Sorption and mineral-promoted transformation of synthetic hormone growth promoters in soil systems.

This work examines the fate of synthetic growth promoters (trenbolone acetate, melengestrol acetate, and zeranol) in sterilized soil systems, focusing on their sorption to organic matter and propensity for mineral-promoted reactions. In organic-rich soil matrices (e.g., Pahokee Peat), the extent and reversibility of sorption did not generally correlate with compound hydrophobicity (e.g., K(ow) values), suggesting that specific binding interactions (e.g., potentially hydrogen bonding through C17 hydroxyl groups for the trenbolone and melengestrol families) can also contribute to uptake. In soils with lower organic carbon contents (1-5.9% OC), evidence supports sorption occurring in parallel with surface reaction on inorganic mineral phases. Subsequent experiments with pure mineral phases representative of those naturally abundant in soil (e.g., iron, silica, and manganese oxides) suggest that growth promoters are prone to mineral-promoted oxidation, hydrolysis, and/or nucleophilic (e.g., H2O or OH(-)) addition reactions. Although reaction products remain unidentified, this study shows that synthetic growth promoters can undergo abiotic transformation in soil systems, a previously unidentified fate pathway with implications for their persistence and ecosystem effects in the subsurface.

Highly potent growth hormone secretagogues.

During an effort to search for more potent growth hormone secretagogues, we discovered a class of compounds of which the best compound 8 was 7-fold more active in vitro than the best compound in the series we revealed before [Tata, J. R.; Lu, Z.; Jacks, T. M.; Schleim, K. D.; Cheng, K.; Wei, L.; Chan, W.-S.; Butler, B.; Tsou, N.; Leung, K.; Chiu, S.-H. L.; Hickey, G. J.; Smith, R. G.; Patchett, A. A. Bioorg. Med. Chem. Lett.1997, 7, 2319.]. Animal studies show that compound 8 can stimulate growth hormone release at the oral dose as low as 0.06 mpk. Chemistry and biological studies are discussed.

Preparation and characterization of recombinant chicken growth hormone (chGH) and its putative antagonist chGH G119R mutein.

Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized after being optimized for expression in E. coli. The respective cDNAs were inserted into expression vector, expressed and found almost entirely in the insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded, and purified to homogeneity by anion-exchange chromatography on Q-Sepharose. The overall yields were 400 to 500 mg from 5 L of fermentation. Both proteins were > 98% pure, as evidenced by SDS-PAGE, and contained at least 95% monomers, as documented by gel-filtration chromatography under non-denaturing conditions. Circular dichroism analysis revealed that both proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGH receptor extracellular domain, but its affinity, as determined by RRA, was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed using FDC-P1 3B9 cells stably transfected with rabbit GHR, was 30-40-fold lower, whereas chGH G119R mutant did not bind to oGHR-ECD and was devoid of any biological activity in FDC-P1 3B9 cells. However, in binding experiments that were carried out using chicken liver membranes, both oGH and chGH showed similar IC(50) values in competition with (125)I-oGH, while the IC(50) of G119R mutein was 10-fold higher. These results emphasize the importance of species specificity and indicate the possibility of antagonistic activity of chGH G119R.

Thiazole-derived potent, highly bioavailable short duration growth hormone secretagogues.

Replacement of the phenyl in 3 with a 2-pyridyl or 4-thiazolyl group resulted in increased potency in the rat pituitary cell GH release assay and in beagles.

Synthesis and biological activities of spiroheterocyclic growth hormone secretagogues.

The synthesis and biological activities of a series of spiroheterocyclic growth hormone secretagogues are reported. Modification of the spiroindane part-structure of the prototypal secretagogue L-162,752 revealed that the spiroindane could be replaced with spirobenzodihydrothiophen derivatives to enhance not only in vitro potency but also oral activity. In this study non-aromatic D-2-amino-4-cyclohexylbutanoic analogs (8a-8d) were also identified to be active secretagogues.

