The degradation of corticotropin-releasing factor by enzymes of the rat brain studied by liquid chromatography-mass spectrometry.

The corticotropin-releasing factor (CRF; 41 amino acid residues) is a major regulatory peptide in the response to stress and is distributed over many regions of the brain. We have studied the enzymatic degradation of CRF and related peptides by the CRF-degrading enzyme(s) of the rat brain (CRF-DA) by liquid-chromatographic-mass spectrometric technique and by online tandem mass spectrometric experiments. Peptide fragments of the human/rat CRF (1-41) generated by the CRF-DA of the particulate cell fraction were separated and structurally assigned. Major sites of enzymatic attack were identified at the P1 positions Ser1, Thr11 , His13, Leu15, Arg23, Arg35, and Lys36 with Leu15 as the site of primary cleavage. The CRF-DA was shown to be dominated by a metalloendopeptidase activity inhibited by O-phenanthroline and EDTA. The cytosolic fraction generated a similar degradation pattern with a pronounced cleavage at the Arg35 position.

Identification and measurement of beta-endorphin levels in the skin during induced hair growth in mice.

We describe new and effective techniques for extracting proopiomelanocortin (POMC)-derived peptides from mammaliar skin. Using this methodology (hot-acid extraction) and two independent HPLC-controlled RIA systems, we identify beta-endorphin peptide in mammalian skin and demonstrate significant hair cycle-dependent fluctuations in both the skin concentration and the in situ expression pattern of beta-endorphin (sebaceous glands) during the entire murine hair cycle. The observed anagen (growth phase) associated increase in beta-endorphin concentration and its decline during the follicle involution (catagen) or resting (telogen) phase raise the possibility of a regulatory function of this neuropeptide in cyclic changes of skin physiology.

Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of beta-endorphin by endothelial cells.

An on-line HPLC-mass spectrometric procedure with an electrospray atmospheric pressure ionization (ESI-API) ion source was developed to identify the enzymatic degradation products (peptides) generated by incubation of human beta-endorphin (h beta E) with cultured aortic endothelial cells. The samples from the complex incubation mixture were prepurified and enriched using a small reversed-phase (RP) perfusion precolumn. Flow switching was applied to transfer the peptides from this precolumn to the analytical RP column of 2 or 0.32 mm I.D. and to separate them by gradient elution. The peptides were detected by means of an on-line coupled triple quadrupole mass spectrometer (TSQ 700) with an ESI-API ion source operated in the positive ion mode. This MS system behaves as a concentration sensitive detector at flow-rates from 5 to 150 microliters/min. MS-MS experiments supported the unambiguous assignment of the peptide structures. Thus most of the peptide fractions were identified and the region 16-17-18 (-L-F-K-) of h beta E was found to be primarily attacked by the enzymes of the endothelial cells.

Pathways of degradation of buserelin by rat kidney membrane.

The pathways of in vitro degradation of the gonadotropin-releasing hormone (GnRH) analog buserelin [pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg- ProNHEt, B1-9] by the rat kidney membrane fraction was investigated using high-performance liquid chromatography for the separation of the peptide products and electrospray mass spectrometry for their identification. The N-terminal peptides B1-4, B1-3, B1-2, C-terminal peptides B3-9, B4-9, B5-9, B6-9, middle sequence B3-4 and the amino acids Trp, Ser and Tyr were found to be formed. However, due to extreme differences in the stability of the peptides toward the battery of membrane enzymes (B1-2, B6-9 >> B1-3, B5-9 >> B1-9 >> B1-4 > B4-9 > B3-9, B3-4), the final products of buserelin degradation were B1-2, B1-3, B5-9, and B6-9 and the amino acids Ser and, corresponding to the formation of B1-2 and B6-9, Trp and Tyr, respectively. The sequences B3-9, B4-9 and B3-4 were clearly detectable only when the inhibitors of aminopeptidases amastatin and bestatin were included in the incubations.(ABSTRACT TRUNCATED AT 250 WORDS)

Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) derivatives are used in cancer therapy, but relatively little is known about their metabolic fate in the organism. This paper describes the application of high-performance liquid chromatography combined with electrospray mass spectrometry to identify the degradation products resulting from the incubation of two GnRH analogues, D-Phe6-GnRH and DSer(OtBu)6-desGly10-GnRH-ethylamide (buserelin) with rat kidney membranes. Reversed-phase columns were applied with gradient elution using a flow-rate of ca. 2 microliters/min to the mass spectrometer. Post- and precolumn stream splitting were employed to adjust the flow-rates for columns of 2 and 0.32 mm I.D. The pattern of peptide degradation products obtained with this method indicates that a defined proteolytic membrane enzyme system is responsible for these catabolic processes.

