Exogenous 20K growth hormone (GH) suppresses endogenous 22K GH secretion in normal men.

The physiological and pharmacological functions of the 20-kDa human GH (20K-hGH) isoform are unknown. We conducted a pharmacokinetic study of recombinant 20K-hGH in human subjects (Phase I clinical trial). Placebo or 20K-hGH was administered sc to normal men (20-31 yr of age, n = 6-8 per group) at 2100 h. Serum 20K- and 22K-hGH levels were monitored every 30 min for 24 h by specific enzyme-linked immunosorbent assays. Serum free fatty acid, insulin-like growth factor I, insulin, and glucose levels were measured for 24 h. In the placebo group, the secretion profiles of endogenous 20K- and 22K-hGH were pulsatile and similar to each other. The proportion of 20K- to 22K-hGH was fairly constant. In the 20K-hGH-treated groups, serum 20K-hGH levels increased in a dose-dependent manner over the dose range of 0.01-0.1 mg/kg. Maximum serum 20K-hGH levels were reached at 3-4 h and decreased with half-lives of 2-3 h. Marked suppression of endogenous 22K-hGH secretion was observed in a time-dependent manner. Serum free fatty acid and insulin-like growth factor I levels were significantly elevated (P < 0.01) at 4, 8, and 12 h and at 8, 12, and 24 h after 20K-hGH administration, respectively. Serum insulin and glucose levels did not change significantly within 24 h. These results suggested that: 1) regulation of 20K-hGH secretion is physiologically the same as that of 22K-hGH; 2) the pharmacokinetics after sc injection of 20K-hGH are comparable with those of 22K-hGH; 3) 20K-hGH regulates hGH secretion through "GH-induced negative feedback mechanisms"; and 4) administration of 20K-hGH is expected to exert GH actions (growth-promoting activity and lipolytic activity). Monitoring of serum 20K- and 22K-hGH levels may be useful in evaluating the effects of administered GH isoforms on their own release from the pituitary.

Pharmacological properties of the new centrally acting muscle relaxant (R)-(+)-3-phenyl-5-[2-(1-pyrrolidinylmethyl)butyryl]isoxazole hydrochloride.

The pharmacological properties of (R)-(+)-3-phenyl-5-[2-(1-pyrrolidinylmethyl)butyryl]isoxazole hydrochloride (CAS 144576-50-1, MS-322), a new centrally acting muscle relaxant, were investigated and compared with those of eperisone-HCl (CAS 56839-43-1) in experimental animals. MS-322 (1.5-6 mg/kg i.v.) reduced both anemic and intercollicular decerebration-induced rigidity in rats. Similar doses were required for these effects. The activity of MS-322 (200 mg/kg p.o.) against intercollicular decerebration-induced rigidity was greater and longer lasting than that of the same dose of eperisone-HCl. MS-322 (0.75-6 mg/ kg i.v.) inhibited the flexor reflex in anesthetized cats in a dose-dependent manner and more strongly than eperisone-HCl. MS-322 had no effect on the neuromuscular junction in anesthetized rats. The muscle relaxant activity of MS-322 in mice, demonstrated by the traction and rota-rod tests was stronger than that of eperisone-HCl, whereas MS-322 affected spontaneous motor activity in mice less than eperisone-HCl. The results of this study suggest that MS-322 is a potent centrally acting muscle relaxant with relatively weak depressant activity at other central nervous system pathways.

Characterization of alpha 1-adrenoceptor subtypes in rat spinal cord.

The alpha 1-adrenoceptor subtypes in ventral and dorsal horns of rat lumbar spinal cord were studied. High concentrations of the alpha 1D-adrenoceptor antagonist, BMY 7378 (8-[2-[4-(2-methoxyphenyl) 1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride), displaced bound [3H]prazosin in a single-site manner and the binding affinity was close to those for alpha 1A- and alpha 1B-adrenoceptor binding sites. 5-Methyl-urapidil displaced bound [3H]prazosin in a two site manner and the high and low affinities were close to those of alpha 1A- and alpha 1B-adrenoceptor binding sites, respectively. The alpha 1-adrenoceptor subtype populations of ventral and dorsal horns did not differ. The alpha 1A- and alpha 1B-adrenoceptor populations comprised 70% and 30%, respectively, of the total and very few alpha 1D-adrenoceptors were detected.

