Stabilization of Resveratrol in Blood Circulation by Conjugation to mPEG and mPEG-PLA Polymers: Investigation of Conjugate Linker and Polymer Composition on Stability,Metabolism, Antioxidant Activity and Pharmacokinetic Profile

Resveratrol is naturally occurring phytochemical with diverse biological activities such as chemoprevention, anti-inflammatory, anti-cancer, anti-oxidant. But undergoes rapid metabolism in the body (half life 0.13h). Hence Polymer conjugation utilizing different chemical linkers and polymer compositions was investigated for enhanced pharmacokinetic profile of resveratrol. Ester conjugates such as α-methoxy-ω-carboxylic acid poly(ethylene glycol) succinylamide resveratrol (MeO-PEGN-Succ-RSV) (2 and 20 kDa); MeO-PEG succinyl ester resveratrol (MeO-PEGO-Succ-RSV) (2 kDa); α-methoxy poly(ethylene glycol)-co-polylactide succinyl ester resveratrol (MeO-PEG-PLAO-Succ-RSV) (2 and 6.6kDa) were prepared by carbodiimide coupling reactions. Resveratrol-PEG ethers (2 and 5 kDa) were synthesized by alkali-mediated etherification. All polymer conjugates were fully characterized in vitro and the pharmacokinetic profile of selected conjugates was characterized in rats. Buffer and plasma stability of conjugates was dependent on polymer hydrophobicity, aggregation behavior and PEG corona, with MeO-PEG-PLAO-Succ-RSV (2 kDa) showing a 3h half-life in rat plasma in vitro. Polymer conjugates irrespective of linker chemistry protected resveratrol against metabolism in vitro. MeO-PEG-PLAO-Succ-RSV (2 kDa), Resveratrol-PEG ether (2 and 5 kDa) displayed improved pharmacokinetic profiles with significantly higher plasma area under curve (AUC), slower clearance and smaller volume of distribution, compared to resveratrol.     

Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.     

Dose-dependent elimination of 5-fluoro-2'-deoxyuridine in the monkey

The kinetics of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluorouracil (FUra) disposition after bolus intravenous injection were determined in anesthetized rhesus and cynomolgus monkeys. FdUrd disappearance from plasma was an apparent triexponential process with average half-lives of 0.5, 2, and 8 min; FUra disappearance was biphasic with average half-lives of 2 and 13 min. After FdUrd injection, FUra reached peak plasma concentrations of 15-30% of the initial FdUrd concentrations within 3 min, and then disappeared more slowly than FdUrd. Total FdUrd clearance fell from 105 to 73 to 56 ml/kg/min as the dose increased from 10 to 20 to 40 mg/kg. Metabolic clearance was about 85% of total clearance and fell similarly with increasing dosage. Total and metabolic FUra clearances were about 30% of FdUrd values at an equimolar dose. Renal FdUrd clearance exceeded glomerular filtration rate and was decreased by probenecid, indicating tubular secretion; renal FUra clearance was close to glomerular filtration rate. There was no apparent correlation between dose and renal clearance or volume of distribution. It was concluded that FdUrd, like FUra, is eliminated primarily by a dose-dependent process. The metabolic basis of the dose-dependent kinetics remains to be determined.     

Capillary endothelial surface layer selectively reduces plasma solute distribution volume

We previously reported that a 0.4- to 0.5-microm-thick endothelial surface layer confines Dextran 70 (70 kDa) to the central core of hamster cremaster muscle capillaries. In the present study we used a variety of plasma tracers to probe the barrier properties of the endothelial surface layer using combined fluorescence and brightfield intravital microscopy. No permeation of the endothelial surface layer was observed for either neutral or anionic dextrans >/=70 kDa, but a neutral Dextran 40 (40 kDa) and neutral free dye (rhodamine, 0.4 kDa) equilibrated with the endothelial surface layer within 1 min. In contrast, small anionic tracers of similar size (0. 4-40 kDa) permeated the endothelial surface layer relatively slowly with half-times (tau(50)) between 11 and 60 min, depending on tracer size. Furthermore, two plasma proteins, fibrinogen (340 kDa) and albumin (67 kDa), moved slowly into the endothelial surface layer at the same rates, despite greatly differing sizes (tau(50) approximately 40 min). Dextran 70, which did not enter the glycocalyx over the course of these experiments, entered at the same rate as free albumin when it was conjugated to albumin. These findings demonstrate that for anionic molecules size and charge have a profound effect on the penetration rate into the glycocalyx. The equal rates of penetration of the glycocalyx demonstrated by the different protein molecules suggests that multiple factors may influence the penetration of the barrier, including molecular size, charge, and structure.     