El uso de la hormona de crecimiento recombinante humana como coadyuvante en el tratamiento de las quemaduras en niños

Pra evaluar el efecto de la hormona de crecimiento humana (hGH) en los niños quemados; el grupo I recibió el manejo convencional más una dosis diaria de hGH a 0.3 Ul/Kg de peso durante 7 días y el grupo II recibió el manejo convencional. Se cálculo el balance nitrogenado y se tomó una muestra sérica al ingreso y el séptimo día para cuantificar el factor de crecimiento similar a la insulina tipo I (IGF-I). 11 pacientes fueron incluidos en el grupo I y 10 en el grupo II. La superficie quemada en el grupo I fue de 23.9ñ6.8 por ciento y en el grupo II fue de 22.0ñ10.8 por ciento (p=0.6). La estadía hospitalaria en el grupo I fue de 18.5ñ7.2 días mientras en el grupo II fue de 27.9ñ16.05 días (p=0.09). El balance nitrogenado en el grupo I fue de 3.9ñ3.6 gramos, mientras en el grupo II fue de 0.4ñ1.4 gramos (p=0.01). Los niveles del IGF-I al ingreso fueron de 44.5ñ38.2 ng/ml en el grupo I y 40.4ñ36.7 en el grupo II (p=0.8); estos niveles aumentaron al séptimo día hasta 146.0ñ120.2 en el grupo I y en el grupo II hasta 47.4ñ41.4 (p=0.02). En cinclusión la hGH en niños quemados mejora el balance nitrogenado, aumenta los niveles de IGF-I y en esta forma puede ser efectiva para disminuir la estadía hospitalaria y las complicaciones

Introduction of spacer peptides N-terminal to a cleavage recognition motif in recombinant fusion proteins can improve site-specific cleavage.

To improve site-specific cleavage of a methionyl porcine growth hormone [[Met1]-pGH(1-46)-IGF-II] fusion protein by the enzyme H64A subtilisin, a series of flexible, unstructured spacer peptides were introduced N-terminal to the cleavage site. When enzymatic digestion preceded refolding of the fusion proteins, IGF-II could only be liberated from substrates which contained spacer peptides. Compared with the parent construct, the yield of IGF-II from refolded fusion proteins containing spacers was improved up to two-fold. Furthermore, this cleavage rate was improved by removing a competing protease recognition motif from the fusion partner. These data show that fusion partners can influence site-specific proteolysis of fusion proteins. Introduction of flexible spacers between the moieties can alleviate these interactions.

Long-acting growth hormones produced by conjugation with polyethylene glycol.

Derivatives of human growth hormone (hGH) of increasing size were produced by reaction with the N-hydroxysuccinimide ester of polyethylene glycol-5000 (PEG5000), a 5-kDa reagent that selectively conjugates to primary amines. By adjusting the reaction conditions and purification procedure, it was possible to isolate hGH derivatives containing up to seven PEG moieties that altered the Stokes radius and thereby the effective molecular masses of the unmodified hormone from 22 to 300 kDa. Fortunately, the most reactive amines were ones that did not lie in either of the two sites important for receptor binding. Nonetheless, increasing the level of PEG modification linearly reduced the affinity of hGH for its receptor and increased the EC50 in a cell-based assay up to 1500-fold. Most of the reduction in affinity was the result of slowing the association rate for the receptor. The clearance rate of hGH in rats was inversely proportional to effective molecular weight and closely fit a filtration model. We have tested the potency of these analogs by injecting them daily or every 6 days into hypophysectomized rats and determining the effects on body and organ growth. The efficacy of these analogs was optimal for hGH conjugated with 5 eq of PEG5000, and the potency was increased by about 10-fold compared with unmodified hGH. Such PEG-hGH derivatives show promise as long-acting alternatives to daily injections of hGH. More generally these studies show that improving hormone clearance properties, even at the expense of reducing receptor binding affinity, can lead to dramatic increases in hormone efficacy.

