Brian Rafferty

Pharmacokinetic evaluation of superactive analogues of growth hormone-releasing factor (1–29)-amide

D-amino acid-substituted analogues of growth hormone-releasing factor 1-29)-amide with superagonist activities in the rat were examined for increased plasma half-life and resistance to degradation in vivo. After IV injection, half-lives of the analogues were in the range 4.7-7.4 min, none of which was significantly different from that of the parent compound (6.2 min). Following SC injection, 4.6-7.2% of the dose of the analogues reached the circulation compared with 5.1% of the parent compound. Conformational restraints introduced into the N-terminal region of the molecule, which gave enhanced potency, did not alter the susceptibility of the peptide to proteolytic degradation.

Radioimmunoassay for human growth hormone-releasing factor (hGRF 1-40): comparison of plasma immunoreactive GRF after intravenous and subcutaneous administration to rats

A homologous radioimmunoassay (RIA) system was developed for human GRF 1-40 and used to measure immunoreactive (IR) concentrations of the peptide in rats to determine some of its pharmacokinetic characteristics after intravenous (i.v.) and subcutaneous (s.c.) administration. A plot of the disappearance of IR-hGRF from plasma after a single intravenous injection was fitted by a biexponential curve, analysis of which gave a half-life of 3.2 +/- 0.2 min for the initial distribution phase and 57.3 +/- 1.5 min for the elimination phase. Comparison of areas under the plasma IR-hGRF/time curves after injection of identical doses of hGRF 1-40 showed that the amount detected in the circulation after it was injected s.c. was only 14-16% of the amount detected after i.v. administration. Such results may indicate degradation of a substantial proportion of the dose of the peptide at the site of injection or during its transfer to plasma; this should be borne in mind when undertaking s.c. administration for clinical purposes or in assessing the effect of GRF analogues.

Corticotropin-induced desensitization: steroidogenic and cyclic AMP responses in supervised adrenocortical cells

The characteristics of the sensitization and desensitization of superfused, rat dissociated adrenal fasciculata cells were examined. The dynamic output of steroids and cyclic AMP was determined following pulsed treatment with various agonists. Repeated doses of identical small amounts of ACTH or ACTH gave gradually increasing responses which were maximal after 3-4 injections, but then desensitized the adrenal cells. An initial dose of 2.5 X 10(-11) moles of ACTH, itself insufficient to stimulate steroidogenesis in this system, had the effect of priming the cells, which showed an enhanced initial response and achieved maximum responsiveness on the second injection of 4 X 10(-13) moles ACTH. Thereafter, although the cells exhibited a diminishing steroid output, a dose at the end of the experiment of 8 X 10(-12) moles ACTH restored the maximum responsiveness, and demonstrated that cell loss or death could not account for the desensitization effect. Only a sensitization of the cells was observed following repeated doses of 5 X 10(-6) moles cyclic AMP. Since no desensitization effect was discernible for this agonist, it was concluded that in this system the lesion giving rise to the desensitization effect occurred prior to the adenylate cyclase catalytic unit for the generation of cyclic AMP within the cell and the receptor-nucleotide-regulatory protein complex is thus implicated in the desensitization mechanism for adrenal steroidogenesis. The studies demonstrate the exquisite sensitivity of adrenal cells to the desensitizing effects of even brief intermittent pulses of ACTH.

Validation of an SEC‐HPLC Method for the Analysis of rhG‐CSF in Pharmaceutical Formulations

Abstract Granulocyte colony‐stimulating factor (G‐CSF) is a hematopoietic cytokine produced by recombinant DNA technology and used clinically to treat neutropenia. An isocratic high performance liquid chromatography procedure was developed for the assay of filgrastim in pharmaceutical formulations. HPLC separation was carried out by size‐exclusion chromatography on a TSK gel G2000 SW column (60 cm × 7.5 mm I.D.). The mobile phase was composed of phosphoric acid (pH 2.5; 0.1 M), run at a flow rate of 1.0 mL min−1 and with UV detection at 214 nm. Method validation investigated parameters such as the range, linearity (r 2 = 0.9998), precision, accuracy, and robustness; the method yielded good results with a quantitation limit of 45 µg mL−1 and a detection limit of 12 µg mL−1. The results demonstrate the validity of the SEC‐HPLC method for the analysis of filgrastim.

