The clinical usefulness of liquid human growth hormone (hGH) (Norditropin SimpleXx in the treatment of GH deficiency

Human growth hormone (hGH) is an essential therapeutic drug for the treatment of GH deficiency. The development of recombinant GH using a pen injection system has enabled easy and safe treatment of GH-deficient patients; however, the process of dissolving hGH in the powder form is complicated and dangerous. In this study, we investigated the usefulness of a newly developed liquid form of hGH (Norditropin((R)) SimpleXx(TM)) in the treatment of 51 patients with GH deficiency. Fifteen previously untreated patients with GH deficiency were treated with liquid hGH (group A), and 36 patients who had previously used hGH in the powder form were changed to the liquid form (group B). Both groups were treated with liquid hGH 0.5 IU/kg per week for 6 months. The growth rate of patients in group A increased from 4.0 +/- 2.4 cm/year to 9.2 +/- 2.9 cm/year. The patients in group B continued to grow at the same rate as before using the liquid hGH therapy. Questionnaires to the patients in group B demonstrated that 85% preferred the convenience of using the new liquid form of hGH. Our results indicate that liquid hGH has similar efficacy to that of powder hGH, but its improved convenience may have a beneficial effect on patient compliance.     

Structure of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor: evidence for strong structural coupling between receptor binding sites

The structure of the ternary complex between the phage display- optimized, high-affinity Site 1 variant of human growth hormone (hGH) and two copies of the extracellular domain (ECD) of the hGH receptor (hGHR) has been determined at 2.6 A resolution. There are widespread and significant structural differences compared to the wild-type ternary hGH hGHR complex. The hGH variant (hGH(v)) contains 15 Site 1 mutations and binds>10(2) tighter to the hGHR ECD (hGH(R1)) at Site 1. It is biologically active and specific to hGHR. The hGH(v) Site 1 interface is somewhat smaller and 20% more hydrophobic compared to the wild-type (wt) counterpart. Of the ten hormone-receptor H-bonds in the site, only one is the same as in the wt complex. Additionally, several regions of hGH(v) structure move up to 9A in forming the interface. The contacts between the C-terminal domains of two receptor ECDs (hGH(R1)- hGH(R2)) are conserved; however, the large changes in Site 1 appear to cause global changes in the domains of hGH(R1) that affect the hGH(v)-hGH(R2) interface indirectly. This coupling is manifested by large changes in the conformation of groups participating in the Site 2 interaction and results in a structure for the site that is reorganized extensively. The hGH(v)- hGH(R2) interface contains seven H-bonds, only one of which is found in the wt complex. Several groups on hGH(v) and hGH(R2) undergo conformational changes of up to 8 A. Asp116 of hGH(v) plays a central role in the reorganization of Site 2 by forming two new H-bonds to the side-chains of Trp104(R2) and Trp169(R2), which are the key binding determinants of the receptor. The fact that a different binding solution is possible for Site 2, where there were no mutations or binding selection pressures, indicates that the structural elements found in these molecules possess an inherent functional plasticity that enables them to bind to a wide variety of binding surfaces.     

Differential effects of hGH and IGF-I on body proportions

The differential growth effects of hGH and IGF-I on the upper/lower (U/L) body segment in relation to height (Ht) were analyzed in 15 patients with isolated Growth hormone deficiency (IGHD,:7M, 8F) mean age 5.0 +/- 3.2 (SD) years treated with hGH; 21 patients with multiple pituitary hormone deficiency including growth hormone (MPHD: 14M, 7F) aged 10.0 +/- 3.8, treated with hGH; 9 patients with Laron Syndrome (LS) (4M,5F) aged 6.9 +/- 5.6 years treated with IGF-I; 9 boys with intrauterine growth retardation (IUGR) aged 6.3 +/- 1.25 years treated by hGH; and 22 boys with idiopathic short stature (ISS) aged 8.0 +/- 1.55 years treated by hGH. The dose of hGH was 33 microg/kg/day, that of IGF-I 180-200 microg/kg/day. RESULTS: the U/L body segment ratio in IGHD patients decreased from 2.3 +/- 0.7 to 1.1 +/- 0.7 (p <0.001), and the Ht SDS increased from -4.9 +/- 1.3 to 2.3 +/- 1 (p < 0.001) following treatment. In MPHD patients the U/L body segment decreased from 1.1 +/- 1.1 to -0.6 +/- 1.0 (p < 0.001), and the Ht SDS increased from -3.3 +/- 1.4 to -2.5 +/- 1.0 (p < 0.009). In the LS group the U/L body segment ratio did not change with IGF-I treatment but Ht improved from -6.1 +/- 1.3 to -4.6 +/- 1.2 (p < 0.001), The differential growth response of the children with IUGR and with ISS resembled that of the children with LS. CONCLUSIONS: hGH and IGF-I act differentially on the spine and limbs.     

