Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflow(TM) preparative electrophoresis technology is described. Gradiflow(TM) electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency. (C) 2005 Elsevier B.V. All rights reserved.

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific beta subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both the in vitro and in vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles, in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greater in vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both alpha 2,3 and alpha 2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greater in vitro biological potency compared to those of the pituitary human follicle stimulating hormone.

Modeling of subcutaneous absorption kinetics of infusion solutions in the elderly using technetium

Absorption kinetics of solutes given with the subcutaneous administration of fluids is ill-defined. The gamma emitter, technitium pertechnetate, enabled estimates of absorption rate to be estimated independently using two approaches. In the first approach, the counts remaining at the site were estimated by imaging above the subcutaneous administration site, whereas in the second approach, the plasma technetium concentration-time profiles were monitored up to 8 hr after technetium administration. Boluses of technetium pertechnetate were given both intravenously and subcutaneously on separate occasions with a multiple dosing regimen using three doses on each occasion. The disposition of technetium after iv administration was best described by biexponential kinetics with a V-ss of 0.30 +/- 0.11 L/kg and a clearance of 30.0 +/- 13.1 ml/min. The subcutaneous absorption kinetics was best described as a single exponential process with a half-life of 18.16 +/- 3.97 min by image analysis and a half-life of 11.58 +/- 2.48 min using plasma technetium time data. The bioavailability of technetium by the subcutaneous route was estimated to be 0.96 +/- 0.12. The absorption half-life showed no consistent change with the duration of the subcutaneous infusion. The amount remaining at the absorption site with time was similar when analyzed using image analysis, and plasma concentrations assuming multiexponential disposition kinetics and a first-order absorption process. Profiles of fraction remaining at the absorption sire generated by deconvolution analysis, image analysis, and assumption of a constant first-order absorption process were similar. Slowing of absorption from the subcutaneous administration site is apparent after the last bolus dose in three of the subjects and can De associated with the stopping of the infusion. In a fourth subject, the retention of technetium at the subcutaneous site is more consistent with accumulation of technetium near the absorption site as a result of systemic recirculation.

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimers

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+T cell recognition

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.

Population pharmacokinetics of magnesium in preeclampsia

OBJECTIVE: The purpose of this study was to determine the population pharmacokinetics of magnesium from sparse observational data in patients with preeclampsia. STUDY DESIGN: Serum magnesium concentrations (1-11 per patient) were obtained retrospectively from the records of 116 patients with preeclampsia who had a loading dose of magnesium sulfate (16 or 20 mmol), followed by a maintenance dose (1 mmol/h) over an average of 28 hours. Population clearance, volume of distribution, and the baseline magnesium concentration were estimated using the NONMEM program. RESULTS: The following population typical values, together with the interpatient variability (expressed as coefficient of variation) were obtained with the use of a 1-compartment model: systemic clearance, 4.28 L/h (37.3%); volume of distribution, 32.3 L (32.1%); baseline concentration, 0.811 mmol/L (18.5%). The average half-life was 5.2 hours. Clonus was not obtunded in 4 patients whose serum magnesium concentrations were similar to the average concentration of 1.7 mmol/L. The variability remaining unexplained after the population model was fitted to the data was 6.5% to 10.8%. CONCLUSION: This study extended knowledge of the pharmacokinetic disposition of magnesium in preeclampsia. The results are potentially useful for the calculation of loading and maintenance doses, particularly when the relationship between serum concentration and effect in preeclampsia is clarified.

Determination of reference values for glucose tolerance, insulin tolerance, and insulin sensitivity tests in clinically normal cats

Objective-To determine reference values and test variability for glucose tolerance tests (GTT), insulin tolerance tests (ITT), and insulin sensitivity tests (IST) in cats, Animals-32 clinically normal cats. Procedure-GTT, ITT, and IST were performed on consecutive days. Tolerance intervals tie, reference values) were calculated as means +/- 2.397 SD for plasma glucose and insulin concentrations, half-life of glucose (T-1/2glucose), rate constants for glucose disappearance (K-glucose and K-itt), and insulin sensitivity index (S-l). Tests were repeated after 6 weeks in 8 cats to determine test variability. Results-Reference values for T-1/2glucose, K-glucose, and fasting plasma glucose and insulin concentrations during GTT were 45 to 74 minutes, 0.93 to 1.54 %/min, 37 to 104 mg/dl, and 2.8 to 20.6 muU/ml, respectively. Mean values did not differ between the 2 tests. Coefficients of variation for T-1/2glucose, K-glucose, and fasting plasma glucose and insulin concentrations were 20, 20, 11, and 23%, respectively. Reference values for K-itt were 1.14 to 7.3%/min, and for S-l were 0.57 to 10.99 x 10(-4) min/muU/ml. Mean values did not differ between the 2 tests performed 6 weeks apart, Coefficients of variation for K-itt and S-l were 60 and 47%, respectively. Conclusions and Clinical Relevance-GTT, ITT, and IST can be performed in cats, using standard protocols. Knowledge of reference values and test variability will enable researchers to better interpret test results for assessment of glucose tolerance, pancreatic beta -cell function, and insulin sensitivity in cats.