Spontaneous chemical degradation of substance P in the solid phase and in solution.

The instability of the undecapeptide substance P (SP), a neuropeptide implicated in several physiological processes, was occasionally observed when the peptide was stored in the solid state or in solution. The aim of the present study was to identify the decomposition products of SP stored as lyophilized peptide or in aqueous neutral solution. The main pathway of the decomposition of SP acetate consists of the subsequent release of N-terminal dipeptides via their diketopiperazines, cyclo(Arg-Pro) and cyclo(Lys-Pro). In contrast to the decomposition of the acetate of SP, the hydrochloride and trifluoroacetate salts were found to be considerably more stable. Under the studied conditions the release of N-terminal dipeptides dominates over other possible routes of spontaneous modifications, such as S-oxidation and deamidation.

In-vivo release of a GnRH agonist from a slow-release poly(lactide-glycolide) copolymer preparation: comparison in rat, rabbit and guinea-pig.

Different batches of 50:50 poly((+-)-lactide-glycolide) copolymer (PLG) were used as biodegradable carriers for D-Phe6-gonadotropin-releasing hormone (GnRHa) in the form of injectable long-acting implants loaded with 10% GnRHa and tracer amounts of [125I]GnRHa. After their injection subcutaneously into rats, rabbits, and guinea-pigs, the release kinetics of the peptide were determined by counting the radioactivity remaining in the implants (i) after recovery from the rats after death or (ii) directly on the skin above the injection site of rabbits and guinea-pigs in-vivo. No significant differences in the release pattern of the peptide amongst the three species whether the release process was controlled by diffusion or by degradation of the polymeric matrix were found. It is concluded that the results of in-vivo release tests using laboratory animals are valid for man and that enzymes are not involved in the degradation of the polymeric matrix. The results may be of general importance for the use of long-term release PLG formulations of highly active drugs, especially peptides and proteins.

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats.

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.

Insulin aggregation in solution.

The process of insulin aggregation in neutral solutions was studied by dynamic light scattering. Solutions of different concentrations were subjected to thermal and mechanical stress (37 degrees, rotation) for a period of 4 weeks. The starting solutions contained exclusively one particle distribution of insulin in the association equilibrium with hexamers as the largest structures. After a lag period of about 8 days the solutions showed continuously increasing scattering intensities but did not evolve perceptible turbidity within the experimental period. A more rapid increase in scattering intensity was observed in diluted than in concentrated solutions. The analysis of scattering data unexpectedly revealed that insulin species did not grow continuously. After the lag period one additional relatively restricted size distribution with particles of a mean radius of about 100 nm was found, the amount of which increased continuously with time. The occurrence of these particles seems to be related to adsorption phenomena of insulin to the solid interface. We assume the 100 nm-class of aggregates to be a transient state in the physical destabilization process of insulin solutions.

Gonadotropin-releasing hormone (GnRH) pharmacokinetics: peptide hormone pharmacokinetics needs clarification.

The plasma level curves of the peptide hormone gonadotropin-releasing hormone (GnRH) after its intravenous, intramuscular, and intraperitoneal administration into rats were fitted according to a two- (i.v.) and one-compartment model (i.m., i.p.), respectively. From the pharmacokinetic parameters it is concluded that urinary excretion and proteolytic degradation by kidney and liver are not sufficient to fully account for the clearance of the hormone and that, therefore, proteolytic degradation by tissues may play a role for the elimination of GnRH. This may be generally true with other short peptide hormones. The GnRH pharmacokinetics is shown as an example to underline that there presently exist problems of interpreting pharmacokinetic data of peptide hormones and that there is a need for a close interplay between biochemical and pharmacokinetic studies on peptide hormones for their pharmacokinetic behaviour to be understood.

Proteolytic inactivation of luteinizing hormone-releasing hormone (LHRH) by the whole rat ovary in vitro.

Using 3H-labeled luteinizing hormone-releasing hormone (LHRH) at low concentrations, the in vitro proteolytic inactivation of the peptide hormone by whole rat ovaries was studied and compared with that by the soluble and particulate rat ovarian fraction. Whole rat ovaries were found to express the three proteolytic activities that were, according to their properties, also observed in rat ovarian homogenates: (1) soluble intracellular activity which was released into the medium, (2) released activity of membrane-bound origin, and (3) firmly membrane-bound activity. It is suggested that in vivo LHRH is largely inactivated extracellularly at least by enzymes that are located in the plasma membrane although the membrane-bound activity comprises only about 1% of the whole LHRH-inactivating capacity of the ovary.

[The purification and characterization of Cordemcura]. / Untersuchungen zur Reinigung und Charakterisierung von Cordemcura.