The World Health Organization reference reagent for vascular endothelial growth factor, VEGF165

Preparations of human sequence recombinant vascular endothelial growth factor-165 (VEGF165) synthesized in Escherichia coli were formulated and lyophilized at NIBSC. Following evaluation at NIBSC, the first preparation, 01/424, has been distributed since 2002 as a NIBSC research reagent, but shows variation between ampoules in the volume and crystalline appearance of the lyophilized plug. A second preparation, 02/286, was subsequently lyophilized in a different formulation. Preparation 02/286 has now been evaluated in a collaborative study for its suitability to serve as a reference standard, and compared with preparation 01/424, by five laboratories using in vitro bioassays or immunoassays. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 02/286 as the WHO reference reagent (RR) for human VEGF165, with an assigned unitage of 13,000 units per ampoule. Details on ordering the WHO RR can be found at www.nibsc.ac.uk.

Human Acoustic Neuromas Secrete Interleukin-6 in Cell Culture

Interleukin-6 (IL-6) secretion by cell cultures of human acoustic neuromas was examined. Secretory rates varied from 0.02 to 5.4 ng/10(5) cells per 4 days, depending on the tumor. The IL-6 immunoreactivity eluted from a Sephadex G-100 column in a major peak corresponding to an M(r) of 30,000 and a lesser peak corresponding to an M(r) of 50,000. Western blot analysis revealed three IL-6 immunoreactive bands with M(r)s corresponding to 53,000, 29,000, and 24,000. Tumor necrosis factor-alpha, interleukin-1-beta, and cholera toxin all stimulated IL-6 secretion. An antisense phosphorothioate oligonucleotide against IL-6 messenger RNA inhibited both [3H]thymidine uptake and IL-6 secretion by acoustic neuroma cells in culture. In addition, [3H]thymidine uptake was inhibited by a specific polyclonal antibody against IL-6. We conclude that human acoustic neuroma cells produce and secrete IL-6, which may act in an autocrine manner to stimulate cellular proliferation.

International Standards for hepatocyte growth factor/scatter factor: initial assessment of candidate materials and their evaluation by multicentre collaborative study

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent paracrine growth factor with motogenic, mitogenic and morphogenic activities, and a potential therapeutic role in hepatic and renal disease, as well as diagnostic and prognostic applications. It is synthesised as an inactive, single-chain precursor that is cleaved by serine proteases to give a biologically active, heterodimeric form. To develop World Health Organization (WHO) International Standards (IS) for HGF/SF, candidate preparations of the two forms were assessed in a multicentre study in which they were compared with local standards by bioassay and immunoassay. Among laboratories, there was a wide variation in the estimates of potencies of the candidate standards in terms of in-house reference preparations, but between-assay and within-assay variabilities were low within laboratories. In some assay systems, the precursor and heterodimer showed different responses. Since both molecular forms are widely used in current assay systems, this suggested that a reference preparation was required for each form of the HGF/SF molecule. Accordingly, the Expert Committee on Biological Standardization of WHO established the heterodimeric material (96/564) as the first IS for HGF/SF, human, recombinant, with an assigned unitage of 4000 IU/ampoule and, for the purpose of immunoassay calibration, a nominal HGF/SF content of 4 microg/ampoule. The precursor preparation (96/556) was established as the first IS for HGF/SF (precursor) with an assigned unitage of 2000 IU/ampoule and, for the purpose of immunoassay calibration, a nominal HGF/SF (precursor) content of 4 microg/ampoule. The preparations can be obtained upon written request to the National Institute for Biological Standards and Control (NIBSC, PO Box 1193), by e-mail (standards@nibsc.ac.uk) or ordered at http://www.nibsc.ac.uk.

Human meningiomas possess muscarinic acetylcholine receptors: stimulation of phosphatidylinositol turnover by carbachol

The effect of carbachol, an acetylcholine receptor agonist,on rate of phosphatidylinositol (PI) turnover in culturedhuman meningioma cells was investigated. Exposure of meningiomacells for 2 h to carbachol (3.12—200 μmol/L) resultedin a dose-dependent stimulation of PI turnover to a maximum of 5.5-foldover basal controls. A time course study showed stimulation ofIP3formation after 30 s followed by increasesin IPIP1 and IPIP2. The stimulatoryeffect of carbachol on PI turnover was completely abolished bythe muscarinic receptor antagonist, atropine, but was unalteredby the nicotinic antagonist, hexamethonium. Reverse-transcriptionof meningioma-derived RNA into cDNA followed by amplification by the polymerase chain reaction using specific primers revealedpresence of m1 type muscarinic receptor mRNA. These results provideevidence that human meningioma cells possess muscarinic acetylcholine receptors the activation of which leads to PI hydrolysis.

Characterization of a rat anterior pituitary cell bioassay.

SummaryWe have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 × 106 cells · ml−1 · well−1 in serum-free medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10−8M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.