Dexamethasone enhances SOX9 expression in chondrocytes

SOX9 is a transcription factor that activates type II procollagen (Col2a1) gene expression during chondrocyte differentiation. Glucocorticoids are also known to promote chondrocyte differentiation via unknown molecular mechanisms. We therefore investigated the effects of a synthetic glucocorticoid, dexamethasone (DEX), on Sox9 gene expression in chondrocytes prepared From rib cartilage of newborn mice. Sox9 mRNA was expressed at high levels in these chondrocytes. Treatment with DEX enhanced Sox9 mRNA expression within 24 h and this effect was observed at least up to 48 h. The effect of DEX was dose dependent, starting at 0.1 nM and maximal at 10 nM. The half life of Sox9 mRNA was approximately 45 min in the presence or absence of DEX. Western blot analysis revealed that DEX also enhanced the levels of SOX9 protein expression. Treatment with DEX enhanced Col2a1 mRNA expression in these chondrocytes and furthermore, DEX enhanced the activity of Col2-CAT (chloramphenicol acetyltransferase) construct containing a 1.6 kb intron fragment where chondrocyte-specific Sry/Sox-consensus sequence is located. The enhancing effect of DEX was specific to SOX9, as DEX did not alter the levels of Sox6 mRNA expression. These data suggest that DEX promotes ch differentiation through enhancement of SOX9.

Tetrahydrofolates are greatly stabilized by binding to bovine milk folate-binding protein

The dietary supply of folates and their measurement are both affected, potentially, by the instability of some folates. Labile folates appear to be stabilized by binding to folate-binding protein (FBP); this paper reports measurements of that stabilization. The degradation rates of the very labile tetrahydrofolate (H(4)folate) and moderately labile 5-methyltetrahydrofolate (5-CH(3)H(4)folate) were measured with the compounds free or bound to either soluble or immobilized bovine milk FBP. Complexation increased stability from 2- to > 1000-fold, depending on buffer and temperature conditions. H(4)folate at 4degreesC and pH 6.7 appeared to be quite stable for > 100 d when bound to soluble FBP but had a half-life of < 1 h when free. Stabilization of milk folates may be a role of FBP and would improve the bioavailability of milk folate to newborns and other consumers.

Dietary chromium tripicolinate supplementation reduces glucose concentrations and improves glucose tolerance in normal-weight cats

The effect of dietary chromium supplementation on glucose and insulin metabolism in healthy, non-obese cats was evaluated. Thirty-two cats were randomly divided into four groups and fed experimental diets consisting of a standard diet with 0 ppb (control), 150 ppb, 300 ppb, or 600 ppb added chromium as chromium tripicolinate. Intravenous glucose tolerance, insulin tolerance and insulin sensitivity tests with minimal model analysis were performed before and after 6 weeks of feeding the test diets. During the glucose tolerance test, glucose concentrations, area under the glucose concentration-time curve, and glucose half-life (300 ppb only), were significantly lower after the trial in cats supplemented with 300 ppb and 600 ppb chromium, compared with values before the trial. Fasting glucose concentrations measured on a different day in the biochemistry profile were also significantly lower after supplementation with 600 ppb chromium. There were no significant differences in insulin concentrations or indices in either the glucose or insulin tolerance tests following chromium supplementation, nor were there any differences between groups before or after the dietary trial. Importantly, this study has shown a small but significant, dose-dependent improvement in glucose tolerance in healthy, non-obese cats supplemented with dietary chromium. Further long-term studies are warranted to determine if the addition of chromium to feline diets is advantageous. Cats most likely to benefit are those with glucose intolerance and insulin resistance from lack of exercise, obesity and old age. Healthy cats at risk of glucose intolerance and diabetes from underlying low insulin sensitivity or genetic factors may also benefit from long-term chromium supplementation. (C) 2002 ESFM and AAFP.

Tungsten isotope evidence from similar to 3.8-Gyr metamorphosed sediments for early meteorite bombardment of the Earth

The 'Late Heavy Bombardment' was a phase in the impact history of the Moon that occurred 3.8-4.0 Gyr ago, when the lunar basins with known dates were formed(1,2). But no record of this event has yet been reported from the few surviving rocks of this age on the Earth. Here we report tungsten isotope anomalies, based on the Hf-182-W-182 system (half-life of 9 Myr), in metamorphosed sedimentary rocks from the 3.7-3.8-Gyr-old Isua greenstone belt of West Greenland and closely related rocks from northern Labrador, Canada. As it is difficult to conceive of a mechanism by which tungsten isotope heterogeneities could have been preserved in the Earth's dynamic crust-mantle environment from a time when short-lived Hf-182 was still present, we conclude that the metamorphosed sediments contain a component derived from meteorites.

