Effect Of Benzyl Alcohol on Recombinant Human Interleukin-1 Receptor Antagonist Structure and Hydrogen–Deuterium Exchange

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen–deuterium exchange rates of individual rhIL-1ra residues. Additionof 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90–97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4215–4224, 2011     

Pharmacokinetics of Recombinant Human Interferon-βser in Healthy Volunteers and Its Effect on Serum Neopterin

The pharmacokinetics of and biologic response modification by recombinant human interferon-_ser (rIFN-_ser) were evaluated in 12 healthy male volunteers. Subjects received a single intravenous (iv) injection of 90 × 10⁶ IU of rIFN-_ser followed by a single or eight consecutive daily 90 × 10⁶ IU subcutaneous (sc) doses. Blood samples collected after the iv, first sc, and last sc doses and prior to each sc dose were assayed for interferon antiviral activity and the inter-feron-inducible marker neopterin. Following iv administration, serum interferon concentrations generally declined biexponentially, with a mean serum clearance of 0.76 ± 0.28 L/hr-kg, a mean steady-state volume of distribution of 2.88 ± 1.81 L/kg, and a mean terminal half-life of 4.29 ± 2.29 hr as determined by noncompartmental analysis. Following sc administration, absorption of rIFN-_ser was prolonged, with serum concentrations generally below 100 IU/mL. No accumulation of rIFN-_ser in serum was noted after eight daily sc injections. In contrast, serum neopterin levels did not increase above baseline levels until 12 hr after iv dosing and 24 hr after sc dosing. The mean increase in serum neopterin at 24 hr post iv injection was significantly greater than that at 24 hr post sc dosing.     

Genotoxicity Assessment of HM10620 Containing Recombinant Human Interferon-α

HM10620 is a recombinant human interferon-α (rhIFN-α) linked to immunoglobulin via N-terminal–specific non-peptidyl polyethylene glycol linker to improve the in vivo stability of interferon. Potential genotoxic effects of HM10620 in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro chromosomal aberration assay, and the in vivo micronucleus assay. HM10620 did not cause any mutation in the Ames assay tested using five tester strains at six concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay and the in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Chromosomal aberration was not induced at the concentrations of 10, 20, and 40 μg/mL. Also, there was no difference in the incidence of micronucleated polychromatic erythrocytes at doses of 10, 20, and 40 mg/kg in male mice compared with the vehicle control group. Therefore, based on the results obtained from the three studies, it is concluded that HM10620 is not a mutagenic agent in bacterial cells and causes no chromosomal damage in mammalian cells both in vitro and in vivo.     

Modification of the P-Glycoprotein Dependent Pharmacokinetics of Digoxin in Rats by Human Recombinant Interferon-α

Purpose This study was conducted to investigate in vivo the impact of interferon-alpha (IFN)-α on P-glycoprotein (P-gp) activity in rats by studying how its administration modifies the bioavailability of digoxin, a fairly pure P-gp substrate. Methods Human recombinant IFN-α was given to rats (n = 5–7 per group) daily for 8 days at different doses (IntronA^? 10⁶, 2.10⁶, or 4.10⁶ IU kg⁻¹, s.c.), whereas pegylated-IFN-α (ViraferonPeg^?, 29 μg kg⁻¹) was given s.c. three times a week. Rats were then given digoxin (32 μg kg⁻¹) i.v. or orally. The pharmacokinetics of digoxin was studied. Intestinal P-gp expression was also examined. Results The pharmacokinetics of i.v. administered digoxin was not modified by IFN-α, but a dose-dependent increase in areas under the curve (AUCs) was observed in the orally administered digoxin parameters in rats (AUCs: 392 ± 83 min μg L⁻¹, p < 0.01 and 550 ± 97 min μg L⁻¹, p < 0.001, respectively, vs. 286 ± 111 min μg L⁻¹ for control). A decrease in P-gp expression in the ileum (relative intensities: 0.70 ± 0.19 for 4 Million International Unit (MIU) kg⁻¹ IFN-α-treated animals vs. 1.00 ± 0.13 for controls, p < 0.05) and mainly in the jejunum (relative intensities: 0.46 ± 0.13 for 4 MIU kg⁻¹ IFN-α-treated animals vs. 1.00 ± 0.08 for controls, p < 0.001) was observed. Conclusion IFN-α induces in vivo a significant dose-dependent inhibitory effect on intestinal P-gp activity related to a local decrease in its expression, thereby predicting important clinical consequences when IFN-α and other P-gp substrates are associated.     

