzymes present at the mucosal barrier and renders the peptides and proteins more lipophilic, resulting in increased bioavailability. From these standpoints, we synthesized lipophilic derivatives of peptides and proteins such as thyrotropin-releasing hormone (TRH), tetragastrin (TG), calcitonin and insulin by chemical modification with fatty acids. The pharmacological activities of these derivatives were relatively high as compared with the native peptides. A significant increase in the intestinal absorption of these derivatives of peptides was observed in comparison with native peptides. Overall, the effects of acylation on the intestinal absorption of these peptides were more predominant in the large intestine than those in the small intestine. In addition, these derivatives were more stable than the parent peptides in homogenates of the various intestinal mucosae. We also examined the intestinal transport characteristics of TG and its acyl derivatives using Caco-2 cell monolayers in order to assess the contribution of enzymatic and transport barriers on their intestinal absorption. The degradation clearance of TG on the apical membrane was decreased by chemical modification with fatty acids. In addition, the permeability clearance of TG was improved by the acylation. On the other hand, the intestinal absorption of thyrotropin releasing hormone (TRH), which is transported by a carrier-mediated process, was also enhanced by chemical modification with lauric acid. In summary, this chemical modification approach may be useful to improve the intestinal absorption of peptide and protein drugs.

Alternate drug delivery routes for A-71623, a potent cholecystokinin-A receptor agonist tetrapeptide.

A-71623 (BOC-Trp-Lys(epsilon-N-2-methylphenylaminocarbonyl)- Asp-(N-methyl)-Phe-NH2) is a tetrapeptide which has high affinity and selectivity for cholecystokinin receptors; it is a potent appetite suppresser in animal studies. Because of its low (< 1%) oral bioavailability, studies were performed to assess the feasibility of delivery of A-71623 by pulmonary, sublingual, and transdermal routes of administration. The pKa was determined to be 4.2 by spectrophotometric titration; aqueous solubility is increased by increasing pH and by increasing ethanol content. The solubility of A-71623 in ethanol/propellant mixtures was investigated; solubility ranged from 1.0 to 2.5 mg/mL in mixtures of ethanol, propellant 11 (trichlorofluoromethane), and propellant 12 (dichlorodifluoromethane). The log apparent octanol/water partition coefficient was 2.8 at pH 5 and 1.0 at pH 8. Maximum stability at 70 degrees C was seen in the range of pH values of 5.5-7.5; hydrolysis of the N-terminal BOC group appears to be the primary route of degradation. Increasing ethanol content increases the stability; Arrhenius analysis indicated a t90 of 150 days under ambient conditions in 25% ethanol. Intratracheal delivery of 3 mumol/kg A-71623 in 50% ethanol to rats showed rapid and efficient absorption of drug from the lungs, with a Cmax of 2.7 microM and an AUC of 85 microM*min. Similar studies in dogs showed bioavailabilities of 59% and 46% for 2 and 3 mumol/kg intratracheal doses, respectively, relative to intravenous administration. Sublingual administration of 1 mumol/kg A-71623 in a vehicle of 80% ethanol/2% Klucel/2.5% peppermint oil gave high prolonged plasma levels of A-71623, with a Cmax of 0.37 microM, indicating high bioavailability and favorable partitioning and distribution effects from the sublingual cavity for this formulation. Transdermal delivery was examined by in vitro diffusion through human skin; the permeability coefficient of A-71623 in 40% ethanol was 2.6 x 10(-5) cm/hr, suggesting that transdermal delivery of up to 2 mg/day may be feasible. In conclusion, the results provide preliminary indications that delivery of efficacious doses of A-71623, and perhaps other CCK analogs, by alternate routes of delivery is probably feasible.

Effect of CI-988 on cholecystokinin tetrapeptide-induced panic symptoms in healthy volunteers.