The distribution of polypeptide YY-like immunoreactivity in rat tissues.

The distribution of peptide YY (PYY)-like immunoreactivity (IR) in rat tissues was determined by specific RIA after extraction with boiled 1 N acetic acid. The high concentration of PYY-IR was observed in the gastrointestinal tract, with concentrations gradually increasing from the duodenum to the end of colon. The concentration of PYY-IR in the colon was 298.7-449.5 pmol/g tissue (approximately 100-200 times more than that in the duodenum). Pituitary and pancreas contained measurable amounts of PYY-IR (6.8 and 6.3 pmol/g tissue). The concentration of PYY-IR in the mucosa was higher than that in the muscular layer in the small intestine, cecum, colon, and rectum. The ratio of the mucosal PYY-IR to the muscular PYY-IR was highest in the distal small intestine (4.7-6.8). Sephadex G-50 gel chromatography of the colon extracts revealed the one PYY-IR peak which corresponds to [125I]PYY. The gradual increase of PYY-IR from the duodenum to the end of the colon is different from the distribution of other known gut peptides.

Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay.

Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the beta-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125iodine. This assay was highly specific for HCG and there was no cross-reactivity with alpha, beta-subunit of HCG, luteinizing hormone and follicle stimulating hormone.

Effect of sultopride on prolactin secretion in rats.

Oral administration of sultopride caused a significant and dose-related increase in serum prolactin levels in the rat. Sultopride was 4-6 times as potent as sulpiride in stimulating prolactin secretion. Both drugs produced much greater stimulations of prolactin release in female rats than in male rats, suggesting the sex difference in response to drugs. CB-154, a dopamine agonist, inhibited the sultopride-induced prolactin release. Stereoselective activity of sultopride-isomers was observed in increasing rat prolactin secretion. Sultopride had no significant effects on LH and FSH basal levels in female rats. In a successive study, basal prolactin levels in the male rat were higher at 2-3 days, but lower at 6 and 14 days. Prolactin response 1 hr after sultopride administration was observed throughout the experiments. Sultopride neutralized the dopamine-mediated inhibition of prolactin secretion from the anterior pituitary in vitro. These results suggest that sultopride, like sulpiride, stimulates prolactin secretion by blocking the dopamine receptor in the pituitary.

Effects of chronic sultopride treatment on endocrine systems in psychotic women.

The effects of chronic sultopride treatment on endocrine systems were studied using five schizophrenic women. Sultopride, an antipsychotic drug, was administered orally three times daily for 5 weeks in a daily dose of 300-600 mg. The serum prolactin levels increased significantly after 1 day of treatment, reaching a maximum at 1 week and remaining elevated during treatment. The serum GH levels declined temporarily after 1 week of treatment and then returned to normal values after 3-5 weeks of treatment. Sultopride had no significant effects on LH, FSH, TSH, insulin, estradiol-17 beta and cortisol basal levels. Serum sultopride levels measured by radioimmunoassay remained steady during treatment. These results showed that sultopride stimulates prolactin secretion in schizophrenic women, probably by blocking pituitary dopamine receptors.

Elevated immunoreactive beta-endorphin level in ventricular fluid after analgesic electrical stimulation of posteromedial hypothalamus.

Immunoreactive beta-endorphin (beta-EP) in the ventricular fluid of six carcinomatous patients was measured using a specific radioimmunoassay. The subjects were undergoing a surgical procedure for relief of chronic intractable pain. This procedure involved the focal stimulation and coagulation of the posteromedial hypothalamus. Samples of ventricular fluid were collected before and after the stimulation and serially after the coagulation. Prior to stimulation, beta-EP-like immunoreactivity (beta-EP-LI) was below 200 pg/ml. In all of the six patients with pain relief, electrical stimulation led to a marked increase in immunoreactive beta-EP. In three patients beta-EP levels remained high after electrical coagulation for 6-24 hrs. These results suggest that beta-EP-like material, released into the ventricular fluid, may contribute to the initial pain blockade that results from stimulation and coagulation of the posteromedial hypothalamus.

