Synthesis of a novel pentagastrin-drug conjugate for a targeted tumor therapy.

The synthesis of the novel pentagastrin seco-CBI conjugate 3, which is based on the highly cytotoxic antitumor antibiotic (+)-duocarmycin SA (1), is reported. A key step in the synthesis is the palladium-catalyzed carbonylation of aryl bromide 7 to give the benzyl ester 16, which is transformed into the new seco-CBI derivative 21 bearing a carboxylic acid ester moiety. Subsequent transformation of 21 into an activated ester followed by the introduction of beta-alanine and tetragastrin led to the new pentagastrin drug 3 that contains a peptide moiety for targeting cancer cells expressing CCK-B/gastrin receptors.

Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides.

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.

[Anxiety-inducing and -inhibiting agents: differential effect of pentagastrins on the white rat behavior]. / Induktory i ingibitory trevozhnosti: differentsial'noe deistvie pentagastrinov na povedenie belykh krys.

Parenteral administration of des-BOC-Pentagastrin induced the anxiety and fear manifestations, depressing also explorative behaviour in open field experiments in rats. Intranasal administration evoked similar effects, whereas pentagastrin reduced the anxiety level, increasing explorative behaviour. Pentagastrin and des-BOC-Pentagastrin displayed antagonism at the receptor level.

Distribution of distances in thiopeptides by fluorescence energy transfer and frequency-domain fluorometry.

Frequency-domain fluorescence spectroscopy was employed to examine the decays of tryptophan in Boc-Trp-Met-Asp-Phe-NH2 (donor) and (Formula: see text) (donor-acceptor pair). The efficiency of energy transfer in the thiopeptide amounted to 60%. The measured dispersion of fluorescence decay times was used to recover the donor-acceptor distance distribution. The parameters of the Gaussian distance distribution obtained for this peptide (r, the mean distance (9 A); hw, the halfwidth (25 A)) indicate the lack of a distinct favorable conformation.

Phospholipid structure determines the effects of peptides on membranes. Differential scanning calorimetry studies with pentagastrin-related peptides.

The effect of phospholipid structure on the interaction between small peptides and phospholipid membranes has been studied by high-sensitivity differential scanning calorimetry. The peptides used, N-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 and N-Boc-beta-Ala-Trp-Met-Lys-Phe-NH2, are basic analogs of the hormone pentagastrin. These peptides split the gel-to-liquid crystalline phase transition of synthetic phosphatidylcholines into two components. For dimyristoyl (DMPC), dipalmitoyl (DPPC) and 1-stearoyl-2-oleoyl (SOPC) phosphatidylcholines, one component remains at the temperature corresponding to that of pure lipid and the other one is shifted towards higher temperatures. With increasing peptide concentration there is a gradual increase in the enthalpy of the high-temperature component at the expense of the low-temperature one, and there is also an increase in the total enthalpy of the transition. A mixture of the peptide with distearoylphosphatidylcholine (DSPC) behaves differently, with the transition occurring at a temperature below that of the pure lipid increasing with peptide concentration. The susceptibility of various phosphatidylcholines to perturbation by the peptides increases in the order DMPC greater than SOPC greater than DPPC greater than DSPC. The effect of these peptides on the phase transitions of acidic phosphatidylglycerols is generally greater than with the corresponding phosphatidylcholines, but the dependence on the length of lipid hydrocarbon chains is similar. Perturbation of the thermotropic phase transition is strongest for dimyristoylphosphatidylglycerol, followed by the dipalmitoyl and the distearoyl analogs. The effect of the peptides on the phase transition of dimyristoylphosphatidylserine is significantly smaller compared to that observed with dimyristoylphosphatidylglycerol and it is further reduced for dimyristoylphosphatidic acid. The phase transition of this latter lipid remains virtually unchanged, even in the presence of high concentrations of the peptide. Similar resistance to the perturbation of the phase transitions by the peptides is observed for synthetic phosphatidylethanolamine. The different susceptibility of various phospholipids to perturbation by the peptides is suggested to be related to different degrees of intermolecular interaction between phospholipid molecules, and particularly to different abilities of phospholipids to form intermolecular hydrogen bonding.

Hormonal activation of single K+ channels via internal messenger in isolated pancreatic acinar cells.