Stable Formulations of Recombinant Human Growth Hormone and Interferon-γ for Microencapsulation in Biodegradable Mircospheres

Purpose. The successful development of controlled release formulations for proteins requires that the protein not be denatured during the manufacturing process. The major objective was to develop formulations that stabilize two recombinant human proteins, human growth hormone (rhGH) and interferon- (rhIFN-), at high protein concentrations (>100 mg/mL) in organic solvents commonly used for microencapsulation, methylene chloride and ethyl acetate. Methods. Several excipients were screened to obtain the maximum solubility of each protein. These formulations (aqueous, lyophilized, milled, spray dried, or isoelectric precipitate) were then rapidly screened by emulsification in the organic solvent followed by recovery into excess buffer. Additional screening was performed with solid protein that was suspended in the organic solvent and then recovered with excess buffer. The recovery of native protein was determined by native size exclusion chromatography (SEC-HPLC) and circular dichroism (CD). The selected formulations were encapsulated in poly-lactic-coglycolic acid (PLGA) microspheres by either water-in-oil-in-water (W/O/W) or solid-in-oil-in-water (S/O/W) methods. The initial protein released from the microspheres incubated at physiological conditions was analyzed by SEC-HPLC, CD, and biological assays. Results. The stability of a given formulation in the rapid screening method correlated well with stability during encapsulation in PLGA microspheres. Formulations of rhGH containing Tween 20 or 80 resulted in lower recovery of native protein, while trehalose and mannitol formulations (phosphate buffer, pH 8.0) yielded complete recovery of native rhGH. Other additives such as carboxymethyl cellulose, gelatin, and dextran 70 were not effective stabilizers, and polyethylene glycol provided some stabilization of rhGH. Trehalose/rhGH (1:4 mass ratio) and mannitol/rhGH (1:2 mass ratio) formulations (potassium phosphate buffer, pH 8.0) were lyophilized, reconstituted to 200 and 400 mg/mL rhGH, respectively, and then encapsulated in PLGA micro-spheres. The protein was released from these microspheres in its native state. Lyophilized formulations of rhGH yielded analogous results indicating the ability of trehalose and mannitol to stabilize the protein. Small solid particles of rhGH generated by spray drying (both air and freeze-drying) formulations containing Tween 20 or PEG were stable in ethyl acetate, but not methylene chloride. Similar results were also obtained with rhIFN- (137 mg/mL in succinate buffer, pH 5.0), where both mannitol and trehalose were observed to stabilize the protein during exposure to the organic solvents resulting in the release of native rhIFN- from PLGA microspheres. Conclusions. The rapid screening method allowed the development of stable concentrated protein solutions or solid protein formulations that could be successfully encapsulated in PLGA microspheres. The excipients observed to stabilize these proteins function by preferential hydration of the protein, and in the dry state (e.g., trehalose) may stabilize the protein via water substitution yielding a protective coating around the protein surface. Studies of other proteins should provide further insight into this mechanism of protein stabilization during encapsulation.     

Effect of von Willebrand Factor on the Pharmacokinetics of Recombinant Human Platelet Glycoprotein Ibα-Immunoglobulin G1 Chimeric Proteins

Purpose Recombinant human platelet glycoprotein Ibα-immunoglobulin G1 chimeric proteins (GPIbα-Ig) have varying levels of anti-thrombotic activities based on their ability to compete for platelet mediated adhesion to von Willebrand Factor (vWF). Valine substituted GPIbα-Ig chimeras, at certain position, increase the binding affinity to vWF over its “wild-type” GPIbα-Ig analog. The purpose of this study was to determine the pharmacokinetics of two valine substituted GPIbα-Ig chimeras, GPIbα-Ig/1V (valine substitution at 239 position) and GPIbα-Ig/2V (double valine substitution at 233 and 239 position), in mice, rats and dogs.Methods Head-to-head comparisons of pharmacokinetics of GPIbα-Ig/1V and GPIbα-Ig/2V were investigated in rats and dogs after intravenous administration. Since vWF precipitates in the serum but not in plasma preparation, the concentration-time profiles of GPIbα-Ig/2V in rats were examined from the same blood samples for determination of matrix effect. The disposition of GPIbα-Ig/2V was also compared in vWF-deficient versus wild-type mice.Results For GPIbα-Ig/2V, the serum clearances were 2.62 ± 0.27 ml/hr/kg in rats and 1.97 ± 0.24 ml/hr/kg in dogs. The serum clearances of less potent GPIbα-Ig/1V were 1.08 ± 0.08 and 0.97 ± 0.19 ml/hr/kg in rats and dogs, respectively. In addition, the serum clearance of GPIbα-Ig/2V of 1.53 ml/hr/kg in vWF-deficient mice was lower than that in wild-type mice of 2.79 ml/hr/kg.Conclusion The difference in disposition for valine substituted forms of GPIbα-Ig in laboratory animals are likely affected by their enhanced binding affinity for circulating vWF.     