Il-8((3-73))K11R is a high affinity agonist of the neutrophil CXCR1 and CXCR2

In studies aimed at developing a high affinity IL-8 antagonist, our first objective was to generate a high-affinity IL-8 analogue. We targeted amino acids within the receptor-binding domain and found that IL-8((3-73))K11R induced significantly more neutrophil beta-glucuronidase release than either IL-8 or the alternate analogues and, in chemotaxis assays, induced 2-3-fold greater neutrophil responses than IL-8. Furthermore, in competitive radio- or biotinylated-ligand binding assays, IL-8((3-73))K11R was more effective than IL-8, IL-8((3-73)), or its T12S, H13F, and K11R/T12S/H13F analogues in blocking IL-8 binding to neutrophils; 1.8 pmol IL-8((3-73))K11R inhibited by 50% the binding of approximately 20 pmol (125)I-IL-8 to neutrophils. Both IL-8 (a CXCR1/CXCR2 ligand) and the CXCR2-specific ligand GROalpha differentially inhibited binding of (125)I-IL-8((3-73))K11R to neutrophils, albeit weakly, suggesting that IL-8((3-73))K11R is a high affinity ligand for both the CXCR1 and CXCR2. Thus IL-8((3-73))K11R is an excellent candidate for further studies aimed at generating a high affinity IL-8 antagonist.     

Detection of human growth hormone doping in urine: out of competition tests are necessary

The misuse of human growth hormone (hGH) in sport is deemed to be unethical and dangerous because of various adverse effects. Thus, it has been added to the International Olympic Committee list of banned substances. Until now, the very low concentration of hGH in the urine made its measurement difficult using classical methodology. Indeed, for routine diagnosis, only plasma measurements were available. However, unlike blood samples, urine is generally provided in abundant quantities and is, at present, the only body fluid allowed to be analysed in sport doping controls. A recently developed enzyme-linked immunosorbent assay (Norditest) makes it now possible, without any extraction, to measure urinary hGH (u-hGH) in a dynamic range of 2–50 ng hGH/1. In our protocol, untreated and treated non-athlete volunteers were followed. Some of them received therapeutical doses of recombinant hGH (Norditropin) for one week either intramuscularly (three increasing doses) or subcutaneously (12 I.U. every day). The u-hGH excretion after treatment showed dramatic increases of 50–100 times the basal values and returned to almost the mean normal level after 24 h. u-hGH was also measured in samples provided by the anti-doping controls at major and minor competitions. Depending on the type of efforts made during the competition, the hGH concentration in urine was dramatically increased. Insulin-like growth factor binding proteins and β2-microglobulins in urine and/or in blood could be necessary for the correct investigation of any hGH doping test procedure.     

Liquid growth hormone: preservatives and buffers

Growth hormone (GH) treatment is a successful medical therapy for children and adults with GH deficiency as well as for growth retardation due to chronic renal disease, Turner syndrome and in children born small for gestational age. For all of these conditions, treatment is long term and patients receive daily subcutaneous injections of GH for many years. Patient compliance is therefore of critical importance to ensure treatment benefit. One of the major factors influencing compliance is injection pain. Besides the injection device used, pain perception and local tissue reaction following injection are dependent on the preservative used in the formulation and the concentration of GH. Injection pain may also be related to the buffer substance and injection volume. A liquid formulation of GH, Norditropi SimpleXx, has been developed that dispenses with the need for reconstitution before administration. The formulation uses phenol (3 mg/ml) as a preservative (to protect product from microbial degradation or contamination) and histidine as a buffer. Alternative preservatives used in other GH formulations include m-cresol (9 mg/ml) and benzyl alcohol (3-9 mg/ml). Buffering agents include citrate and phosphate. Phenol has been successfully used as a preservative in drug formulations for more than 50 years and is considered a safe and effective agent which complies with strict international requirements for preservatives in drug formulations. In toxicological studies, no or only mild local reactions have been observed following subcutaneous administration of phenol (7.5 mg/ml), m-cresol (3-4 mg/ml) and benzyl alcohol (9 mg/ml). No general toxicity reactions were observed after subcutaneous administration of these agents. Clinical evaluation of the preservatives and buffers used in Norditropin SimpleXx showed that pain perception was similar between formulations containing phenol and benzyl alcohol, whereas m-cresol was associated with more painful injections than benzyl alcohol. Furthermore, patients reported more pain following injection of a citrate-buffered solution than after a histidine-buffered solution. More pain was also reported following large volume injections and following injections with solutions containing high protein concentrations. In summary, optimization of the preservative and buffer content of a liquid GH formulation may reduce injection pain and lead to improved patient compliance.     