Immunological effect of a synthetic growth hormone peptide on the growth performance in swine.

A mouse monoclonal antibody (mAb), designated PS-7.6, was previously shown to enhance the activity of porcine growth hormone (pGH) in promoting the growth of hypophysectomized (hypox) rats. Epitope mapping studies indicated that the region recognized by PS-7.6 resided within an amino acid sequence 54-95 of pGH. A peptide corresponding to this sequence was synthesized and found to induce swine antibodies capable of augmenting pGH activity in hypox rats. On the basis of these previous observations, an attempt was made in this study to determine whether or not the peptide pGH(54-95) could be used as a vaccine to elicit antibodies functionally similar to PS-7.6 mAb, thus potentiating the efficacy of endogenous GH in swine. Young pigs (15-20 kg) were immunized with pGH(54-95) that had been conjugated with ovalbumin (OVA) and boosted twice at 4-week intervals. Control animals were similarly immunized with OVA. The weight gains and feed consumption of these animals were closely monitored throughout the trials. A number of carcass parameters were also examined when these animals reached 110-120 kg, at which time they were killed. Results indicated that immunization with peptide significantly accelerated the daily weight gain during the growing phase of growth. However, this effect disappeared during the finishing phase of growth. The failure to prolong the initial growth effect by the peptide immunization apparently correlated with the kinetics of antibody production, because antibodies immunoreactive to the peptide and pGH were detected in these animals after immunization but gradually diminished. This idea was supported by the fact that antibodies obtained from pigs 5 and 9 weeks after the initial immunization potentiated the activity of pGH in hypox rats, whereas antibodies harvested at week 16 did not. Furthermore, carcass evaluation was performed at time of killing and showed that the leaf fat and loin eye muscle were also significantly improved by peptide immunization. Taken together, the present findings suggest that pGH(54-95) peptide can be employed as a potential growth-promoting vaccine to improve the performance of swine.

Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis.

The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D-Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse-phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D-isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.

Reduction of cumulative body weight gain and adipose tissue mass in obese mice: response to chronic treatment with synthetic hGH 177-191 peptide.

The effects of long-term treatment of C57BL/6J (ob/ob) mice with a synthetic carboxylterminal sequence of human growth hormone, hGH 177-191, were investigated. Results indicate that the hGH 177-191 reduced the cumulative body weight gain, and decreased the adipose tissue mass. The lipogenesis in adipose tissues was significantly inhibited by the treatment with hGH 177-191. These findings support the suggestion that hGH 177-191 is the functional domain of hGH for the antilipogenic actions of the intact hormone both in vivo and in vitro. The hGH 177-191 peptide has the potential to be an effective compound for the treatment of human obesity and for the improvement of meat qualities in farm animals.

Conjugation of synthetic peptides to carrier iscoms: factors affecting the immunogenicity of the conjugate.

This study was designed to explore optimal conditions for the conjugation of a synthetic peptide to preformed influenza virus iscoms using MHS (maleimidohexanoyl-N-hydroxy-succinimide ester) as coupling agent. The peptide used in this study comprised amino acids 122-138 of porcine growth hormone (pGH). Different ratios of peptide to carrier iscoms were tested and the resulting conjugates were analysed for composition, antigenicity and immunogenicity. The problem of low solubility and poor immunogenicity of high-density peptide conjugates is discussed and a general protocol for conjugation of peptides to carrier iscoms is proposed.

The effects of synthetic growth hormone on spinal cord injury.

The aim of this study was to determine the effect of synthetic growth hormone on healing process of reversible injured spinal cord. The rat was selected for the experiment in preference to larger animals for economy and availability. Under general anesthesia one level laminectomy was carried out at T-1 with the dura mater intact. The injury was created by Rivlin and Tator's clip method. Recovery of motor function was assessed for up to 4 weeks using inclined plane test of hind limb motor function. Although the effect of the drug is evident after 3 weeks, the study must be held for longer periods for a sufficient regeneration time to maintain a significant statistical evaluation.

