Comparison between the biological effects of LH-RH and buserelin on the induction of LH release from hemi-pituitary glands of female rats in vitro

The biological effects of LH-RH and the agonist [D-Ser(But)6-des Gly10]-LH-RH(1-9)-ethylamide (buserelin) were compared during 8 h of incubation with female rat hemi-pituitary glands. Similar dose-response relationships were found for LH-RH and buserelin as concerns the release of luteinizing hormone (LH) by pituitary glands from intact and ovariectomized rats. Also the LH secretion patterns from glands of intact rats were similar: an initial low response was followed by a fast increase (priming effect) after which the response declined again (desensitization). In a subsequent experiment pituitary glands from ovariectomized rats were first exposed to LH-RH or buserelin for 4 h and then further incubated in medium only. After discontinuation of the stimuli the rate of LH release decreased in all cases, but this decrease was significantly greater when the glands had been exposed to LH-RH. Short-term (1/2, 1 or 2 h) exposures to LH-RH or buserelin followed by an intervening period (1 1/2, 1 or 0 h, respectively) of incubation in medium only resulted in an almost similar, significant increase in the subsequent protein synthesis-independent LH response to LH-RH (priming effect). Only preincubation with LH-RH for 2 h was significantly more effective. The results demonstrate equal intrinsic activities for LH-RH and buserelin. Differences in the biopotencies for LH-RH and buserelin in vivo and in vitro may occur only after discontinuation of the external stimuli.     

The effects of luteinizing hormone releasing hormone (LH-RH) in pregnant rats. I. Postnidatory effects.

The effects of LH-RH on pregnancy in rats were investigated. A single 500 mcg injection of LH-RH on Days 9, 10, or 11 of pregnancy terminated pregnancy, whereas injection on Days 6-8 or 13-16 had little or no effect. The ED 50 on Day 10 for b.i.d. administration was 150 mcg and 550 mcg for a single injection. Administration on Day 9 was followed by a decrease in circulating progesterone levels on Days 10 and 11. The administration of large doses of progesterone reversed the effects of LH-RH administration on Days 7-12. Treatment with estradiol-17beta did not potentiate the effect of progesterone, but appeared to slightly retard fetal resorption when administered alone. The results suggest that the antifertility effect of LH-RH is mediated via functional luteolysis.     

Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats

The recombinant cytokines IFN-gamma and TNF-alpha stimulate several macrophage-mediated functions important in host defense. However, systemic administration of cytokines may be limited by significant host toxicity. We investigated whether aerosolized cytokines can stimulate alveolar macrophage and blood monocyte function, and whether they induce an inflammatory response in the lungs of normal rats. We found that aerosolized murine rIFN-gamma or recombinant human TNF-alpha increased IL-1 production by both alveolar macrophages and blood monocytes for at least 5 days after administration. Furthermore, murine rIFN-gamma increased the expression of Ia Ag on alveolar macrophages and human rTNF-alpha increased alveolar macrophage- and blood monocyte-mediated tumor lysis. Sequential aerosolization of IFN-gamma and TNF-alpha significantly increased both IL-1 release and Ia expression compared to either cytokine administered alone. Aerosolized human rTNF-alpha achieved lung levels comparable to those produced by an i.v. TNF-alpha dose reported to cause diffuse organ injury and death in rats. However, plasma TNF-alpha levels were several thousand-fold lower after aerosol administration. Aerosolized cytokines did not induce lung edema or an inflammatory cell infiltrate within the airways or alveoli. Aerosolized human rTNF-alpha alone, or murine rIFN-gamma and human rTNF-alpha, induced margination of leukocytes in pulmonary blood vessels 1 day after aerosolization, and a few small foci of pulmonary hemorrhage 5 days later. We conclude that aerosol administration of IFN-gamma or TNF-alpha enhances both pulmonary and systemic monocyte function, and that the combination of IFN-gamma and TNF-alpha produce additive or synergistic effects. Aerosolized cytokines induce only a minimal pulmonary inflammatory response. Aerosolized TNF-alpha produces high cytokine levels in the lung but very low uptake into the circulation.     

Orexin receptor type-1 antagonist SB-334867 inhibits the development of morphine analgesic tolerance in rats

Herein the effect of orexin receptor type-1 antagonist SB-334867 on the development of tolerance to analgesic effects of morphine was studied in rats. To incite tolerance, morphine sulfate was injected intraperitoneally (i.p., 10mg/kg) once a day for 7 days. The tail flick test was used to evaluate antinociceptive effects of the morphine. A selective OxR1 receptor antagonist, SB-334867, was microinjected (i.c.v.) into the right cerebral ventricle (10 μg/10 μl) immediately before each morphine injection. Repeated morphine application resulted in tolerance to morphine analgesic effects as a decreasing trend during 7 days. Also, repeated administration of SB-334867 (i.c.v.) alone was without significant effect on the nociception as compared to control. Microinjection of SB-334867 prior to each morphine injection inhibited the development of tolerance, so that the analgesic effects of morphine were significantly higher in SB-334867 plus morphine treated rats than that of vehicle plus morphine treated ones on days 4-7. It is concluded that orexin receptor type-1 might be involved in the development of tolerance to morphine analgesic effects.     