Regional distribution of sultopride and sulpiride in rat brain measured by radioimmunoassay.

Sensitive and specific radioimmunoassays for both sultopride and sulpiride were developed. Using these radioimmunoassays, the regional distributions of sultopride and sulpiride in rat brain after intraperitoneal administration were investigated. Although relatively small amounts of both drugs were detected in the brain, sultopride appears to pass the blood-brain barrier more easily than sulpiride. Relatively high concentrations of sultopride were seen in hypothalamus, striatum, the mesolimbic area and hippocampus, while sulpiride accumulated mainly in brain areas such as hypothalamus, medulla oblongata and cerebellum, where the blood-brain barrier is less effective. Both drugs seem to be concentrated by the pituitary and pineal body. These differences between sultopride and sulpiride in penetration to the brain may depend on their different lipid solubilities, since sultopride has a higher lipid solubility compared with sulpiride.

Characteristics of [3H]sultopride binding to rat brain.

The binding of [3H]sultopride, a benzamide drug, to rat brain was investigated in vitro. Specific [3H]sultopride binding was observed in dopaminergic regions: striatum, nucleus accumbens, olfactory tubercle, substantia nigra, frontal cortex and anterior pituitary. Specific [3H]sultopride binding to striatum was saturable and had one high affinity binding site with a KD of 5.8 nM and a total density of receptors 25.7 pmol/g. [3H]Sultopride binding was stereoselectively displaced by (-)- and (+)-sultopride. Inhibition studies indicated that all neuroleptic drugs and dopamine were capable of displacing sultopride from its binding sites. A highly significant correlation was observed between IC50 values against [3H]sultopride and those against [3H]spiperone binding. Specific [3H]sultopride binding was highly dependent on the presence of sodium ions. The results suggest that the characteristics of sultopride binding sites seem to be similar to those of the D2-receptor labeled by spiperone and haloperidol. The sultopride binding site was highly dependent on the presence of sodium ions and may thus be characterized as a sodium-dependent D2-receptor.

Development of radioimmunoassay for sultopride and sulpiride.

Sensitive radioimmunoassays for both sultopride and sulpiride in human serum have been developed. The antiserum used was obtained by immunizing rabbits with sultopride albumin conjugate. Using tritiated sultopride as the ligand, a sensitive standard curve was established with a useful range from 0.05 to 5 ng. The antiserum showed less than 1% cross reactivity with sultopride metabolites but 4.5% with sulpiride, which enabled us to establish a sulpiride standard curve between 0.1 and 500 ng. Serum concentrations of sultopride or sulpiride after single oral administration to normal volunteers have been determined using these radioimmunoassays. Serum sultopride levels reached peak 1 hr after the administration and declined with an apparent half life of 3.5 hr, while serum sulpiride levels increased gradually and the high serum level at 2 hr remained for up to 6 hr. Such differences in sultopride and sulpiride pharmacokinetics may depend on their different lipid solubilities.

Separation of serum prednisolone and prednisolone-21-hemisuccinate by extraction and their concurrent determination by radioimmunoassay.

We describe a simple, sensitive, and reliable radioimmunoassay for prednisolone and prednisolone-21-hemisuccinate in serum. The antiserum produced in rabbits to prednisolone-21-hemisuccinate/bovine serum albumin was specific for prednisolone and prednisolone-21-hemisuccinate. A simple dichloromethane extraction permitted the separation of prednisolone from prednisolone-21-hemisuccinate in the serum samples. Interference by cortisol, although not insignificant, is minimized in this assay. We used the method to measure prednisolone and prednisolone-21-hemisuccinate concentrations after a bolus injection of prednisolone-21-hemisuccinate into human beings and mice.

[Radioimmunoassay for plasma betamethasone and its application for clinical study (author's transl)].

