Solid state chemistry of proteins: I. glass transition behavior in freeze dried disaccharide formulations of human growth hormone (hGH)

Although freeze dried formulations are commonly characterized using differential scanning calorimetry (DSC), a protein-rich system behaves as a "strong glass", and the glass transition temperature, T(g), cannot be directly determined by DSC. A strong glass means a small heat capacity change at T(g), triangle upC(p), and a very broad glass transition region, or a large triangle upT(g). However, direct experimental evidence for a small triangle upC(p) and a large triangle upT(g) have been lacking. Here, we utilize extrapolation of thermal analysis data in protein:disaccharide mixtures to evaluate T(g), triangle upT(g), and triangle upC(p) for "pure" human growth hormone (hGH) from low to moderate residual water. We find that triangle upT(g) is indeed large and triangle upC(p) is very small. Also, the T(g) for pure hGH decreases from a value of about 136 degrees C when dry to around 25 degrees C at 12% water. This glass transition is not the onset of mobility within the protein molecule but rather signals onset of whole molecule rotation and translation. We also observe complex pre-T(g) thermal events in the DSC data, which are interpreted as consequences of relaxation events, largely due to the disaccharide, and are characteristic of freeze dried systems having a broad distribution of relaxing substates.     

Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid

This research presents storage stability of human growth hormone, hGH, in lyophilized di-saccharide formulations. Stability via HPLC assay was assessed at 40 and 50 degrees C. Structure of the protein in the solids was assessed by infrared spectroscopy. Molecular mobility was characterized by structural relaxation times estimated from DSC data and by measurement of atomic motion on a nanosecond time scale by neutron scattering. Very large stability differences were observed among the various formulations, with both chemical and aggregation stability showing the same qualitative trends with formulation. Near the T(g), T(g) appeared to be a relevant stability parameter, but for storage well below T(g), stability seems unrelated to T(g). Stability (chemical and aggregation) was weakly correlated with secondary structure of the protein, and there was a partial quantitative correlation between degradation rate and the structural relaxation time. However, at equivalent levels of disaccharide relative to protein, sucrose systems were about a factor of two more stable than trehalose formulations, but yet had greater mobility as measured by structural relaxation time. Secondary structure was equivalent in both formulations. Neutron scattering results documented greater suppression of fast dynamics by sucrose than by trehalose, suggesting that well below T(g), fast dynamics are important to stability.     

Solid state characterization of progesterone in a freeze dried 1:2 progesterone/HPBCD mixture

Progesterone rapidly dissolves in an aqueous solution containing hydroxypropyl-beta-cyclodextrin (HPBCD) at 1:2 molar ratio, forming a soluble inclusion complex. After filtration and freeze-drying of the final solution, the final powder was examined by SEM, DSC, TGA, XRD, Raman, and FTIR spectroscopy. Experimental results confirm that an inclusion complex exists in the solid state and possible structure of the complex is briefly discussed.     

Structure characterization of human serum proteins in solution and dry state

Abstract: The present report describes application of advanced analytical methods to establish correlation between changes in human serum proteins of patients with coronary atherosclerosis (protein metabolism) before and after moderate beer consumption. Intrinsic fluorescence, circular dichroism (CD), differential scanning calorimetry and hydrophobicity (S_o) were used to study human serum proteins. Globulin and albumin from human serum (HSG and HSA, respectively) were denatured with 8 m urea as the maximal concentration. The results obtained provided evidence of differences in their secondary and tertiary structures. The thermal denaturation of HSA and HSG expressed in temperature of denaturation (T_d, °C), enthalpy (ΔH, kcal/mol) and entropy (ΔS kcal/mol K) showed qualitative changes in these protein fractions, which were characterized and compared with fluorescence and CD. Number of hydrogen bonds (n) ruptured during this process was calculated from these thermodynamic parameters and then used for determination of the degree of denaturation (%D). Unfolding of HSA and HSG fractions is a result of promoted interactions between exposed functional groups, which involve conformational changes of α‐helix, β‐sheet and aperiodic structure. Here evidence is provided that the loosening of the human serum protein structure takes place primarily in various concentrations of urea before and after beer consumption (BC). Differences in the fluorescence behavior of the proteins are attributed to disruption of the structure of proteins by denaturants as well as by the change in their compactability as a result of ethanol consumption. In summary, thermal denaturation parameters, fluorescence, S_o and the content of secondary structure have shown that HSG is more stable fraction than HSA.     