Anti-L-selectin aptamers: binding characteristics, pharmacokinetic parameters, and activity against an intravascular target in vivo

Therapeutic and diagnostic applications have been envisioned for aptamers, a class of oligonucleotide ligands that bind their target molecules with high affinity and specificity (Gold, J. Biol. Chem. 270, 13581-13584, 1995). To identify parameters that are important for the in vivo activity of aptamers acting on intravascular targets, we have studied binding characteristics in vitro, pharmacokinetic parameters in Sprague-Dawley rats, and inhibitory activity in a SCID mouse/human lymphocyte model of lymphocyte trafficking for both 2'F pyrimidine 2'OH purine RNA and ssDNA anti-human L-selectin aptamers. The data indicate that aptamers with low nanomolar affinity are suitable candidates for use as in vivo reagents and that nonspecific binding to vascular cells is not an issue for efficacy. As is often observed for other reagents, plasma clearance is biphasic. Both the distribution phase and the clearance rate strongly affect in vivo activity. Pharmacokinetic parameters and in vivo activity are significantly improved by conjugating aptamers to a carrier molecule, such as polyethylene glycol (PEG). Most active in vivo is 1d40, a 2'F pyrimidine 2'OH purine aptamer conjugated to 40 kDa PEG. At a dose of 5.4 nmol/kg body weight, its duration of effect (time to 50% inhibition) is 11.2 hours, and at 1 mg or 90 nmol/kg, its plasma clearance rate (CL) is 0.4 ml/min/kg. Its ED50 is estimated to be 80 pmol/kg in preinjection dose-response experiments, compared with 4 pmol/kg for the dimeric anti-L-selectin antibody DREG56. Further improvement of in vivo activity is expected from nucleotide modifications that increase resistance to nuclease digestion for aptamers where mass is not rate limiting for clearance. Because the relationship of clearance to conjugate molecular weight (MW) is not the same for all aptamers, it is advisable to determine the relationship at the outset of in vivo studies. In summary, the data suggest that properly formulated aptamers have the capacity to be effective therapeutic agents against intravascular targets.     

Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein that binds sex steroids with high affinity. Variations in hSHBG glycosylation contribute to its electrophoretic microheterogeneity, but the functional significance of different SHBG glycoforms is unknown. Carbohydrates may influence the biological activities and half-lives of glycoproteins and we have examined how oligosaccharides at specific sites influence the plasma clearance of hSHBG in vivo. To accomplish this, fully-glycosylated hSHBG, or hSHBG mutants lacking specific oligosaccharides chains, were expressed in Chinese hamster ovary (CHO) cells and purified by immunoaffinity chromatography. The purified recombinant proteins were then biotinylated to study their plasma half-lives after intravenous injection into rabbits. When compared to hSHBG isolated from serum, recombinant hSHBG migrates with a slightly larger average molecular size during denaturing polyacrylamide gel electrophoresis. This is due to a greater proportion (33–39% vs. 3%) of more highly branched N-linked oligosaccharides on the recombinant proteins. When injected into rabbits, the disappearance of recombinant hSHBG showed two exponential components, as previously shown for natural hSHBG in the same animal model. The mean±S.E.M. plasma half-lives of recombinant hSHBG (t_1/2 0.11±0.03 h and t_1/2β 18.94±1.65 h) are shorter than previously measured for natural hSHBG (t_1/2 3.43±0.72 h and t_1/2β 38.18±7.22 h) and this is likely due to differences in the composition of their N-linked oligosaccharides. An O-linked chain at Thr⁷ does not influence the plasma clearance of hSHBG in the presence or absence of N-linked carbohydrates at Asn³⁵¹ and Asn³⁶⁷. However, a 1.5–1.6 fold (p<0.03) increase in plasma half-life of variants lacking both N-glycosylation sites was observed and this is probably due to the fact these variants are not recognized by the asialoglycoprotein receptor-mediated clearance system. Removal of either N-glycosylation consensus site also increased (p<0.0001) the plasma half-life of hSHBG by 2.3–2.4 fold. Thus, the metabolic clearance of hSHBG appears to be determined by the number of N-linked oligosaccharides rather than their location.     