In vivo and in vitro effects of LH-RH on cAMP content of anterior pituitary in rats.

Changes in the cAMP content of anterior pituitary of male rats were studied after in vivo administration of LH-RH or in vitro incubation in the presence of LH-RH. Although a significant rise was observed 5 min after in vivo injection of LH-RH, the cAMP level increased after only a two-hour lag period in vitro. Maximum level was reached in vivo with 1 microgram LH-RH/100 g b.w., but there was no maximal production in vitro even in the presence of 5 x 10(-6) M LH-RH. A summary of the available observations on the effect of LH-RH on pituitary cAMP level is given in the present paper.     

Development of a new aerosol delivery system for systemic pulmonary delivery in anaesthetized and orotracheal intubated rats.

Over the last decade, the systemic absorption of a broad range of therapeutics after pulmonary application has been demonstrated in animals as well as in humans. The most common method used in the laboratory is the intratracheal instillation of drugs in solution. This method is, however, unsatisfactory, because of discrepancies in particle distribution, clearance, kind of injury and bioavailability between instillation and inhalative application. On the other hand, a precise determination of the amount of drug applied by aerosol, and of the aerosol volume retained within the lungs is rather difficult, and is not possible for use with small animals such as mice or rats. We describe a system which allows the delivery of aerosols directly into the animal's lungs, and calculation of the amount of drug retained in the lungs. Our system was tested in vitro and in vivo and was shown to allow precise and efficient pharmacokinetic and toxicological studies to be carried out.     

Synthesis and biological activity of position 1 analogs of LH-RH.

(Formyl-sarcosine)1-LH-RH (I), (acetyl-sarcosine)1-LH-RH (II), (2-pyrrolidone-4-carboxylic acid)1-LH-RH (III), (N-methyl-2-pyrrolidone-4-carboxylic acid)1-LH-RH (IV, hydroxyproline1-LH-RH (V) and (cylcopentane-carboxylic acid)1-LH-RH (VI) were synthesized by solid phase methods on a benzhydrylamine resin support. Peptides I-IV were assayed for LH- and FSH-releasing activity over a 4-h period after subcutaneous infection into immature male rats in order to detect any prolongation ofactivity. The peptides were found to have the following integrated LH-releasing activities compared with LH-RH: I, 64%; II, 72%; III, 19%; IV, 58%. None of the peptides were found to be longer acting than LH-RH. Peptides V and VI were far less active, 0.001% and 1.4%, respectively.     

Development of a peptide-containing chewing gum as a sustained release antiplaque antimicrobial delivery system

The objective of this study was to characterize the stability of KSL-W, an antimicrobial decapeptide shown to inhibit the growth of oral bacterial strains associated with caries development and plaque formation, and its potential as an antiplaque agent in a chewing gum formulation. KSL-W formulations with or without the commercial antibacterial agent cetylpyridinium chloride (CPC) were prepared. The release of KSL-W from the gums was assessed in vitro using a chewing gum apparatus and in vivo by a chew-out method. A reverse-phase high-performance liquid chromatography method was developed for assaying KSL-W. Raw material stability and temperature and pH effects on the stability of KSL-W solutions and interactions of KSL-W with tooth-like material, hydroxyapatite discs, were investigated. KSL-W was most stable in acidic aqueous solutions and underwent rapid hydrolysis in base. It was stable to enzymatic degradation in human saliva for 1 hour but was degraded by pancreatic serine proteases. KSL-W readily adsorbed to hydroxyapatite, suggesting that it will also adsorb to the teeth when delivered to the oral cavity. The inclusion of CPC caused a large increase in the rate and extent of KSL-W released from the gums. The gum formulations displayed promising in vitro/ in vivo release profiles, wherein as much as 90% of the KSL-W was released in a sustained manner within 30 minutes in vivo. These results suggest that KSL-W possesses the stability, adsorption, and release characteristics necessary for local delivery to the oral cavity in a chewing gum formulation, there-by serving as a novel antiplaque agent. Published: March 30, 2007     

Acute stimulatory effects of an LH-RH agonist on the ovary in pseudopregnant rats