The impact of drying method and formulation on the physical properties and stability of methionyl human growth hormone in the amorphous solid state

The objective of this work was to investigate the impact of drying method and formulation on the physical stability (aggregation) and selected important physical properties of dried methionyl human growth hormone (Met-hGH) formulations. Solutions of Met-hGH with different stabilizers were dried by different methods (freeze drying, spray drying, and film drying), with and without surfactant. Properties of the dried powders included powder morphology, specific surface area (SSA), protein surface coverage, thermal analysis, and protein secondary structure. Storage stability of Met-hGH in different formulations was also studied at 50 degrees C and at 60 degrees C for 3 months. The dried powders displayed different morphologies, depending mainly on the method of drying and on the presence or absence of surfactant. Film dried powders had the lowest SSA (approximately 0.03 m(2)/g) and the lowest total protein surface accumulation (approximately 0.003%). Surfactant caused a reduction in the SSA of both spray dried and freeze dried powders. Spray dried powders showed greater protein surface coverage and SSA relative to the same formulations dried by other means. Greater in-process perturbations of protein secondary structure were observed with polymer excipients. Formulation impacted physical stability. In general, low molecular weight stabilizers provided better stability. For example, the aggregation rate at 50 degrees C of Met-hGH in a freeze dried trehalose-based formulation was approximately four times smaller than the corresponding Ficoll-70-based formulation. Drying method also influenced physical stability. In general, the film dried preparations studied showed superior stability to preparations dried by other methods, especially those formulations employing low molecular weight stabilizers.     

Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor

Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.     

PEGylation of somatropin (recombinant human growth hormone): impact on its clearance in humans

1. PHA-794428 is a PEGylated version of somatropin (human growth hormone). The pharmacokinetics of PHA-794428 have been studied in humans following single subcutaneous administration (dose range 10-500 microg kg(-1)). In the same study the pharmacokinetics of somatropin were also determined following a 3.6 mg (51 microg kg(-1)) subcutaneous dose. Comparison of the pharmacokinetics of both molecules indicates that PEGylation of somatropin with a 40 kD PEG results in a ten- to 20-fold increase in area under the curve and a similar increase in half-life when compared with somatropin in human (at equivalent subcutaneous doses). 2. Literature data indicate that somotropin is cleared by two mechanisms. The first processes is clearance by glomerular filtration. This is a passive, non-capacity-limited process. A second, capacity-limited, process is mediated by interaction with growth hormone receptors present in a number of tissues including the liver. It is hypothesized that PHA-794428 shares the same clearance mechanisms. However, the addition of the PEG moiety has modulated the clearance by both of these processes. Pharmacokinetic modelling of human serum concentration data obtained for these molecules strongly supports this hypothesis. The renal clearance is reduced due to the increased size of the molecule (Cl/F reduced from 9.6 to 0.1 l h(-1) for somatropin and PHA-794428, respectively). In addition, the reduction in growth hormone receptor affinity has reduced the clearance mediated by interaction with this receptor (somatropin Km = 3.6 microg l(-1) and Vmax = 104 microg h(-1)/PHA-794428 Km = 53 microg l(-1) and Vmax = 84 microg h(-1)).     

PEGylation of somatropin (recombinant human growth hormone): Impact on its clearance in humans

1. PHA-794428 is a PEGylated version of somatropin (human growth hormone). The pharmacokinetics of PHA-794428 have been studied in humans following single subcutaneous administration (dose range 10–500 µg kg^?1). In the same study the pharmacokinetics of somatropin were also determined following a 3.6 mg (51 µg kg^?1) subcutaneous dose. Comparison of the pharmacokinetics of both molecules indicates that PEGylation of somatropin with a 40 kD PEG results in a ten- to 20-fold increase in area under the curve and a similar increase in half-life when compared with somatropin in human (at equivalent subcutaneous doses).