Lung specific stealth liposomes as antitubercular drug carriers in guinea pigs

The problem of patient non-compliance in the management of tuberculosis (TB) can be overcome by reducing the dosing frequency of antitubercular drugs (ATD) employing drug carriers. This study reports on the intravenous (iv) administration of lung specific stealth liposomes encapsulating ATD (rifampicin and isoniazid in combination) to guinea pigs and the detailed pharmacokinetic/chemotherapeutic studies. Following a single iv administration of liposomal drugs, the latter were found to exhibit sustained therapeutic levels in plasma for 96-168 hr with half-lives of 24-70 hr, mean residence time (MRT) of 35-81 hr and organ drug levels up to day 7. The relative bioavailability (as compared to oral free drugs) was increased by 5.4-8.9 folds, whereas the absolute bioavailability (as compared to iv free drugs) was increased by 2.9-4.2 folds. Weekly therapy with liposomal ATD for 6 weeks produced equivalent clearance of Mycobacterium tuberculosis from organs as did daily therapy with oral free drugs. Hence, intravenous liposomal ATD offer the therapeutic advantage of reducing the dosing frequency and improving the patient compliance in the management of TB.     

Insulin-degrading neutral proteinases of the plasma membrane of loach liver and embryo cells

Insulin-degrading, Ca2+-activated, neutral proteinases of molecular weight about 150 kDa and 70 kDa were purified from plasma membranes of the loach liver and embryo cells. It was shown that dithiothreitol and cysteine enhanced the enzyme activity, whereas p-chloromercuribenzoate and iodoacetic acid inhibited its level. Incubation of isolated plasma membranes with 5'[gamma 32P]ATP resulted in phosphorylation of these proteinases. The intensity of the process increased under the influence of insulin (100 microU/ml), that correlated with a decrease in the activity of proteinase with molecular weight of 150 kDa and an increase in 70 kDa enzyme activity. The data suggest the existence of common regulatory pathways of insulin degradation in plasma membranes of the loach liver and embryo cells.     

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. I. Clearance profiles

Marked differences were observed between the clearance profiles of immunoreactive plasma gonadotropins in gonadectomized and intact male bullfrogs (Rana catesbeiana). The disappearance patterns of endogenously secreted follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from plasma of intact animals following chronic (1-4 days) infusion with gonadotropin releasing hormone (GnRH) showed multiple components, but the initial few half-lives were relatively short (less than 1 h) and about 90% of both gonadotropins were cleared from the plasma within 6 h. Hypophysectomy had no effect on gonadotropin clearance rates following the termination of GnRH infusion. Clearance profiles of exogenous gonadotropins after chronic (6 h) infusion of bullfrog pituitary extract were similar to those observed after GnRH infusion. Gonadectomized frogs also cleared these infused pituitary gonadotropins at the same rate as intact animals, confirming that gonadectomy did not impair peripheral clearance mechanisms. Relatively rapid clearance rates were also observed for endogenous FSH and LH in normal untreated frogs. By comparison, the disappearance rates of FSH and LH from plasma of six long-term gonadectomized males following hypophysectomy were extremely slow: first half-lives for FSH and LH were 25.6 h and 17.2 h, respectively, and subsequent half-lives were even longer. Several weeks were required to clear fully the FSH and LH from the circulation in these males. Thus, a significant change in the physicochemical form of the circulating gonadotropins after gonadectomy in the male bullfrog is postulated; the corresponding changes in clearance rates were considerably greater than have been observed in any other species.     