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.     

Purification of a pyrogen-free human growth hormone antagonist

Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kilodaltons. With the aid of computer molecular simulation, an hGH analog was created by altering an hGH gene to reflect the change of one amino acid (glycine [G] 120 to arginine [R]) within the third alpha-helix of the hGH molecule. This hGH analog, named hGHG120R, was found to be an hGH antagonist. It may have important implications in treating human conditions in which hGH levels are abnormally high, as found in type I diabetics. Several hundred milligrams of purified hGHG120R were needed to determine the biological activity of the antagonist in animal models. A multistep downstream process was developed to purify hGHG120R from cultured mouse L cells transfected with the hGHG120R gene. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, reversed phase high performance liquid chromatography, phase separation, and lyophiliation. This work discusses the rationale for the design of the process and experimental results on the purification of hGHG120R using the process. (c) 1995 John Wiley & Sons, Inc.     

Growth hormone regulation of growth hormone-releasing hormone gene expression

Slot-blot hybridization technique was used to evaluate growth hormone-releasing hormone (GHRH) mRNA levels in the hypothalamus of long-term (14 days) hypophysectomized (HPX) rats treated or not with 125 micrograms hGH/rat, twice daily IP, since the first day postsurgery. In addition, mRNA levels were determined in the hypothalamus of short-term (4 days) GH-treated (250 micrograms hGH/rat, twice daily IP) intact rats. GHRH mRNA levels were increased in HPX rats, and GH treatment partially counteracted this rise. Short-term administration of GH decreased GHRH mRNA levels in intact rats. These results, evaluated together with previous findings showing decreased hypothalamic GHRH-like immunoreactivity in both HPX rats and intact rats given GH (6, 7, 9), indicate that GH exerts a negative feedback action on the synthesis and release of GHRH.     

CXCL8((3-73))K11R/G31P antagonizes ligand binding to the neutrophil CXCR1 and CXCR2 receptors and cellular responses to CXCL8/IL-8

We recently reported that CXCL8((3-73))K11R is a high affinity agonist of neutrophil activation and chemotactic responses. In this report we employed CXCL8((3-73))K11R as a template to generate CXCL8/IL-8 analogues with antagonist activities, using site-directed mutagenesis to introduce conservative amino acid substitutions into the first turn within the molecule's beta-pleated sheet region (G31P, P32G) and, in association with these, into the putative receptor-recognition site (T12S, H13F, F17S). We then examined their impact on the analogues' biological activities and found that a G31P substitution rendered CXCL8((3-73))K11R a high affinity antagonist of CXCL8/IL-8. The ranking (in the order of decreasing CXCL8/IL-8 antagonist activities) of the CXCL8((3-73))K11R analogues we generated was, G31P>T12S/G31P>H13F/G31P>T12S/H13F/G31P>P32G approximately T12S/P32G approximately H13F/P32G>T12S/H13F/P32G; CXCL8((3-73))K11R/F17S did not inhibit CXCL8/IL-8-dependent responses. CXCL8((3-73))K11R/G31P had no discernible agonist (beta-glucuronidase release, chemotactic) activity, but at 12.5 ng/ml it bound to purified neutrophils more avidly than did 1.25 microg/ml CXCL8/IL-8. Furthermore, CXCL8((3-73))K11R/G31P competitively antagonized the binding of CXCR1- and CXCR2-specific antibodies to these receptors. Taken together, these data thus provide further impetus to the study of the potential efficacy of CXCL8((3-73))K11R/G31P as a broad-spectrum antagonist of the ELR-CXC chemokines in experimental and clinical settings.     