2. Literature data indicate that somotropin is cleared by two mechanisms. The first processes is clearance by glomerular filtration. This is a passive, non-capacity-limited process. A second, capacity-limited, process is mediated by interaction with growth hormone receptors present in a number of tissues including the liver. It is hypothesized that PHA-794428 shares the same clearance mechanisms. However, the addition of the PEG moiety has modulated the clearance by both of these processes. Pharmacokinetic modelling of human serum concentration data obtained for these molecules strongly supports this hypothesis. The renal clearance is reduced due to the increased size of the molecule (Cl/F reduced from 9.6 to 0.1 l h^?1 for somatropin and PHA-794428, respectively). In addition, the reduction in growth hormone receptor affinity has reduced the clearance mediated by interaction with this receptor (somatropin K_m = 3.6 µg l^?1 and V_max = 104 µg h^?1/PHA-794428 K_m = 53 µg l^?1 and V_max = 84 µg h^?1).     

Isolation and characterization of growth hormone from a marine fish,bonito (Katsuwonus pelamis)

Growth hormone (GH) was extracted under alkaline conditions (pH 10) from pituitary glands (6.3 g) of bonito (Katsuwonus pelamis), and subsequently purified by gel filtration, ion exchange chromatography, and reversed-phase HPLC. The GH was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with yellowtail GH antiserum at each step of purification. GH activity was determined by an in vivo bioassay. The yield of this hormone was 4.8mg/g wet tissue. Intraperitoneal injection of bonito GH at doses of 0.1 and 1 μg/g body wt at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. Bonito GH antiserum exhibited both species and hormone specificity in radioimmunoassay. However, the bonito GH antiserum as well as yellowtail GH antiserum exhibited hormone specificity but not species specificity in immunoblotting. A molecular weight of 21 000 and an isoelectric point of 7.0 for bonito GH were estimated by SDS-PAGE and gel electrofocusing, respectively. The complete amino acid sequence of 185 residues was determined by sequencing fragment peptides prepared by chemical and enzymatic cleavages. Sequence comparison of bonito GH with other GHs revealed that there is a significant deletion in the middle of the molecule.     

A novel long-acting human growth hormone fusion protein (VRS-317): enhanced in vivo potency and half-life

A novel recombinant human growth hormone (rhGH) fusion protein (VRS-317) was designed to minimize receptor-mediated clearance through a reduction in receptor binding without mutations to rhGH by genetically fusing with XTEN amino acid sequences to the N-terminus and the C-terminus of the native hGH sequence. Although in vitro potency of VRS-317 was reduced approximately 12-fold compared with rhGH, in vivo potency was increased because of the greatly prolonged exposure to the target tissues and organs. VRS-317 was threefold more potent than daily rhGH in hypophysectomized rats and fivefold more potent than daily rhGH in juvenile monkeys. In juvenile monkeys, a monthly dose of 1.4?mg/kg VRS-317 (equivalent to 0.26?mg/kg rhGH) caused a sustained pharmacodynamic response for 1 month equivalent to 0.05?mg/kg/day rhGH (1.4?mg/kg rhGH total over 28 days). In monkeys, VRS-317, having a terminal elimination half-life of approximately 110?h, was rapidly and near-completely absorbed, and was well tolerated with no observed adverse effects after every alternate week subcutaneous dosing for 14 weeks. VRS-317 also did not cause lipoatrophy in pig and monkey studies. VRS-317 is currently being studied in GH-deficient patients to confirm the observations in these animal studies.     