The mechanism underlying hormonal activation of potassium channels was investigated in pig pancreatic acinar cells by patch-clamp single-channel and whole-cell current recordings. It was shown directly that a peptide hormone belonging to the cholecystokinin-gastrin family, CCK5, can activate single voltage-sensitive potassium channels which can be blocked by tetraethylammonium. The single-channel currents were recorded from electrically isolated cell-attached membrane patches to which the hormone had no access and the activation must therefore involve an intracellular messenger. The hormonal response requires external Ca2+ in the isolated membrane-patch area indicating that calcium gating is not directly linked to hormone-receptor interaction.

Role of peptide structure in lipid-peptide interactions: high-sensitivity differential scanning calorimetry and electron spin resonance studies of the structural properties of dimyristoylphosphatidylcholine membranes interacting with pentagastrin-related pentapeptides.

The effects of amino acid substitutions in the pentapeptide pentagastrin on the nature of its interactions with dimyristoylphosphatidylcholine (DMPC) are assessed by differential scanning calorimetry and electron spin resonance. In two peptide analogues, the Asp at position 4 in pentagastrin (N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2) is replaced by Gly or Phe. These uncharged, more hydrophobic peptides have little effect on the transition temperature of DMPC, but they broaden the transition and lower the transition enthalpy as do integral membrane proteins. These peptides also mimic the behavior of integral membrane proteins in decreasing the order of a 5-doxylstearic acid spin probe below the transition temperature and in exhibiting a second immobilized lipid component using a 16-doxylstearic acid spin probe in DMPC. Three charged peptides were studied: pentagastrin, an analogue with positions 4 and 5 reversed (i.e., ending in Phe-Asp-NH2), and one with Asp replaced by Arg at position 4. All three of these charged peptides altered the phase transition behavior of DMPC to give two components, one above and one below the transition temperature of the pure lipid. With increasing peptide concentration, the higher melting transition became more prominent. The arginine-containing peptide produced the largest shifts in melting temperature followed by pentagastrin and then the "reversed" peptide. The arginine-containing peptide also increased the enthalpy of the transition. These peptides also increased the ordering of DMPC below the phase transition as measured with both 5- and 16-doxylstearic acid. The ordering effect was most pronounced with the arginine-containing peptide using the 5-doxylstearic acid probe. The results demonstrate that even the zwitterionic DMPC can interact more strongly with positively charged peptides than with negatively charged ones. In addition, peptide sequence as well as composition is important in determining the nature of peptide-lipid interactions. The markedly different effects of these pentagastrin peptides on the phase transition and motional properties of DMPC occur despite the similar depth of burial of these peptides with DMPC.

Role of peptide structure in lipid-peptide interactions: a fluorescence study of the binding of pentagastrin-related pentapeptides to phospholipid vesicles.

The binding of pentagastrin and three other structurally related pentapeptides to phospholipid vesicles has been studied by fluorescence spectroscopy. The fluorescence of the tryptophan residues of these peptides exhibits an increased quantum yield upon binding to phospholipid vesicles. This is accompanied by a blue shift of the maximum emission, indicative of the incorporation of the tryptophan residue into a more hydrophobic environment. The affinity of the peptides for a zwitterionic phospholipid, dimyristoylphosphatidylcholine (DMPC), increases in the following order: N-t-Boc-beta-Ala-Trp-Met-Gly-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2. Comparison of the interaction of these various peptides with this phospholipid indicates that although the interaction is largely of hydrophobic nature, the structure of the polar amino acids and their electrostatic charge have significant influence on the nature of the bindings. In addition, the sequence of polar and apolar amino acids appears to be of importance. The higher affinity for DMPC of N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 as compared to its "reversed" analogue N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2 suggests that the ability of the peptides to fold into amphiphatic structures can enhance their lipid binding affinity. For all peptides the interaction with DMPC is greater at 8 degrees C, i.e., below the lipid phase transition temperature, than at 40 degrees C, i.e., above the lipid phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentagastrin analogues containing alpha-aminooxy acids. VII. Catabolism in vivo and in vitro of a pentagastrin analogue containing N-terminal aminooxyacetyl residue.

In rat, BOC-NHO-1-14C-Ac-Trp-Met-Asp-Phe-NH2 (14C-I) is metabolised more slowly than BOC-1-14C-Gly-Trp-Met-Asp-Phe-NH2 (14C-II). Of the cytosol fractions of the rat organs the lung and pancreas exhibited a lower activity in the catabolism of BOC-NHOAc-Trp-Met-Asp-Phe-NH2 (I) than in that of BOC-Gly-Trp-Met-Asp-Phe-NH2 (II). In contrast, the cytosol fractions of the dog's lung, small intestine and pancreas hydrolysed I at a faster rate than they hydrolysed II.