PEG-albumin plasma expansion increases expression of MCP-1 evidencing increased circulatory wall shear stress: an experimental study

Treatment of blood loss with plasma expanders lowers blood viscosity, increasing cardiac output. However, increased flow velocity by conventional plasma expanders does not compensate for decreased viscosity in maintaining vessel wall shear stress (WSS), decreasing endothelial nitric oxide (NO) production. A new type of plasma expander using polyethylene glycol conjugate albumin (PEG-Alb) causes supra-perfusion when used in extreme hemodilution and is effective in treating hemorrhagic shock, although it is minimally viscogenic. An acute 40% hemodilution/exchange-transfusion protocol was used to compare 4% PEG-Alb to Ringer's lactate, Dextran 70 kDa and 6% Hetastarch (670 kDa) in unanesthetized CD-1 mice. Serum cytokine analysis showed that PEG-Alb elevates monocyte chemotactic protein-1 (MCP-1), a member of a small inducible gene family, as well as expression of MIP-1α, and MIP-2. MCP-1 is specific to increased WSS. Given the direct link between increased WSS and production of NO, the beneficial resuscitation effects due to PEG-Alb plasma expansion appear to be due to increased WSS through increased perfusion and blood flow rather than blood viscosity.     

Liver uptake and hepato-biliary transfer of galactosylated proteins in rats are determined by the extent of galactosylation

The effect of molecular mass and surface density of galactose residues on hepatic uptake and subsequent biliary excretion of galactosylated proteins was investigated in rats. Several proteins with different molecular weights (15-70 kDa) and different numbers of galactose units were synthesized and radiolabeled with 111In. Galactosylated proteins were administered i.v. to anaesthetized rats and samples of plasma and bile were collected for 3 h. Liver was harvested at the end of the experiments and the radioactivity of all samples was measured. Galactosylated proteins accumulated primarily in the liver and 2-10% of the administered dose appeared in the bile, mainly in undegraded form. The hepatic uptake clearance (Cl liver) and biliary excretion rate constant (kbile) of galactosylated proteins were calculated. No direct effect of molecular weight was observed, however, on increasing the galactose density, Cl liver increased from about 4 to 400 ml/h whereas kbile gradually decreased from about 0.057 to 0.007 (h-1). In conclusion, both hepatic uptake and biliary excretion of galactosylated proteins were found to be affected by the extent of galactosylation.     

In vitro and in vivo evaluation of hydrophilic dendronized linear polymers

Rigid-rod dendronized linear polymers consisting of a poly(4-hydroxystyrene) backbone and fourth-generation polyester dendrons were evaluated in vitro and in vivo to determine their suitability as drug delivery vectors. Cytotoxicity assays indicated that the polymers were well tolerated by cells in vitro. Biodistribution studies of the polymers in both nontumored and tumored mice revealed that as for random coil linear polymers, renal clearance was a function of polymer size, with significant urinary excretion observed for a 67 kDa dendronized polymer. High accumulation in organs of the reticuloendothelial system was exhibited by a dendronized polymer with a very high molecular weight (M(n) = 1740 kDa), but was not as significant for smaller polymers with M(n) = 67 kDa and M(n) = 251 kDa. The rank order for tumor accumulation of the polymers on a percent injected dose per gram tumor basis was 251 kDa approximately 1740 kDa > 67 kDa. These data will help guide the selection of highly functionalizable rigid-rod dendronized polymers with pharmacokinetic properties appropriate for use as drug carriers.     

Immunogenicity of recombinant IL-2 modified by covalent attachment of polyethylene glycol

Human rIL-2, expressed and purified from Escherichia coli, is currently being tested as an anticancer therapeutic agent. Some of the patients undergoing clinical trials with rIL-2 have developed antibodies to rIL-2. We describe a chemical modification of rIL-2 that reduces its immunogenicity. rIL-2 has been chemically modified with a water soluble polymer, monomethoxy polyethylene glycol (PEG). This covalent conjugate PEG-rIL-2 has enhanced solubility and extended in vivo circulation. Attachment of PEG to rIL-2 reduces its immunogenicity when tested in rabbits and in mice. Ag-specific IgG antibody titers were 100 to 1000-fold lower when PEG-rIL-2 was used as the Ag, compared to rIL-2. In a long term study, 7 of 10 rabbits injected with PEG-rIL-2 had no Ag-specific IgG antibody response. In these seven rabbits, the in vivo behavior of the injected PEG-rIL-2 remained essentially unchanged after repeated immunizations. PEG-rIL-2 injected before rIL-2 injections, immunosuppressed the antibody response to rIL-2 in rabbits. The maintenance of the systemic exposure of PEG-rIL-2 after repetitive dosing is related to its decreased immunogenicity. Thus, the PEGylation (covalent attachment of PEG) of rIL-2-enhances its potential as an anticancer therapeutic.     