Synthetic human growth hormone releasing factor (h-GRF-I-44-NH2) dose response effect on growth hormone and prolactin secretion in healthy adult men

Summary The dose-effect relationship of an i.v. bolus of synthetic h-GRF-44 on growth-hormone and prolactin secretion has been studied. Seven healthy adult volunteers received in a random order h-GRF-44 2.5, 5, 10, 20, 40 and 80 µg and a placebo. Plasma growth hormone (GH) was determined between 30 min before and 240 min after injection, the area under the curve (AUC) and the peak GH level being used to assess the response. For both parameters a dose-effect relationship was observed. Doses as low as 2.5 µg were capable of eliciting a rise in GH plasma levels in few patients. Above 40 µg the dose-effect curves tended to plateau, although the decrease in the slope of the dose-effect curve at the peak was more marked. Intersubject variability was large, so precise determination of the minimal effective dose as well as the lowest dose giving a maximal effect was not possible. The available evidence suggests that the i.v. dose of synthetic h-GRF-44 (SR 95228) which is likely to promote GH release into the blood stream in most healthy adults is within the range 40–100 µg. In these healthy adults unwanted effects were infrequent with these low doses. Unlike previous experience with higher doses of another synthetic h-GRF-44, prolactin secretion in this study was not affected.     

Influence of intravenous administration of growth hormone releasing peptide-2 (GHRP-2) on detection of growth hormone doping: growth hormone isoform profiles in Japanese male subjects

Administration of exogenous 22 kDa recombinant human growth hormone (rhGH) suppresses the non-22 kDa pituitary growth hormone (GH) secretion by negative feedback; then, the elevated 22 kDa GH to non-22 kDa GH ratio (Rec/Pit ratio) can be utilized to detect doping with rhGH (isoform differential immunoassay). The influence of intravenous administration of growth hormone releasing peptide GHRP-2 on the isoform differential immunoassay for detecting rhGH doping has been investigated.In this study, a reference population (n=100) was used, with 0.04 mg/kg rhGH subcutaneous administration (n=5), 100 μg of GHRP-2 intravenous administration (n=10) and 0.04 mg/kg rhGH combined with 100 μg of GHRP-2 (n=10) in Japanese male subjects. The results indicated that the low dose (0.04 mg/kg) of rhGH led to significantly increased Rec/Pit ratio compared with the Japanese reference limit (P < 0.001). Because GHRP-2 dose led to increases in concentrations of both recombinant GH (recGH) and pituitary GH (pit GH), no significant change in the Rec/Pit ratio was observed (P > 0.05). In a combined administration study, after GHRP-2 dose the Rec/Pit ratios decreased to 39.9-43.9% compared with the elevated ratio caused by the rhGH dose.The results indicated that GHRP-2 administration cannot only be detected by the isoform differential immunoassay but also masks rhGH doping. The analysis of GHRP-2 was found to be suitable for compensating for the disadvantages of the isoform differential immunoassay because GHRP-2 and its metabolite (AA-3) in urine could be detected during the periods of masking of the Rec/Pit ratio by means of liquid chromatography/tandem mass spectrometry.     

Intranasal delivery of recombinant human growth hormone (somatropin) in sheep using chitosan-based powder formulations.

The effectiveness of chitosan in promoting the intranasal bioavailability of recombinant human growth hormone (hGH) has been evaluated. hGH was formulated with chitosan to produce a powder blend (Formulation A) and granules (Formulation B) for intranasal administration. The in vivo pharmacokinetic performance of the formulations was evaluated in a group of six sheep in a randomised crossover study. A subcutaneous injection of hGH solution was administered as a control. The intranasal and subcutaneous doses of hGH were 0.3 and 0.03 mg/kg, respectively. The intranasal formulations appeared to be well tolerated. Mean bioavailabilities of hGH from Formulations A and B were 14 and 15%, respectively relative to subcutaneous injection. It is concluded that chitosan-based intranasal powder formulations may provide a practical means for non-injectable delivery of hGH and, potentially, other therapeutic protein molecules.     

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. CySl82-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (T^v_{18 + 19}) and the corresponding tryptic peptide from rhGH (T_{18 + 19}) were identical. Electrospray mass spectrometry (ES/MS) of T^v_{18 + 19} isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T_{18 + 19} from rhGH. A synthetic T_{18 + 19} peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragments in ES/MS compared to T^v_{18 + 19} isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cysl82-SSS-Cys189) bridge not found in the corresponding T_{18 + 19} disulfide peptide. Furthermore, the synthetic T^v_{18 + 19} co-eluted in RP-HPLC with T_{18 + 19} isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T_{18 + 19} and T^v_{18 + 19} peptides were performed. Using these data all protons were assigned. The major chemical shift changes (δ§>0.05 ppm) observed were for the β-protons of Cys182 and Cys189 in T^v_{18 + 19} as compared to T_{18 + 19}. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data, rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore^TM. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties. © Munksgaard 1996.     

