Burkhard Mehlis

Identification and measurement of β-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.

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.

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.

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.

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.

In-vivo Release of a GnRH Agonist from a Slow-release Poly(lactide-glycolide) Copolymer Preparation: Comparison in Rat, Rabbit and Guinea-Pig

Abstract— 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.

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.

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.