3-Amino-5-(4-pyridinyl)-1,2-dihydro-pyrid-2-one (1) is an amphoteric compound and forms one crystalline sodium salt and two hydrochlorides. Physicochemical properties UV, NMR and MS are described. TLC has been used mainly and is the most sensitive method for estimation of 1-byproducts. Coloured byproducts, generated by hypochlorite or air oxidation during synthesis and handling in solution, are monitored by vis-spectra, diminished by sulfite addition and removed by alkaline precipitation. The purification procedure is able to produce 1 with only 0.1% of precursors or byproducts.

[The influence of viscosity-increasing pharmaceutic aids on the liberation of the peptide gonadotropin releasing hormone (GnRH) in solution]. / Untersuchungen über den Einfluss viskositätserhöhender pharmazeutischer Hilfsstoffe auf die Liberation des Peptids Gonadotropin Releasing Hormon (GnRH) in Lösung.

The in vitro liberation of the peptide hormone GnRH from polymer solutions was studied as a function of the viscosity of the polymer solutions. The liberation experiments were performed with a flow through dialysis apparatus. From solutions of dextran, carboxymethylcellulose, hydroxyethylcellulose, and polyvinylpyrrolidone the release of GnRH is prolonged whereas the release of GnRH from highly viscous solutions of methylcellulose and polyacrylic acid is not. The delivery process was found to be diffusion-controlled in solutions of dextran, carboxymethylcellulose, and hydroxyethylcellulose. In solutions of polyvinylpyrrolidone the diffusion-controlled prolongation of the GnRH release is additionally influenced by binding of the peptide to the polymer. The results imply that in these polymer solutions the diffusion coefficient of GnRH does not obey the classical relation D approximately 1/eta which is often used to predict liberation from viscous solutions. Thus, the measured macroscopic viscosity of the polymer solutions is not relevant for the diffusion-controlled prolongation of the in vitro release of GnRH. This phenomenon can be explained by the obstruction effect and the microviscosity of the polymer solutions. The described rapid and simple method can be applied for the quantitative determination of the prolonged release in polymer solutions based on diffusion-controlled processes and is useful in the comparison of the efficacy of various polymeric additives. Furthermore the method allows the quantitative proof of the decrease of diffusion in viscous low-molecular solutions and the proof of prolongations of delivery due to the binding of the investigated drug to the polymer.

[Synthesis of cyclic and non-cyclic tachykinin partial sequences. 2. Synthesis of the homocyclic substance P(6-11) hexapeptide]. / Synthese cyclischer und cyclisch-verzweigter Tachykinin-Teilsequenzen. Teil 2: Darstellung des homodet-cyclischen Substanz P(6-11)-Hexapeptides.

The authors describe the synthesis of Gln-Phe-Phe-Gly-Leu-Met by cyclization of H-Leu-Met-Gln-Phe-Phe-Gly using three different methods. The linear sequence was obtained by a (2+4)-segment condensation. The resulting cyclopeptide showed only a small kinin activity on isolated guinea pig ileum compared to substance P, but it is a full agonist.

Effect of dextran on the release of gonadotropin-releasing hormone (GnRH) injected into rats: plasma GnRH and gonadotropin response.

Prolonged release of the peptide gonadotropin-releasing hormone (GnRH) from its aqueous solution was achieved by addition of the polymer dextran (Mw ∼ 500,000). This effect observed in an in vitro system was caused by a decrease of the diffusion coefficient of the peptide. When GnRH was intramuscularly injected into male rats, the addition of dextran to the injected peptide solution led to a prolongation of the GnRH plasma level at the expense of its peak value. This change can be explained by a decrease of the absorption rate of GnRH into blood, which parallels the in vitro observation. As a result, the gonadotropin response to GnRH was stronly increased.

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown.

Analogs of luteinizing hormone-releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct 3H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe6,desGly10]-LHRH ethylamide and [D-Ser(But)6,desGly10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al2O3 catalyst to high specific radioactivities. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various tissues of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe6,desGly10]- and [D-Ser(But)6,desGly10]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of the analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.

Proteolytic degradation of gonadotropin-releasing hormone (GnRH) by rat ovarian fractions in vitro.

GnRH in physiological concentrations is highly degradable by both soluble and particulate fractions of rat ovarian homogenate in vitro. The two proteolytic enzyme activities differ strongly by the soluble activity showing a dithiothreitol optimum, high inhibition by diisopropyl fluorophospate (ki = 0.7 microM), and a relatively high affinity (Km = 1.1 microM) as opposed to the particulate fraction (Ki = 3.5 mM and Km = 150 microM, respectively). The results of this study show that the rat ovary is differently endowed with GnRH-degrading activity at different sites. The involvement of these in terminating the biological activity of the hormone on the ovary may possibly depend on its exact pathway in this GnRH-target organ.