Gallium(III) and iron(III) complexes of alpha-N-heterocyclic thiosemicarbazones: synthesis, characterization, cytotoxicity, and interaction with ribonucleotide reductase

A series of gallium(III) and iron(III) complexes with five different 4N-substituted alpha-N-heterocyclic thiosemicarbazones, viz., 2-acetylpyridine N,N-dimethylthiosemicarbazone (1), 2-acetylpyridine N-pyrrolidinylthiosemicarbazone (2), acetylpyrazine N,N-dimethylthiosemicarbazone (3), acetylpyrazine N-pyrrolidinylthiosemicarbazone (4), and acetylpyrazine N-piperidinylthiosemicarbazone (5), with the general formula [GaLCl2] (HL = 1 and 2) and [ML2][Y] (M = Ga, HL = 1-5, Y = PF6; M = Fe, HL = 1-5, Y = FeCl4 and PF6) were synthesized and characterized by elemental analysis, a number of spectroscopic methods (NMR, IR, UV-vis), mass spectrometry, and X-ray crystallography. The in vitro antitumor potency was studied in two human cancer cell lines (41M and SK-BR-3). The central metal ions exert pronounced effects in a divergent manner: gallium(III) enhances, whereas iron(III) weakens the cytotoxicity of the ligands. The capacity of ligand 1 and its Ga(III) and Fe(III) complexes to destroy the tyrosyl radical of the presumed target ribonucleotide reductase is reported.     

Self-assembly of Ophiopogonis polysaccharide-iron (III) complex in aqueous solution and solid state

Ophiopogonis polysaccharide-iron (III) (OPI) was prepared and characterized in the present study. The optimum condition for preparing OPI was as follows: OP and trisodium citrate were mixed at a weight ratio of 4:1 and reacted in a water bath at 70 °C for 3 h within the pH range of 8.0–8.5. Aggregation morphology or structure of OPI in aqueous solution and solid state was studied by scanning electron microscopy, transmission electron microscopy and small-angle X-ray diffraction. In aqueous solution, OPI could self-assemble into micron vesicles with flower-shaped morphology. Results of X-ray diffraction showed OPI with layered structure. A core-shell model was proposed for OPI.     

Thiomers in noninvasive polypeptide delivery: in vitro and in vivo characterization of a polycarbophil-cysteine/glutathione gel formulation for human growth hormone

This study was aimed at investigating the potential of a new polycarbophil-cysteine (PCP-Cys)/glutathione (GSH) gel formulation to enhance the permeation of the model drug human growth hormone (hGH) across nasal mucosa in vitro and in vivo. The aqueous nasal gel contained PCP-Cys, GSH, and hGH in a final concentration of 0.3%, 0.5%, and 0.6% (m/v), respectively. In vitro permeation studies were performed in Ussing chambers on freshly excised bovine nasal mucosa using fluorescence-labeled dextran (molecular mass: 4.3 kDa; FD-4) and hGH (FITC-hGH). The release profile of FITC-hGH from the gel formulation and an unmodified PCP control formulation was determined. Furthermore, in vivo studies in rats were performed comparing the PCP-Cys/GSH/hGH gel with PCP/hGH control gel and physiological saline. The permeation of FD-4 and FITC-hGH across the nasal mucosa was improved two-fold and three-fold, respectively, in the presence of PCP-Cys/GSH. The PCP-Cys/GSH/hGH gel and the PCP/hGH control gel showed the same biphasic and matrix-controlled drug release. The nasal administration of the PCP-Cys/GSH/hGH gel formulation to rats resulted in a significantly increased and prolonged hGH plasma concentration-time profile versus unmodified PCP gel and physiological saline. According to these results, PCP-Cys gels might represent a promising new strategy for systemic nasal polypeptide delivery.     