A sensitive and specific radioimmunoassay for plasma betamethasone has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone-3-oxime-human serum albumin (BM-3-oxime-HSA) conjugate. The sensitivity was 20 pg and a standard curve was established with a useful range from 20 pg to 4 ng. The cross reactivity of all tested endogenous steroids was less than 0.2%. Cortisol with a cross reactivity of 0.14% caused slight interference at very high concentrations, but this factor is negligible when plasma cortisol level is less than 30 microgram/dl. The calculated interference by cortisol was less than 10%, and plasma not receiving BM consistently gave blanks which were less than 20 pg/tube. Reliability criteria were satisfactory. By this method plasma BM could be measured directly in dichloromethane extract of plasma. The plasma concentrations of BM were measured in normal subjects and patients with liver diseases following oral administration of BM. The peaks of the plasma concentrations for 1.0 mg and 1.5 mg of BM were 345 +/- 40 (n = 3) and 710 +/- 200 ng/dl (n = 5) respectively within 2 hours after administration in normal subjects. After the peak level, plasma BM rapidly fell and disappeared 24 hours after administration in all examined normal subjects. In patients with chronic active hepatitis, the peak levels for 1.0 mg and 1.5 mg of BM were 428 +/- 48 (n = 4) and 837 +/- 83 ng/dl (n = 7) respectively within 2 hours after administration. However, the peak levels of plasma BM were higher than those of normal subjects, and the disappearance of BM from the blood was markedly delayed, reaching a level of 318 +/- 88 nad 622 +/- 148 ng/dl respectively for 1.0 mg and 1.5 mg of BM at 5 hours. The relatively high plasma concentration of BM, ranging from 135 to 170 ng/dl was maintained even 24 hours after administration in all patients with chronic active hepatitis. The disappearance of cortisol from the blood also rapidly fell in normal subjects, but was markedly delayed in patients with chronic active hepatitis. There was good correlation between the severity of the liver disease as measured by the ICG retention at fifteen minutes and removals of BM and cortisol from the blood.

Percutaneous absorption of betamethasone 17-benzoate measured by radioimmunoassay.

Percutaneous absorption was studied in patients following topical application of betametahsone 17-benzoate cream and gel with occlusion by means of a sensitive and specific radioimmunoassay method. Concentrations of betamethasone 17-benzoate in plasma were between 0.3 and 5 ng/ml, indicating approximately 0.05 to 0.3% of the steroid applied to the skin was detected in plasma. Plasma betamethasone 17-benzoate levels increased in proportion to the amount of the steroid applied to the skin. High correlation between plasma betamethasone 17-benzoate levels and percent inhibition of plasma cortisol was also observed. Approximately 3 ng/ml levels of betamethasone 17-benzoate in plasma induced 90% inhibition of plasma cortisol. The data suggest that betamethasone 17-benzoate in gel base was more readily absorbed than in cream base.

Enzymatic radioiodination of succinyl cyclic AMP tyrosine methyl ester by lactoperoxidase and radioimmunoassay for cyclic AMP.

A highly specific and simple radioimmunoassay for cyclic AMP with a sensitivity of 0.04 picomoles/tube has been developed according to the method of Steiner et al., using 125I-succinyl cyclic AMP tyrosine methyl ester as a tracer. The tracer with higher immunoactivities could be simply and constantly prepared by an enzymatic iodination procedure utilizing lactoperoxidase, radioactive iodide and hydrogen peroxide generated by glucose-glucose oxidase system, rather than by chloramine-T procedure.

Radioimmunoassay for plasma betamethasone 17-benzoate.

A sensitive radioimmunoassay for plasma betamethasone 17-benzoate has been developed. The antiserum used was obtained by immunizing rabbits with betamethasone 17-benzoate-21-hemisuccinate-bovine-serum-albumin conjugate. All of the endogenous steroids tested cross reacted less than 0.10%. A standard curve was established with a useful range from 0.05-5 ng. Reliability criteria were satisfactory. Measurement of plasma concentrations of betamethasone 17-benzoate was performed in patients and in rabbits following occlusive dressing of betamethasone 17-benzoate cream and gel base.