Metabolism of C-terminal pentapeptide of gastrin in the rat. Part IV. Catabolic pathways of the N-terminal BOC-14C-glycine fragment.

The catabolism of the 14C-labelled pentagastrin was investigated in rats. The following results were found: -- the labelled BOC-glycine fragment is split off the pentagastrin in the small intestine -- it is absorbed through the intestinal wall, and -- enters first the portal and then the systemic circulation.

Effects of pentagastrin, G17, and G34 on the electrical and mechanical activities of canine antral smooth muscle.

Intracellular microelectrode technique and standard organ bath technique were used to investigate the effects of pentagastrin, G17, and G34 on the electrical and mechanical activities of canine antral circular muscle. All three molecular forms increased the amplitude and duration of the plateau of the gastric action potential and the frequency of spontaneous action potentials. They also increased the amplitude and frequency of spontaneous contractions. G17 was equal to or less potent than pentagastrin in all of its actions on this tissue. G34 had an equal or greater activity than G17. The electrical studies indicate that G17 is active in this tissue in a physiological range of concentrations. The ED50 for the effect of G17 to increase the amplitude of the plateau potential is less than that for the effect of G17 on gastric secretion, indicating that this is a physiological action of gastrin. Atropine studies indicate that only part of the in vitro inotropic action of gastrin is caused by the release of acetylcholine from nerve terminals, but that the chronotropic action is attributable to a direct effect on the smooth muscle membrane.

Pentagastrin analogs containing alpha-aminooxy acids, I. Synthesis of analogs substituted at the N-terminus.

Nine new pentagastrin analogs containing a free or protected alpha-aminooxy acid at the N-terminus were synthesized stepwise. Analogs containing leucyl, norleucyl, norvalyl, L- and D-2-aminodecanoyl residues instead of methionyl residue were also prepared. The peptides were synthesized by the active ester method with subsequent removal of the protecting groups. The purification of the end products was performed by crystallization or column chromatography on silica gel.

Pentagastrin analogs containing alpha-aminooxy acids, II. Simultaneous substitutions at the N- and C-terminus.

Twenty-seven new pentagastrin analogs containing free or protected aminooxy acids at the N-terminus and D-2-aminooxy-3-phenyl-propionic acid, L-(4-chlorophenyl)glycine, L- and D-phenylglycine, or L- or D-cyclohexyglycine instead of the C-terminal L-phenylalanine and analogs extended by aminooxyacetic acid at the C-terminus, were synthesized stepwise. The end products were purified by chromatography on DEAE-cellulose or silica gel.

Pentagastrin analogs containing alpha-aminooxy acids, III. Biological studies and structure-activity relationships.

The stimulation of gastric acid flow by thirty-six new pentagastrin analogs administered intravenously, intrajejunally and intrarectally was determined in rats. Some of the analogs are several times more active than the pentagastrin (Peptavlon, I.C.I.) used as control. Unlike the control, some analogs are absorbed in active form from the jejunum. The N-terminal substitution with alpha-aminooxy acids seems to increase the gastric secretory response, probably by improving the enzymatic resistance of the molecule to various enzymes. None of the analogs tested significantly inhibit the gastric acid output in rats induced by pentagastrin. Structure-activity relationships are discussed.

[The effect of pentagastrin and ornithine-tetragastrin on motor activity of the stomach in skates and scorpion-fish]. / Vliianie pentagastrina i ornitintetragastrina na motoriku zheludka u skatov i skorpen

Low doses of pentagastrin (50 mug/kg b. w.) have no physiological effect on motor activity of the stomach in the skate Dasyatis pastinaca. Average doses (100-200 mug/kg) stimulate the activity, whereas high ones (300 mug/kg) inhibit the frequency of stomach contractions, slightly increasing their amplitude. Ornithine tetragastrin in a dose 2000 mug/kg does not affect motor activity of the stomach in skates. In the scorpion-fish Scorpaena porcus, ornithine tetragastrin (1000 and 2000 mug/kg) inhibits motor activity of the stomach.

Response to pentagastrin in man. III. Comparison of two formulations.

Gastric secretion has been studied in response to the standard preparation of pentagastrin and a new, more stable preparation (pentagastrin meglumine). The sensitivity to, and maximal outputs of acid and pepsin elicited by, the two preparations of pentagastrin did not differ. However, the pentagastrin meglumine evoked a better sustained acid secretion with all dose rates than did the standard pentagastrin.