Stability of immobilized soybean lipoxygenases: influence of coupling conditions on the ionization state of the active site Fe

The potential application of lipoxygenase as a versatile biocatalyst in enzyme technology is limited by its poor stability. Two types of soybean lipoxygenases, lipoxygenase-1 and -2 (LOX-1 and LOX-2) were purified by a two step anion exchange chromatography. Four different commercially available supports: CNBr Sepharose 4B, Fractogel((R)) EMD Azlactone, Fractogel((R)) EMD Epoxy, and Eupergit((R)) C were tested for immobilization and stabilization of the purified isoenzymes. Both isoenzymes gave good yields in enzyme activity and good stability after immobilization on CNBr Sepharose 4B and Fractogel((R)) EMD Azlactone. Rapid decay in activity associated with change in the ionization state of Fe, as shown by EPR measurements was observed within the first 5 days after immobilization on epoxy activated supports (Eupergit((R)) C and Fractogel((R)) EMD Epoxy) in high ionic strength buffers. Stabilization of the biocatalyst on these supports was achieved by careful adjustment of the immobilization conditions. When immobilized in phosphate buffer of pH 7.5 and low ionic strength (0.05 M), the half-life time of the immobilized enzyme increased 20 fold. The dependence of the stability of LOX immobilized on epoxy activated supports on the coupling conditions was attributed to a modulation of the ligand environment of the iron in the active site and consequently its reactivity.     

The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alanine-scanning mutagenesis: insights into the mechanisms responsible for improved affinity

A high-affinity variant of human growth hormone (hGH(v)) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) approximately 400-fold tighter than does wild-type (wt) hGH (hGH(wt)). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions of individual residues within the hGH(v) binding epitope and placed them in context with previously determined structural information. In all, the effects of alanine substitutions were determined for 35 hGH(v) residues that are directly contained in or closely border the binding interface. We found that the distribution of binding energy in the functional epitope of hGH(v) differs significantly from that of hGH(wt). The residues that contributed the majority of the binding energy in the wt interaction (the so-called binding "hot spot") remain important, but their contributions are attenuated in the hGH(v) interaction, and additional binding energy is acquired from residues on the periphery of the original hotspot. Many interactions that inhibited the binding of hGH(wt) are replaced by interactions that make positive contributions to the binding of hGH(v). These changes produce an expanded and diffused hot spot in which improved affinity results from numerous small contributions distributed broadly over the interface. The mutagenesis results are consistent with previous structural studies, which revealed widespread structural differences between the wt and variant hormone-receptor interfaces. Thus, it appears that the improved binding affinity of hGH(v) site 1 was not achieved through minor adjustments to the wt interface, but rather, results from a wholesale reconfiguration of many of the original binding elements.     

Plasma renin activity and aldosterone response to furosemide after chronic growth hormone administration to deficient subjects

The plasma renin activity (PRA) and aldosterone responses to furosemide-induced diuresis were measured in seven children with growth hormone deficiency prior to, during and after the admistration of clinical grade human growth hormone (hGH). The furosemide-stimulated increases in PRA were unchanged from baseline values after one and eight months of hGH administration, but the plasma aldosterone concentrations were significantly increased over control values after eight months of hGH administration.Plasma cortisol concentrations as determined by competitive protein binding assay were measured with the 30-minute cosyntropin (Cortrosyn) test. A normal response as defined by a final level of at least 20 ug/100 ml was found in all children. Resting cortisol concentrations were unchanged during treatment with hGH but the mean increment and final levels were significantly diminished after 8 months of hGH administration. These data have not elucidated the mechanism by which clinical grade hGH improves diuretic-induced aldosterone responsiveness but diminishes cosyntropin stimulation of the adrenal in childhood.     

Melanocortin-4 receptor gene mutations in a Dutch cohort of obese children

The most common monogenic form of obesity is caused by mutations in the gene encoding the melanocortin-4 receptor (MC4R). We have screened the MC4R coding sequence in 291 patients of a Dutch outpatient pediatric obesity clinic. We analyzed the minimal promoter region of the gene in a random subgroup of 217 children. Our aims were (i) to determine the frequency of MC4R mutations in a cohort of Dutch clinically obese children and (ii) to search for mutations in the promoter of the gene. Eleven MC4R coding variants were detected. Five children had mutations that have been shown to affect receptor function by other research groups (p.Y35X, p.I251fs, p.G231S). These children did not have earlier onset of obesity or higher BMI-SDS than the remainder of the cohort. One child had a novel nonsynonymous coding mutation (p.L304F). This variant showed a markedly decreased cell surface expression in in vitro experiments and is thus expected to be pathogenic. We detected 12 variants in the MC4R flanking regions. Five of these were not previously described (c.-1101C>T, c.-705A>T, c.-461A>G, c.-312T>C, c.-213A>G). We investigated these mutations by family studies and a bioinformatic approach. We conclude that rare heterozygous mutations in the coding sequence of MC4R account for some severe obesity cases in the Dutch population. These patients are difficult to recognize in a clinical setting. We generated a list of all MC4R variants that were described in the literature so far, which can aid the interpretation of mutations found in a diagnostic setting.     