Effect of an antilipogenic fragment of human growth hormone on glucose transport in rat adipocytes.

The effect of a synthetic C-terminal peptide sequence of human growth hormone, Leu-Arg-Ile-Val-Gln-Cys-Arg-Val-Ser-Glu-Gly-Ser-Cys-Gly-Phe (hGH 177-191) on glucose transport in adipocytes isolated from genetically-obese Zucker rats was investigated. The results showed that the synthetic peptide induced a reduction of basal and insulin-stimulated D[1-14C]-2-deoxyglucose uptake in isolated adipocytes. In comparison with the intact molecule of human growth hormone (hGH), the synthetic peptide at equimolar concentrations was found to be more potent. These findings are consistent with the suggestion that the functional domain responsible for the antilipogenic activity of hGH resides in the C-terminal region of the hGH molecule and the effect on glucose transport may contribute, at least in part, to the antilipogenic property of the peptide hGH 177-191 as well as of the intact hormone.

Development, production and pharmacodynamics of human growth hormone.

With the advent of recombinant DNA technology, it is possible to produce biosynthetic human growth hormone (B-hGH). Novo Nordisk A/S has developed a method for manufacturing B-hGH which is identical to the 22K fraction of pituitary human growth hormone (P-hGH), using a nonpathogenic strain of Escherichia coli as host. B-hGH has been investigated extensively in physical, chemical and biological studies and found to be identical to P-hGH. Pharmacological studies have revealed that B-hGH possesses the same pharmacokinetic and short-term metabolic profiles as P-hGH. Long term clinical studies have shown that B-hGH induces a significant increase in height velocity in children with growth hormone deficiency (GHD) and is characterized by a low antigenicity.

Synthesis and evaluation of constrained peptide analogues related to the N-terminal region of human growth hormone.

The synthesis and incorporation of two different isomeric gamma-lactam structures into peptide analogues related to hGH [6-13] are described. These peptide analogues and the corresponding aspartimide analogue have been tested for hypoglycemic activity with the intravenous insulin tolerance test. One lactam structure is of the type developed by Freidinger and co-workers, while the isomeric gamma-lactam structure represents a new constrained synthon for use in peptide synthesis. We have found that the hGH [6-13] peptide analogue incorporating the Freidinger lactam was more potent and longer lasting than the aspartimide peptide analogue. The hGH [6-13] peptide analogue incorporating the new gamma-lactam has diminished hypoglycemic activity. The relative biological activities of the three peptide analogues and the possible conformational implications at the physiological site of action are discussed.

Activity of artificial mutant variants of human growth hormone deficient in a disulfide bond between Cys53 and Cys165.

In order to understand the role of Cys53 and Cys165 of human growth hormone (hGH) in receptor-binding and biological activity, artificial mutant variants of hGH were prepared in Escherichia coli by in vitro mutagenesis. Variants of hGH were constructed by replacement of Cys165 with Ala ([Ala165]hGH) or Ser ([Ser165]hGH), by replacement of Cys53 with Ala ([Ala53]hGH), by replacement of Cys53 and Cys165 with Ala ([Ala53, Ala165]hGH), or by replacement of Cys53 with Ala and Cys165 with Ser ([Ala53,Ser165]hGH). All of the variants constructed as well as reduced hGH exhibited less biological activity than that of intact hGH, and the decreases in biological activity were almost equal, as measured by a sensitive biological assay for growth hormone: adipose conversion assay using 3T3-F442A cells. These variants also showed less receptor-binding activity than that of intact hGH. These results suggest that it is possible neither the residue Cys53 nor Cys165 is directly involved in the receptor binding, and that the disulfide bridge between Cys53 and Cys165 in hGH may not always be crucial for the biological activity, though necessary to express full hGH activity.

Solution conformation of a synthetic fragment of human pituitary growth hormone. Two-dimensional NMR of an alpha-helical dimer.

Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.