Effects of charge on antibody tissue distribution and pharmacokinetics

Antibody pharmacokinetics and pharmacodynamics are often governed by biological processes such as binding to antigens and other cognate receptors. Emphasis must also be placed, however, on fundamental physicochemical properties that define antibodies as complex macromolecules, including shape, size, hydrophobicity, and charge. Electrostatic interactions between anionic cell membranes and the predominantly positive surface charge of most antibodies can influence blood concentration and tissue disposition kinetics in a manner that is independent of antigen recognition. In this context, the deliberate modification of antibodies by chemical means has been exploited as a valuable preclinical research tool to investigate the relationship between net molecular charge and biological disposition. Findings from these exploratory investigations may be summarized as follows: (I) shifts in isoelectric point of approximately one pI unit or more can produce measurable changes in tissue distribution and kinetics, (II) increases in net positive charge generally result in increased tissue retention and increased blood clearance, and (III) decreases in net positive charge generally result in decreased tissue retention and increased whole body clearance. Understanding electrostatic interactions between antibodies and biological matrices holds relevance in biotechnology, especially with regard to the development of immunoconjugates. The guiding principles and knowledge gained from preclinical evaluation of chemically modified antibodies will be discussed and placed in the context of therapeutic antibodies that are currently marketed or under development, with a particular emphasis on pharmacokinetic and disposition properties.     

Protein recovery from enzyme‐assisted aqueous extraction of soybean

Enzyme‐assisted aqueous oil extraction from soybean is a “green” alternative to hexane extraction that must realize potential revenues from a value‐added protein co‐product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m² hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran‐grafted agarose quaternary‐amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Size comparison between proteins PEGylated with branched and linear poly(ethylene glycol) molecules

Therapeutic proteins conjugated with branched poly(ethylene glycol) (PEG) have extended in vivo circulation half-lives compared to linear PEG-proteins, thought to be due partly to a greater hydrodynamic volume of branched PEG-proteins, which reduces the glomerular sieving coefficient. In this paper, viscosity radii of PEGylated alpha-lactalbumin (M(r) = 14.2 kDa) and bovine serum albumin (M(r) = 67 kDa) prepared with linear and branched PEGs (with nominal molecular weights 5, 10, 20 and 40 kDa) were compared experimentally using size exclusion chromatography (SEC). PEG adduct:protein molecular weight ratios of the PEGylated proteins covered the range 1:12 to 6:1. Direct comparisons of experimentally measured viscosity radii were found to be misleading due to differences between actual and nominal molecular weights of the PEG reagents used. Comparison with predicted viscosity radii shows that there is no significant difference between the viscosity radii of branched and linear PEG-proteins having the same total molecular weight of PEG adducts. Therefore, longer in vivo circulation half-lives of branched PEG-proteins compared to linear PEG-proteins are not explained by size difference. It is also calculated that the molecular size cut-off for glomerular filtration, 60 A for a 30 kDa PEG, matches the 30-50 A size range for the pores of the glomerular basement membrane. Finally, it is confirmed that prediction of PEG-protein viscosity radii should be based upon conservation of the total PEG adduct surface area to volume ratio for both linear and branched PEG-proteins regardless of PEGylation extent.     

Modeling of Microvascular Permeability Changes after Electroporation

Vascular endothelium selectively controls the transport of plasma contents across the blood vessel wall. The principal objective of our preliminary study was to quantify the electroporation-induced increase in permeability of blood vessel wall for macromolecules, which do not normally extravasate from blood into skin interstitium in homeostatic conditions. Our study combines mathematical modeling (by employing pharmacokinetic and finite element modeling approach) with in vivo measurements (by intravital fluorescence microscopy). Extravasation of fluorescently labeled dextran molecules of two different sizes (70 kDa and 2000 kDa) following the application of electroporation pulses was investigated in order to simulate extravasation of therapeutic macromolecules with molecular weights comparable to molecular weight of particles such as antibodies and plasmid DNA. The increase in blood vessel permeability due to electroporation and corresponding transvascular transport was quantified by calculating the apparent diffusion coefficients for skin microvessel wall (D [μm²/s]) for both molecular sizes. The calculated apparent diffusion coefficients were D = 0.0086 μm²/s and D = 0.0045 μm²/s for 70 kDa and 2000 kDa dextran molecules, respectively. The results of our preliminary study have important implications in development of realistic mathematical models for prediction of extravasation and delivery of large therapeutic molecules to target tissues by means of electroporation.     