Epigenetic control of a VDR-governed feed-forward loop that regulates p21(waf1/cip1) expression and function in non-malignant prostate cells

In non-malignant RWPE-1 prostate epithelial cells signaling by the nuclear receptor Vitamin D Receptor (VDR, NR1I1) induces cell cycle arrest through targets including CDKN1A (encodes p21((waf1/cip1))). VDR dynamically induced individual histone modification patterns at three VDR binding sites (R1, 2, 3) on the CDKN1A promoter. The magnitude of these modifications was specific to each phase of the cell cycle. For example, H3K9ac enrichment occurred rapidly only at R2, whereas parallel accumulation of H3K27me3 occurred at R1; these events were significantly enriched in G(1) and S phase cells, respectively. The epigenetic events appeared to allow VDR actions to combine with p53 to enhance p21((waf1/cip1)) activation further. In parallel, VDR binding to the MCM7 gene induced H3K9ac enrichment associated with rapid mRNA up-regulation to generate miR-106b and consequently regulate p21((waf1/cip1)) expression. We conclude that VDR binding site- and promoter-specific patterns of histone modifications combine with miRNA co-regulation to form a VDR-regulated feed-forward loop to control p21((waf1/cip1)) expression and cell cycle arrest. Dissection of this feed-forward loop in a non-malignant prostate cell system illuminates mechanisms of sensitivity and therefore possible resistance in prostate and other VDR responsive cancers.     

Trend in Height of Turkish and Moroccan Children Living in The Netherlands

ObjectivesTo study trends in height of Turkish and Moroccan immigrant children living in The Netherlands, to investigate the association between height and background characteristics in these children, and to calculate height-for-age-references data for these groups.DesignNationwide cross-sectional data collection from children aged 0 to 18 years by trained professionals in 1997 and 2009. The study population consisted of 2,822 Turkish 2,779 Moroccan, and 13,705 Dutch origin children in 1997and 2,548 Turkish, 2,594 Moroccan, and 11,255 Dutch origin children in 2009. Main outcome measures: Mean height in cm, and mean height standard deviation scores.ResultsIn 2009, mean height at the age of 18y was similar for Turkish and Moroccan children: 177 cm for boys and 163 cm for girls, which was 2 to 3 cm taller than in 1997. Still, Turkish and Moroccan adolescents were 5.5 cm (boys) to 7 cm (girls) shorter than their Dutch peers. No significant differences were found in mean height standard deviation scores across the educational level of the parents, geographical region, primary language spoken at home, and immigrant generation.ConclusionsWhile the secular height increase in Dutch children came to a halt, the trend in Turkish and Moroccan children living in The Netherlands continued. However, large differences in height between Turkish and Moroccan children and Dutch children remain. We found no association with the background characteristics. We recommend the use of the new growth charts for children of Turkish and Moroccan origin who have a height-for-age below -2SD on the growth chart for Dutch children.     

Use of a GnRH agonist to prevent the endogenous LH surge and injection of exogenous LH to induce ovulation in heifers superstimulated with FSH: a new model for superovulation