A randomized, placebo-controlled, double-blind, three-way crossover design was used to evaluate the effectiveness of single oral 100 mg doses of CI-988, a cholecystokinin B (CCKB) antagonist, in attenuating panic symptoms induced by intravenous injection of cholecystokinin-tetrapeptide (CCK-4). Thirty healthy men received the following treatments on three separate occasions: placebo capsules/placebo, placebo capsules/CCK-4, or CI-988 capsules/CCK-4. There was no marked difference in the number, time to onset, or duration of panic symptoms between CI-988/CCK-4 and placebo/CCK-4. There was, however, a 14% difference in sum intensity scores between these treatments that was statistically significant (p = 0.039). The symptoms most affected by CI-988 were cold chills/hot flushes, chest pain/discomfort, and anxiety/fear/apprehension. Panic attack frequency also decreased following CI-988 treatment (8/30 vs. 16/30; p = 0.035). This decrease, amid otherwise modest effects, could be explained by a preferential effect of CI-988 on the subjective experience of anxiety/fear/apprehension. Possible reasons for the relatively modest effects of CI-988 on CCK-4-induced panic symptoms are discussed.

Improvement of transdermal delivery of tetragastrin by lipophilic modification with fatty acids.

The in-vitro permeability of chemically modified tetragastrin with fatty acids through the rat skin was studied. The permeability of these compounds through intact skin and stripped skin of rat was determined with a Franz-type diffusion cell. The permeation of tetragastrin across the intact skin was improved by chemical modification with acetic acid and butyric acid. However, tetragastrin and caproyl-tetragastrin did not permeate across the intact skin up to the end of experiment. The permeation of tetragastrin across the stripped skin was improved by chemical modification, the skin flux of these acyl derivatives being in the order:acetyl > butyroyl > caproyl. The stability of tetragastrin in skin homogenate was also significantly improved by chemical modification with fatty acids. These results suggest that chemical modifications of tetragastrin with fatty acids increases its lipophilicity, which makes it permeable across the stratum corneum. Moreover, the chemical modification reduced the degradation of tetragastrin in the viable skin, resulting an increase in permeation of tetragastrin across the skin.

Development of new lipophilic derivatives of tetragastrin: physicochemical characteristics and intestinal absorption of acyl-tetragastrin derivatives in rats.

In order to improve the intestinal absorption of tetragastrin (TG), we synthesized lipophilic derivatives of TG by acylation of its N-terminal amino group with acetic acid, caproic acid, and lauric acid. The purified TG derivatives, acetyl-tetragastrin (Ac-TG), caproyl-tetragastrin (Cap-TG), and lauroyl-tetragastrin (Lau-TG), were confirmed to be more lipophilic than the parent TG by high-performance liquid chromatography (HPLC). The pharmacological activities and the intestinal absorption of TG and its derivatives were examined by measuring gastric acid secretion. Stimulation of gastric acid secretion by these derivatives after intravenous administration was stronger than with native TG. When the acetyl- and caproyl-derivatives were administered into the large intestinal loops, a marked increase in gastric acid secretion was observed in comparison with TG, while no significant effect occurred following administration of the TG derivatives into the small intestines. These results indicated that chemical modification of TG with fatty acids improves the absorption of TG from the large intestines.

Neuroleptic-like properties of cholecystokinin analogs: distinctive mechanisms underlying similar behavioral profiles depending on the route of administration.

Rats were trained to discriminate vehicle injections from intraperitoneal injections of 3 micrograms/kg caerulein, a cholecystokinin (CCK) neuropeptide analog. The reward that reinforced correct choices was an electrical brain stimulation self-administered by bar pressing. Dose-response quantitative generalization was obtained by using 1 and 2 micrograms/kg caerulein. Qualitative generalization to the vehicle occurred after injecting 10, 20 and 200 micrograms/kg unsulfated CCK-8, 10, 20 and 200 micrograms/kg CCK-4, 5 micrograms/kg CCK-8 and 1 microgram/kg caerulein, neurotensin or bombesin and 200 micrograms/kg apomorphine or 320 micrograms/kg amphetamine. Total generalization to the caerulein cue was obtained with 20 micrograms/kg sulfated CCK-8 or gastrin 2-17, 25 micrograms/kg somatostatin, 50 micrograms/kg haloperidol and 2 mg/kg chlorpromazine. The previous 5 mg/kg injection of an antiemetic drug such as chlorhydrate of trimethobenzamide did not eliminate the discriminative properties of a subsequent injection of caerulein. Our data thus tend to show that IP injection of caerulein produces effects similar to those of IP neuroleptics.