Novel long-lasting interferon alpha derivatives designed by glycoengineering

Human alpha interferons (hIFN-alpha) comprise a family of closely related proteins that block viral infection, inhibit cell proliferation and modulate cell differentiation. Recombinant hIFN-alpha2 has proved useful for the treatment of a variety of human viral diseases and cancers. However, the clinical use of this cytokine has been restricted due to its short circulating half-life, which makes frequent dosing over an extended period necessary. To circumvent this problem, a glycoengineering strategy was carried out using site-directed mutagenesis. Fourteen mutants were constructed by the insertion of one N-glycosylation consensus sequence into different positions of the cytokine. Mutations were focused on amino acid positions that were believed not to be critical for the protein's structure or function. Taking into account the retained specific in vitro bioactivity and the higher carbohydrate content, five N-glycosylation positions were selected to be introduced into the molecule. Successive increases in molecular weight were observed after each addition of a functional consensus sequence, resulting in analogs with 4 and 5 N-linked carbohydrates (4N- and 5N-IFN) with increased size and charge, factors that reduce renal clearance of proteins. Pharmacokinetic experiments showed a similar behavior of 4N- and 5N-IFN variants, with a 25-fold increase in the elimination half-life and a 20-fold decrease in the systemic clearance rate compared with the non-glycosylated rhIFN-alpha2 following subcutaneous administration to rats. Besides, both distribution and elimination half-lives of the 4N analog were longer in comparison with the non-glycosylated cytokine, determining a 10-fold increase in the area under the curve after intravenous inoculation. Thus, herein we describe for the first time heavily glycosylated IFN analogs with a remarkable improvement in pharmacokinetic properties, which allow us to project drugs that combine less frequency of administration with enhanced therapeutic efficacy.     

Insulin in methionine and homocysteine kinetics in healthy humans: plasma vs. intracellular models

Methionine is a sulfur-containing amino acid that is reversibly converted into homocysteine. Homocysteine is an independent cardiovascular risk factor frequently associated with the insulin resistance syndrome. The effects of insulin on methionine and homocysteine kinetics in vivo are not known. Six middle-aged male volunteers were infused with L-[methyl-2H3,1-13C]methionine before (for 3 h) and after (for 3 additional hours) an euglycemic hyperinsulinemic (150 mU/l) clamp. Steady-state methionine and homocysteine kinetics were determined using either plasma (i.e., those of methionine) or intracellular (i.e., those of plasma homocysteine) enrichments. By use of plasma enrichments, insulin decreased methionine rate of appearance (Ra; both methyl- and carbon Ra) by 25% (P < 0.003 vs. basal) and methionine disposal into proteins by 50% (P < 0.0005), whereas it increased homocysteine clearance by approximately 70% (P < 0.025). With intracellular enrichments, insulin increased all kinetic rates, mainly because homocysteine enrichment decreased by approximately 40% (P < 0.001). In particular, transmethylation increased sixfold (P < 0.02), transsulfuration fourfold (P = 0.01), remethylation eightfold (P < 0.025), and clearance eightfold (P < 0.004). In summary, 1) physiological hyperinsulinemia stimulated homocysteine metabolic clearance irrespective of the model used; and 2) divergent changes in plasma methionine and homocysteine enrichments were observed after hyperinsulinemia, resulting in different changes in methionine and homocysteine kinetics. In conclusion, insulin increases homocysteine clearance in vivo and may thus prevent homocysteine accumulation in body fluids. Use of plasma homocysteine as a surrogate of intracellular methionine enrichment, after acute perturbations such as insulin infusion, needs to be critically reassessed.