The absorption and excretion of fluoride and arsenic in humans

The absorption and excretion of fluoride and arsenic were measured in a group of healthy volunteers given drinking water with naturally high concentration of fluoride (F 2.3 mg/l), or high concentration of arsenic (As 0.15 mg/l), or high concentrations of both fluoride and arsenic (F 2.25 mg/l, As 0.23 mg/l and F 4.05 mg/l, As 0.58 mg/l), respectively. The results indicated that, for arsenic, the absorption rate, the proportion of urinary excretion and the biological-half-life did not show statistically significant differences between drinking water containing high arsenic alone and drinking water containing different levels of high arsenic and fluoride. Excretion and retention of arsenic were positively correlated to the total arsenic intake. Similar results were observed for fluoride. This suggests that there are different metabolic processes for arsenic and fluoride in respect to absorption and excretion; and no joint action can be attributed by these two elements. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula)

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8 +/- 1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximate to 65 kDa, similar to that identified in other mammalian species, and binding of I-125-labelled human GH (hGH) was displaced by excess hGH (20 mug). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with I-125-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K-a) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96 +/- 0.2 x 10(9)/M, n = 6). Binding capacity (B-max) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4 +/- 0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.

Attenuation of cardiac fibrosis by pirfenidone and amiloride in DOCA-salt hypertensive rats

1 This study has administered pirfenidone (5-methyl-l-phenyl-2-[1H]-pyridone) or amiloride to attenuate the remodelling and associated functional changes, especially an increased cardiac stiffness, in DOCA-salt hypertensive rats. 2 In control rats, the elimination half-life of pirfenidone following a single intravenous dose of 200 mg kg(-1) was 37 min while oral bioavailability at this dose was 25.7%. Plasma pirfenidone concentrations in control rats averaged 1.9 +/- 0.1 mug ml(-1) over 24 It after 14 days' administration as a 0.4% mixture in food. 3 Pirfenidone (approximately 250-300 mg kg(-1) day(-1) as 0.4% in food) and amiloride (I mg kg-1 day(-1) sc) were administered for 2 weeks starting 2 weeks post-surgery. Pirfenidone but not amiloride attenuated ventricular hypertrophy (2.69 +/- 0.09, UNX 2.01 +/- 0.05. DOCA-salt 3.11 +/- 0.09 mg kg(-1) body wt) without lowering systolic blood pressure. 4 Collagen deposition was significantly increased in the interstitium after 2 weeks and further increased with scarring of the left ventricle after 4 weeks; pirfenidone and amiloride reversed the increases and prevented further increases. This accumulation of collagen was accompanied by an increase in diastolic stiffness constant; both amiloride and pirfenidone, reversed this increase. 5 Noradrenaline potency (positive chronotropy) was decreased in right atria (neg log EC50: control 6.92 +/- 0.06; DOCA-salt 6.64 +/- 0.08); pirfenidone but not amiloride reversed this change. Noradrenaline was a more potent vasoconstrictor in thoracic aortic rings (neg log EC50: control 6.91 +/- 0.10; DOCA-salt 7.90 +/- 0.07); pirfenidone treatment did not change noradrenaline potency. 6 Thus, pirfenidone and amiloride reverse and prevent cardiac remodelling and the increased cardiac stiffness without reversing the increased vascular responses to noradrenaline.

Leaf litter decomposition of Piper aduncum, Gliricidia sepium and Imperata cylindrica in the humid lowlands of Papua New Guinea

No information is available on the decomposition and nutrient release pattern of Piper aduncum and Imperata cylindrica despite their importance in shifting cultivation systems of Papua New Guinea and other tropical regions. We conducted a litter bag study (24 weeks) on a Typic Eutropepts in the humid lowlands to assess the rate of decomposition of Piper aduncum, Imperata cylindrica and Gliricidia sepium leaves under sweet potato (Ipomoea batatas). Decomposition rates of piper leaf litter were fastest followed closely by gliricidia, and both lost 50% of the leaf biomass within 10 weeks. Imperata leaf litter decomposed much slower and half-life values exceeded the period of observation. The decomposition patterns were best explained by the lignin plus polyphenol over N ratio which was lowest for piper (4.3) and highest for imperata (24.7). Gliricidia leaf litter released 79 kg N ha(-1), whereas 18 kg N ha(-1) was immobilised in the imperata litter. The mineralization of P was similar for the three species, but piper litter released large amounts of K. The decomposition and nutrient release patterns had significant effects on the soil. The soil contained significantly more water in the previous imperata plots at 13 weeks due to the relative slow decomposition of the leaves. Soil N levels were significantly reduced in the previous imperata plots due to immobilisation of N. Levels of exchangeable K were significantly increased in the previous piper plots due to the large addition of K. It can be concluded that piper leaf litter is a significant and easily decomposable source of K which is an important nutrient for sweet potato. Gliricidia leaf litter contained much N, whereas imperata leaf litter releases relatively little nutrients and keeps the soil more moist. Gliricidia fallow is more attractive than an imperata fallow for it improves the soil fertility and produces fuelwood as additional saleable products.