The invention provides processes for obtaining a precursor for insulin, analogs or derivatives th... more The invention provides processes for obtaining a precursor for insulin, analogs or derivatives thereof having correctly bonded cystine bridges. The process involves solubilizing a precursor of insulin, insulin analog or derivatives in an aqueous solution or a buffer containing cysteine or cysteine hydrochloride and one or more of chaotropic auxiliary. The solubilized precursors are refolded by adding diluent to the solubilized mixture (reverse dilution). Further, the solubilized precursors, wherein the concentration of precursor in reaction mixture is more than 0.65g/litre can also be refolded by diluting the reaction mixture with a diluent optionally comprising about 5-40 % v/v of one or more of alcoholic or aprotic solvents.
Interaction of [ 125 I]-insulin with intact hepatocytes and its correlation with circulatory insu... more Interaction of [ 125 I]-insulin with intact hepatocytes and its correlation with circulatory insulin level was examined. The hepatocytes from newborn rats bound lowest amount of [ 125 I]-insulin (1.39±0.41 pM/mg cell protein) when circulatory insulin level was high (8±1.5 µU/ml). Hepatocytes from 7 day and 21 day old animals demonstrated a more or less similar relationship, Cells from 31 day old animals exhibited maximum insulin binding, activity (5.13±0.18.pM/mg cell protein) against a low serum insulin level (4.25±0.25 µU/ ml). Scatchard analysis of insulin binding shows that the affinity is higher in the hepatocytes from newborn animals than in the hepatocytes of 31 day old animals. Higher binding observed in the latter case may be due to a greater number of binding sites. Hepatocytes from one year old rats bound very little insulin (2.50±0.36 pM/mg cell protein) against a high circulatory insulin level (9.25±0.85 µU/ml). In view of these results, it appears that the down-regulation hypothesis holds true during ontogeny too.
In vivo and in vitro studies were carried out to evaluate the clinical application of glycosylate... more In vivo and in vitro studies were carried out to evaluate the clinical application of glycosylated hemoglobin and plasma proteins in the diagnosis and management of diabetes mellitus. Glycosylated hemoglobin registered an almost 80% fall in diabetic patients following controlled glycemia for two months while glycosylated plasma protein level registered an 80% fall in the patients after fifteen days of blood glucose homeostasis. Human serum proteins were glycosylated in vitro and glycosylation was linearly proportional to the glucose concentration and incubation time. Polyacrylamide gel electrophoresis of glycosylated serum proteins revealed that albumin and transferrin are the major proteins that are significantly glycosylated. Glycosylated hemoglobin and plasma protein levels were also increased in chronic renal failure patients without any history of diabetes.
Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas admi... more Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas administration of cortisol, cyclic AMP, glucagon, insulin, tryptophan or tyrosine does not. The results of these and other experiments with pyridoxine are discussed in relation to the mechanisms of action of this vitamin on the activity of the enzyme.
Human alpha fetoprotein (AFP) binds bilirubin with an affinity somewhat lower than albumin. Free ... more Human alpha fetoprotein (AFP) binds bilirubin with an affinity somewhat lower than albumin. Free bilirubin was found to have an extinction maximum at 440 nm with an extinction coefficient of 4.97 x 10(4) M-1cm-1. AFP binding with the bile pigment elicits a blue shift while albumin interaction produced red spectral shift.
The objective of the study was to compare the pharmacokinetic (PK) and pharmacodynamic (PD) prope... more The objective of the study was to compare the pharmacokinetic (PK) and pharmacodynamic (PD) properties of an insulin glargine formulation, Glaritus(test) with the innovator's formulation Lantus(reference) using the euglycemic clamp technique in a single-dose, double-blind, randomized, two sequences, four-period replicate crossover study in healthy volunteers (n = 40). Subjects received subcutaneous administration of the insulin glargine (0.4 IU/kg) formulation at two occasions for test and reference and a 20% glucose solution was infused at variable rate to maintain euglycemia for 24 h. Both PK [area under the plasma concentration time curve (AUC) and maximum insulin concentration (C)] and PD endpoints [area under glucose infusion rate time curve () and maximum glucose infusion rate (GIR)] demonstrated bioequivalence of Glaritus to Lantus with the 90% confidence interval of geometric mean ratio of test to reference entirely contained within 0.80-1.25. Both formulations showed eq...
Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated... more Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was ob-MATERIALS AND METHODS Sepharose-4B was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. DEAE cellulose (D-52), cytochrome c , and bovine serum albumin were obtained from Sigma Chemical Co.
Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryp... more Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryptophan was studied in groups of rats fed on diets containing 18 or 5% protein. The basal activity of hepatic tyrosine aminotransferase of rats receiving 5% protein gradually increased with the age of the animals but that of rats receiving 18% protein did not. l-Tyrosine induced hepatic tyrosine aminotransferase in rats receiving 18% protein when tested at ages from 4 to 20 weeks. When induction by l-tyrosine was carried out in rats receiving the 5% protein diet, significant induction of tyrosine aminotransferase occurred only in 4- or 6-week-old rats. Induction by l-tryptophan of tryptophan oxygenase in liver or the basal activity of this enzyme in liver did not differ between the groups fed on 5 and 18% protein. On changing the diet from 0 to 18% protein, the above-mentioned effects on the induction of hepatic tyrosine aminotransferase were reversed.
Hyperglycemia was induced in Sprague-Dawley rats by alloxan and maintained for 2, 6 and 10 weeks ... more Hyperglycemia was induced in Sprague-Dawley rats by alloxan and maintained for 2, 6 and 10 weeks without insulin treatment. Levels of glycosylated plasma proteins, hemoglobin and glycosylated tissue proteins of aorta, heart, kidney, liver and brain were estimated at these intervals. Protein glycosylation was observed to increase linearly as a function of period of hyperglycemia in all the tissues studied, except brain, in the following order: aorta > heart > kidney > liver. Protein glycosylation, leading to formation of covalently modified advanced glycosylated products and protein-protein adducts, may reflect disposition of these tissues to diabetic complications.
The invention provides processes for obtaining a precursor for insulin, analogs or derivatives th... more The invention provides processes for obtaining a precursor for insulin, analogs or derivatives thereof having correctly bonded cystine bridges. The process involves solubilizing a precursor of insulin, insulin analog or derivatives in an aqueous solution or a buffer containing cysteine or cysteine hydrochloride and one or more of chaotropic auxiliary. The solubilized precursors are refolded by adding diluent to the solubilized mixture (reverse dilution). Further, the solubilized precursors, wherein the concentration of precursor in reaction mixture is more than 0.65g/litre can also be refolded by diluting the reaction mixture with a diluent optionally comprising about 5-40 % v/v of one or more of alcoholic or aprotic solvents.
Interaction of [ 125 I]-insulin with intact hepatocytes and its correlation with circulatory insu... more Interaction of [ 125 I]-insulin with intact hepatocytes and its correlation with circulatory insulin level was examined. The hepatocytes from newborn rats bound lowest amount of [ 125 I]-insulin (1.39±0.41 pM/mg cell protein) when circulatory insulin level was high (8±1.5 µU/ml). Hepatocytes from 7 day and 21 day old animals demonstrated a more or less similar relationship, Cells from 31 day old animals exhibited maximum insulin binding, activity (5.13±0.18.pM/mg cell protein) against a low serum insulin level (4.25±0.25 µU/ ml). Scatchard analysis of insulin binding shows that the affinity is higher in the hepatocytes from newborn animals than in the hepatocytes of 31 day old animals. Higher binding observed in the latter case may be due to a greater number of binding sites. Hepatocytes from one year old rats bound very little insulin (2.50±0.36 pM/mg cell protein) against a high circulatory insulin level (9.25±0.85 µU/ml). In view of these results, it appears that the down-regulation hypothesis holds true during ontogeny too.
In vivo and in vitro studies were carried out to evaluate the clinical application of glycosylate... more In vivo and in vitro studies were carried out to evaluate the clinical application of glycosylated hemoglobin and plasma proteins in the diagnosis and management of diabetes mellitus. Glycosylated hemoglobin registered an almost 80% fall in diabetic patients following controlled glycemia for two months while glycosylated plasma protein level registered an 80% fall in the patients after fifteen days of blood glucose homeostasis. Human serum proteins were glycosylated in vitro and glycosylation was linearly proportional to the glucose concentration and incubation time. Polyacrylamide gel electrophoresis of glycosylated serum proteins revealed that albumin and transferrin are the major proteins that are significantly glycosylated. Glycosylated hemoglobin and plasma protein levels were also increased in chronic renal failure patients without any history of diabetes.
Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas admi... more Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas administration of cortisol, cyclic AMP, glucagon, insulin, tryptophan or tyrosine does not. The results of these and other experiments with pyridoxine are discussed in relation to the mechanisms of action of this vitamin on the activity of the enzyme.
Human alpha fetoprotein (AFP) binds bilirubin with an affinity somewhat lower than albumin. Free ... more Human alpha fetoprotein (AFP) binds bilirubin with an affinity somewhat lower than albumin. Free bilirubin was found to have an extinction maximum at 440 nm with an extinction coefficient of 4.97 x 10(4) M-1cm-1. AFP binding with the bile pigment elicits a blue shift while albumin interaction produced red spectral shift.
The objective of the study was to compare the pharmacokinetic (PK) and pharmacodynamic (PD) prope... more The objective of the study was to compare the pharmacokinetic (PK) and pharmacodynamic (PD) properties of an insulin glargine formulation, Glaritus(test) with the innovator's formulation Lantus(reference) using the euglycemic clamp technique in a single-dose, double-blind, randomized, two sequences, four-period replicate crossover study in healthy volunteers (n = 40). Subjects received subcutaneous administration of the insulin glargine (0.4 IU/kg) formulation at two occasions for test and reference and a 20% glucose solution was infused at variable rate to maintain euglycemia for 24 h. Both PK [area under the plasma concentration time curve (AUC) and maximum insulin concentration (C)] and PD endpoints [area under glucose infusion rate time curve () and maximum glucose infusion rate (GIR)] demonstrated bioequivalence of Glaritus to Lantus with the 90% confidence interval of geometric mean ratio of test to reference entirely contained within 0.80-1.25. Both formulations showed eq...
Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated... more Monospecific anti-rat serum alpha-fetoprotein (AFP) IgG was coupled to cyanogen bromide-activated Sepharose-4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. The immunoaffinity column was used to isolate AFP from feto-neonatal rat brain. The purified AFP was immunologically and electrophoretically similar to serum AFP. It yielded a single band with a molecular weight of 70,000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the protein under nondenaturing conditions yielded two charge variants of AFP, reminiscent of AFP from feto-neonatal rat serum. The AFP was ob-MATERIALS AND METHODS Sepharose-4B was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. DEAE cellulose (D-52), cytochrome c , and bovine serum albumin were obtained from Sigma Chemical Co.
Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryp... more Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryptophan was studied in groups of rats fed on diets containing 18 or 5% protein. The basal activity of hepatic tyrosine aminotransferase of rats receiving 5% protein gradually increased with the age of the animals but that of rats receiving 18% protein did not. l-Tyrosine induced hepatic tyrosine aminotransferase in rats receiving 18% protein when tested at ages from 4 to 20 weeks. When induction by l-tyrosine was carried out in rats receiving the 5% protein diet, significant induction of tyrosine aminotransferase occurred only in 4- or 6-week-old rats. Induction by l-tryptophan of tryptophan oxygenase in liver or the basal activity of this enzyme in liver did not differ between the groups fed on 5 and 18% protein. On changing the diet from 0 to 18% protein, the above-mentioned effects on the induction of hepatic tyrosine aminotransferase were reversed.
Hyperglycemia was induced in Sprague-Dawley rats by alloxan and maintained for 2, 6 and 10 weeks ... more Hyperglycemia was induced in Sprague-Dawley rats by alloxan and maintained for 2, 6 and 10 weeks without insulin treatment. Levels of glycosylated plasma proteins, hemoglobin and glycosylated tissue proteins of aorta, heart, kidney, liver and brain were estimated at these intervals. Protein glycosylation was observed to increase linearly as a function of period of hyperglycemia in all the tissues studied, except brain, in the following order: aorta > heart > kidney > liver. Protein glycosylation, leading to formation of covalently modified advanced glycosylated products and protein-protein adducts, may reflect disposition of these tissues to diabetic complications.
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