New analogs of human growth hormone-releasing hormone (1-29) with high and prolonged antagonistic activity

Based on our previous results, in conjunction with various structural considerations, 19 new analogs of the GHRH antagonist [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Abu¹⁵,Nle²⁷,Agm²⁹]hGHRH(1-29) (MZ-5-156) were synthesized by the solid-phase method. These compounds were designed to develop further analogs of this class with increased receptor-binding affinity. All analogs had Abu¹⁵ and Nle²⁷ modifications and were acylated with phenylacetic acid at the N-terminus. Most of the analogs had d -Arg² and Phe(pCl)⁶ substituents and Agm²⁹ or Arg²⁹-NH₂ at the C-terminus. Additional single substitutions consisted of the incorporation of d - or l -Tic¹, d -Tic², Tic⁶ or Phe(pNO₂)⁶ and Arg²⁹-NH₂. The Arg²⁹-NH₂ analog of MZ-5-156 (KT-48) was further modified by single substitutions using Pal¹; d -Tpi²; d - or l -Phe⁴; Phe(pX)⁶×= F, Cl, I; Tyr⁷; Aib⁸; Tyr(Me)¹⁰ or Phe(pCl)¹⁰. Four peptides had multiple substitutions. All the analogs were evaluated for their ability to inhibit GH release induced by hGHRH(1-29)NH₂in vitro and some were also tested in vivo. Peptides [PhAc-Tyr¹,D-Arg²,Phe(pI)⁶,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-30), [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Aib⁸,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-50) and [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Tyr(Me)¹⁰,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-40) with Phe(pI)⁶, Aib⁸ or Tyr(Me)¹⁰modifications, respectively, showed high and prolonged inhibitory effect in superfused rat pituitary system. Analog KT-50 also exhibited a strong and long-term inhibitory activity in vivo in rats. Most of the new analogs showed high binding affinities to rat pituitary GHRH receptors.     

N-biotinyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide as a potential model for S-(1,2-dichlorovinyl)-L-cysteine sulfoxide: characterization of stability and reactivity with glutathione and kidney proteins in vitro

S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide (DCVCS) is a reactive and potent nephrotoxic metabolite of the human trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC). Because DCVCS covalent binding to kidney proteins likely plays a role in its nephrotoxicity, in this study biotin-tagged DCVCS, N-biotinyl-DCVCS (NB-DCVCS), was synthesized, and its stability in buffer alone and in the presence of rat blood or plasma was characterized in vitro. In addition, reactivity toward GSH and covalent binding to selected model enzymes and isolated kidney proteins were characterized. The half-lives of NB-DCVCS (39.6 min) and the DCVCS (diastereomer 1, 14.4 min; diastereomer 2, 6 min) in the presence of GSH were comparable. Incubating the model enzymes glutathione reductase and malate dehydrogenase with 10 μM NB-DCVCS for 3 h at 37 °C followed by immunoblotting using antibiotin antibodies demonstrated that glutathione reductase and malate dehydrogenase were extensively modified by NB-DCVCS. When rat kidney cytosol (6 μg/μL) was incubated with NB-DCVCS (312.5 nM to 5 μM) for 3 h at 37 °C followed by immunoblotting, a concentration-dependent increase in signal with multiple proteins with different molecular weights was observed, suggesting that NB-DCVCS binds to multiple kidney proteins with different selectivity. Incubating rat kidney cytosol with DCVCS (10-100 μM) prior to the addition of NB-DCVCS (2.5 μM) reduced the immunoblotting signal, suggesting that NB-DCVCS and DCVCS compete for the same binding sites. A comparison of the stability of NB-DCVCS and DCVCS in rat blood and plasma was determined in vitro, and NB-DCVCS exhibited higher stability than DCVCS in both media. Collectively, these results suggest that NB-DCVCS shows sufficient stability, reactivity, and selectivity to warrant further investigations into its possible use as a tool for future characterization of the role of covalent modification of renal proteins by DCVCS in nephrotoxicity.