Pseudopregnant rats were anaesthetized on day 5 of pseudopregnancy with pentobarbital and hypophysectomy or sham operation was performed. Polyethylene cannulas were inserted into one of the femoral arteries and utero-ovarian veins. Five-minute blood samples were collected from the ovary for 40 minutes. Following the first five minute blood fraction LH-RH, an agonistic analogue (D-Phe6, des Gly-NH10(2)-LH-RH-ethylamide) or an antagonistic analogue (Formyl-D-Tryp1,3, D-pCl-Ph2, D-Phe6LH-RH) were superfused into the ovarian bursa for 5 minutes in a volume of 50 microliters. Blood pressure, ovarian venous outflow and haematocrit were continuously measured. From the blood samples progesterone and oestradiol-17 beta were determined by RIA and the secretion rate was calculated. It was found, that LH-RH and its antagonistic analogue has no effect on the blood flow, progesterone (P) and oestradiol-17 beta (E2) secretion of ovary. However, an agonistic analogue of LH-RH induced a rapid elevation of blood flow, diminished vascular resistance in the ovary, and in a dose of 50 ng increased the secretion rate of E2. In sham operated animals the effects of agonistic analogue was similar to the effects of 1.0 IU of hCG.     

Experience with a radioimmunoassay of luteinizing hormone-releasing hormone (LH-RH) in biological material of man.

A radioimmunoassay of LH-RH with a sensitivity of 7.8 pg/ml is described. Labelling and purification techniques, methods for extraction of LH-RH and separation techniques of bound and free labelled hormone are compared. Determination of LH-RH levels in serum after administration of synthetic LH-RH by different routes and measurements of endogenous LH-RH levels in serum of normal subjects and patients with different endocrine diseases as well as in cerebrospinal fluid of normal men are performed. The measurement of exogenously administered LH-RH in serum reflects the disappearance of synthetic LH-RH from peripheral circulation in dependence upon the kind of administration route. The level of endogenous LH-RH was found to be under the limit of the assay in all samples of cerebrospinal fluid and of serum of normal male subjects. The results obtained in the patient groups show that the radioimmunological estimation of endogenous LH-RH in peripheral body fluids does not reflect the hypophysiotrophic role of this hypothalamic peptide.     

Hydrolytic instability of the important orexin 1 receptor antagonist SB-334867: Possible confounding effects on in vivo and in vitro studies

SB-334867 has been an important ligand for the study of the orexin 1 (OX1) receptor due to its high OX1/OX2 selectivity and bioavailability. This ligand however, contains a 2-methylbenzoxazole ring system which is known to undergo hydrolysis, particularly under acidic or basic conditions. The possibility that SB-334867 would be susceptible to significant hydrolysis was evaluated in various formulations and in the solid state. SB-334867 was found to be unstable under conditions commonly employed to prepare stock solutions for in vitro and in vivo studies. In addition, and most alarmingly, the hydrochloride salt of SB-334867 was found to quantitatively decompose to an OX1-inactive product even in the solid state. These findings combine to suggest that studies using SB-334867 (and any other 2-methylbenzoxazole-containing compound) should be performed with great care to avoid the confounding effects of the rapid hydrolytic decomposition of this susceptible structure.     

Synthesis and biological activity of LH-RH antagonists modified in position 1

As part of our studies on the design of more potent antagonists of the LH-RH (luteinizing hormone-releasing hormone) decapeptide, twelve new highly soluble D-Arg⁶-analogs have been synthesized. These peptides contain modifications in position 1 and are typified by the general formula (N-acetyl-X¹, D-p-Cl-Phe², D-Trp³, D-Arg⁶, D-Ala¹⁰) LH-RH. We have found that a lypophilic, aromatic substituent is required in position 1 in order to elicit antiovulatory activity at a dose as low as 3 μg. The larger the hydrophobic amino acid (X: p-Br-Phe, β-Nal-2) in position 1, the higher is the antiovulatory activity that can be attained. Analogs with non-aromatic or hydrophilic amino acids (X: Gly, Leu, Arg, His, Glu) in position 1 generally have much lower activities in this series of LH-RH antagonists.     

Comparison of methods for enumeration of interacting species in a biological system

Matrix rank analysis, a method extensively used to enumerate interacting species or components in various biological systems, has been compared with the more readily available technique of factor analysis. Data previously analyzed by matrix rank analysis has been subjected to factor analysis. The results show that there are some agreements and disagreements between the two methods. The sources and nature of the disagreements are discussed. Our work indicates that factor analysis may be preferable to matrix rank analysis.     