A new protocol for superovulating cattle which allows for control of the timing of ovulation after superstimulation with FSH was developed. The preovulatory LH surge was blocked with the GnRH agonist deslorelin, and ovulation was induced by injection of LH. In Experiment 1, heifers (3-yr-old) were assigned to a control group (Group 1A, n = 4) or a group with deslorelin implants (Group 1B, n = 5). On Day -7, heifers in Group 1A received a progestagen CIDR-B((R))device, while heifers in Group 1B received a CIDR-B((R))device + deslorelin implants. Both groups were superstimulated with twice daily injections of FSH (Folltropin((R))-V): Day 0, 40 mg (80 mg total dose on Day 0); Day 1, 30 mg; Day 2, 20 mg; Day 3, 10 mg. On Day 2, heifers were given PGF (a.m.) and CIDR-B((R)) devices were removed (p.m.). Three heifers in Group 1A had a LH surge and ovulated, whereas neither of these events occurred in Group 1B (with deslorelin implants) heifers. In Experiment 2, heifers (3-yr-old) were assigned to 1 of 4 equal groups (n = 6). On Day -7, heifers in Group 2A received a norgestomet implant, while heifers in Groups 2B, 2C and 2D received norgestomet + deslorelin implants. Heifers were superstimulated with FSH starting on Day 0 as in Experiment 1. On Day 2, heifers were given PGF (a.m.) and norgestomet implants were removed (p.m.). Heifers in Groups 2B to 2D were given 25 mg LH (Lutropin((R))): Group 2B, Day 4 (a.m.); Group 2C, Day 4 (p.m.); Group 2D, Day 5 (a.m.). Heifers in Group 2A were inseminated at estrus and 12 and 24 h later, while heifers in Groups 2B to 2D were inseminated at the time of respective LH injection and 12 and 24 h later. Injection of LH induced ovulation in heifers in Groups 2B to 2D. Heifers in Group 2C had similar total ova and embryos (15.2 +/- 1.4) as heifers in Group 2A (11.0 +/- 2.8) but greater (P < 0.05) numbers than heifers in Group 2B (7.0 +/- 2.3) and Group 2D (6.3 +/- 2.0). The number of transferable embryos was similar for heifers in Group 2A (5.8 +/- 1.8) and Group 2C (7.3 +/- 2.1) but lower (P < 0.05) for heifers in Group 2B (1.2 +/- 0.8) and Group 2D (1.3 +/- 1.0). The new GnRH agonist-LH protocol does not require observation of estrus, and induces ovulation in superstimulated heifers that would not have an endogenous LH surge.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.     

Absorption, fluorescence, and cd spectroscopic study of chiral recognition by a binaphthyl-derived chromogenic calixcrown host

The (R)- and (S)-enantiomers of a binaphthyl-appended calix[4]crown-6 ether with two 2,4-dinitrophenylazo chromophore units ((R)-1 and (S)-1) as chiral hosts were tested in their reactions with the enantiomers of alpha-methylbenzylamine ((R)-MBA, (S)-MBA)) and phenylglycinol ((R)-PGL, (S)-PGL) as chiral guests. The visible absorption spectra indicate a two-step process: the first is a nonenantioselective proton transfer from the host to the guest, which is followed by the enantioselective real complexation. In the visible range of the CD spectra a positive/negative band belongs to the absorption of pure (R)-1/(S)-1, and a negative/positive exciton couplet to the absorption of (R)-1-(S)-MBA/(S)-1-(R)-MBA complexes. The latter phenomenon suggests that the complexation of amines is accompanied by a chiral arrangement of the two chromophore units in the hosts. The UV fluorescence of (R)-1/(S)-1 arising from the binaphthyl moiety is quenched by K+ ions, but not by the amine guests, showing that the interaction between the binaphthyl group and the complexed amines is weak.     

Stereoselective pharmacokinetics of RS-8359, a selective and reversible MAO-A inhibitor, by species-dependent drug-metabolizing enzymes

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine selectively and reversibly inhibits monoamine oxidase A (MAO-A). After oral administration of rac-RS-8359 to rats, mice, dogs, monkeys, and humans, plasma concentrations of the (R)-enantiomer were greatly higher than were those of the (S)-enantiomer in all species studied. The AUC((R)) to AUC((S)) ratios were 2.6 in rats, 3.8 in mice, 31 in dogs, and 238 in monkeys, and the (S)-enantiomer was almost negligible in human plasma. After intravenous administration of RS-8359 enantiomers to rats, the pharmacokinetic parameters showed that the (S)-enantiomer had a 2.7-fold greater total clearance (CL(t)) and a 70% shorter half-life (t(1/2)) than those for the (R)-enantiomer but had no difference in distribution volume (V(d)). No significant difference in the intestinal absorption rate was observed. The principal metabolites were the 2-keto form, possibly produced by aldehyde oxidase, the cis-diol form, and the 2-keto-cis-diol form produced by cytochrome P450 in rats, the cis-diol form in mice, RS-8359 glucuronide in dogs, and the 2-keto form in monkeys and humans. Thus, the rapid disappearance of the (S)-enantiomer from the plasma was thought to be due to the rapid metabolism of the (S)-enantiomer by different drug-metabolizing enzymes, depending on species.