Comparison of an accelerometer and a condenser microphone for mechanomyographic signals during measurement of agonist and antagonist muscles in sustained isometric muscle contractions

The purpose of this study was to investigate the influence of the force tremor (FT) on mechanomyographic (MMG) signals recorded by a condenser microphone (MIC) and an accelerometer (ACC) during measurement of agonist and antagonist muscles in sustained isometric contractions. Surface electromyographic (EMG) signals and MMG signals by MIC (MMG-MIC) and ACC (MMG-ACC) were recorded simultaneously on biceps brachii (BB) and triceps brachii (TB). Following determination of the isometric maximum voluntary contraction (MVC), 10 male subjects were asked to perform sustained elbow flexion and extension contractions at 30% MVC until exhaustion. We analyzed the root mean square (RMS) for all signals and compared the sum of the power spectrum (SPA) for 3-6 Hz and 8-12 Hz and the ratio of the sum of SPA for 3-6 Hz and 8-12 Hz in SPA for 3-100 Hz (SPA-FT/SPA-(3-100 Hz)) between MMG-MIC and MMG-ACC. During all sustained muscle contractions, the RMS of EMG and MMG-(MIC) was significantly (p<0.05) increased in antagonistic muscle pairs, while the increase was more noticeable for the agonist than for the antagonist. In addition, the antagonist had a significantly (p<0.05) smaller amplitude than the agonist muscle. The RMS of MMG-ACC, however, showed no significant (p>0.05) difference in RMS amplitude and slope between agonist and antagonist muscles during flexion. In extension, the MMG-ACC-RMS amplitude showed a tendency to be higher in the antagonist than in the agonist, while their slopes showed no significant (p>0.05) difference. The SPA for 3-6 Hz and 8-12 Hz in MMG-(MIC) showed a tendency to be higher in the agonist than the antagonist, and the slopes of the agonist were significantly (p<0.05) higher than those of the antagonist in all contractions. In MMG-ACC, SPA and slopes for 3-6 Hz and 8-12 Hz tended not to differ between agonist and antagonist. The SPA-FT/SPA-(3-100 Hz) in MMG-ACC showed that the antagonist was higher than that of the agonist in all contractions. The MMG-(MIC), however, showed a tendency toward no difference between the agonist and antagonist. In the assessment of muscle activity during simultaneous measurement of the agonist and antagonist during sustained muscle contractions, the MMG signal detected by MIC appeared to be less affected by FT than by ACC due to the different inherent characteristics of the two transducers.     

Inhibitory control of the pituitary LH secretion by LH-RH in male rats.

Luteinizing hormone-releasing hormone (LH-RH) was administered to prepubertal male rats (intact, castrate or castrate-adrenalectomized, 60 g body weight) for 28 days (1 microgram LH-RH/day, s.c.), at a 10-fold physiological dose, as compared to the minimal FSH-releasing dose of 100 ng/rat s.c. In intact rats, serum LH and weight of androgen-dependent organs (vented prostate, seminal vesicles) were reduced after 14 days of treatment. In castrate rats, the postcastration rise in serum LH was abolished by treatment. Pituitary LH content, FSH secretion and prolactin secretion were not suppressed. Hypothalamic LH-RH was increased at 14 and 21 days. In castrate adrenalectomized male rats, LH secretion was also suppressed by 1 microgram LH-RH s.c. x 28 days. The hypothalamic LH-RH content did not increase. The pituitary LH-RH receptor level was not down-regulated after 14 days treatment either in intact or castrate male rats. Pituitary inhibition (LH release) in rats by a supraphysiological dose of LH-RH given for 28 days indicates that the optimal regime for chronic treatment has to be determined by monitoring LH release at regular intervals. Direct pituitary inhibition by LH-RH may explain some of the unexpected antifertility effects observed with high doses of LH-RH.     

The orexin-1 receptor antagonist SB-334867 attenuates anxiety in rats exposed to cat odor but not the elevated plus maze: An investigation of Trial 1 and Trial 2 effects

The orexins are hypothalamic neuropeptides most well known for their roles in regulating feeding and sleeping behaviors. Recent findings suggest that orexin-A may also modulate anxiety, although how and when the orexin system is involved remains unclear. To address this, we investigated the dose-dependent effects of the orexin-1 receptor antagonist SB-334867 in two rodent models of anxiety: the cat odor avoidance model and the elevated plus maze. In both models we tested the effects of SB-334867 when anxiety is novel (Trial 1) and familiar (Trial 2). In the first experiment, Wistar rats were treated with vehicle or SB-334867 (5, 10 or 20 mg/kg, i.p.) prior to their first or second exposure to cat odor. During Trial 1, rats treated with 10 mg/kg of SB-334867 approached the cat odor stimulus more than vehicle-treated rats. During Trial 2 the effects were more marked, with 10 mg/kg of SB-334867 increasing approach times, increasing the number of times rats exited the hide box to engage in exploratory behavior, and decreasing overall hide times. In addition, the 20 mg/kg dose decreased general activity during Trial 2. In the second experiment, the effects of SB-334867 (10 and 20 mg/kg) were tested in the elevated plus maze. There were no significant differences produced by drug treatment during either Trial 1 or Trial 2. Results suggest that SB-334867 decreases anxiety induced by some, but not all, stressors.