The Effect of Chronic Alcohol Ingestion on TGF-β1 and bFGF mRNA of Lung Tissues in Rats

目的:观察慢性饮酒后大鼠肺组织中转化生长因子-β1(TGF-β1)和碱性成纤维细胞生长因子(bFGF)mRNA的表达,探讨慢性饮酒对肺间质的影响.方法:健康雄性Sprague-Dawley大鼠20只随机分为无乙醇液体饲料的对照组和乙醇液体饲料的乙醇组,每组10只.喂养16周后观察所有大鼠肺泡内炎性细胞浸润程度并评分,Masson染色观察肺间质胶原沉积情况,电镜观察肺组织超微结构变化,实时荧光定量PCR检测肺组织TGF-β1和bFGF mRNA的表达量.结果:光镜下肺泡内炎性细胞浸润程度评分乙醇组高于对照组,差异有统计学意义(Z=50,P＜0.05).乙醇组电镜可见线粒体膜和嵴均有不同程度融合、模糊不清,细胞间隔均可见不同程度的胶原纤维沉积.乙醇组肺组织中TGF-β1和bFGF mRNA表达量高于对照组,差异有统计学意义(t分别为3.702和3.487,均P＜0.01).结论:慢性饮酒可增加大鼠肺组织中TGF-β1、bFGF mRNA表达量,使肺组织胶原沉积,引起肺泡炎症和肺间质疾病.     

Therapeutic Solutions of Human Albumin – The Possible Effect of Process-Induced Molecular Alterations on Clinical Efficacy and Safety

Human albumin solutions were developed as therapeutic during the Second World War to address blood loss due to battlefield injury. This indication was based on the recognition that albumin provided most of the oncotic capacity of human plasma. For the succeeding sixty years, this formed the basis for the use of albumin in traumatology and emergency medicine. In more recent times, the pharmacological properties arising from albumin's complex structure have become a focus of attention by clinical researchers. In particular, albumin, through anti-inflammatory and anti-oxidant properties, has been proposed as an agent for the treatment of sepsis, cirrhosis and other inflammatory states. Some evidence for these indications has accrued from a number of small clinical trials and observational studies. These studies have not been confirmed in other large trials. Together with other investigators, we have shown that the process of plasma fractionation results in alterations in the structure of albumin, including those parts of the molecule involved in anti-oxidant and anti-inflammatory effects. Albumin products from diverse manufacturers show heterogeneity in their ability to address these effects. In this article, we review the historical development of albumin solutions, pointing out the variations in fractionation chemistries which different manufacturers have adopted. We suggest ways by which the manufacturing processes have contributed to variations in the physico-chemical properties of molecule. We review the outcomes of clinical studies assessing the role of albumin in ameliorating conditions such as sepsis and cirrhosis, and we speculate as to the extent which heterogeneity in the products may have contributed to variable clinical outcomes. Finally, we argue for a change in the perception of the plasma product industry and its regulatory overseers. Historically, albumin has been viewed as a generic commodity, with different preparations being interchangeable in their clinical application. We suggest that this implied biosimilarity is not necessarily applicable for different albumin solutions. The use of albumin, in indications other than its historical role as a plasma expander, can only be validated by clinical investigation of each separate albumin product.     

Temperature- and pH-Induced Multiple Partially Unfolded States of Recombinant Human Interferon-α2a: Possible Implications in Protein Stability

PURPOSE: To study the effect of solution conditions on the structural conformation of recombinant human interferon-alpha2a (IFNalpha2a) to investigate its tendency to form partially unfolded intermediates. METHODS: The structural properties of IFNalpha2a were studied at various pH values (2.0-7.4) and temperatures (5 degrees C-80 degrees C) using Trp fluorescence emission, fluorescence quenching, near- and far-UV circular dichroism (CD) spectroscopy, and DSC. RESULTS: Fluorescence intensity measurements as a function of temperature indicated the onset of the thermal unfolding of IFNalpha2a, denoted by Td, around 60 degrees C above pH 4.0. Td was not observed at pH 3.5 and below. Acrylamide and iodide quenching studies indicated partial unfolding of protein with decrease in pH and with increase in temperature up to 50 degrees C. Near-UV CD studies indicated a significant loss in the tertiary structure of protein on increase in temperature from 15 degrees C to 50 degrees C at all solution pHs. DSC scans supported results obtained from fluorescence and CD studies at pH 4.0 and below. DSC, however, was insensitive to changes that occurred at moderate temperatures at pH 5.0 and 7.4. CONCLUSIONS: IFNalpha2a has a tendency to acquire multiple partially unfolded states with structural conformations sensitive to solution pH and temperature. These states were formed at moderate temperatures, and it is speculated that these partially unfolded states could play an important role in the aggregation of proteins during the long-term storage of aqueous protein formulations.     

Effect of Reactive‐Aldehydes on the Modification and Dysfunction of Human Serum Albumin

Advanced glycation end products (AGEs) are generated not only from glucose but also from several aldehydes such as methylglyoxal, glyoxal, and glycolaldehyde. The aim of the present study was to investigate the effect of several aldehydes on human serum albumin (HSA) in terms of the physicochemical properties and formation of AGE structures. HSA modified with methylglyoxal, generated by the glycolysis pathway and degradation of the Schiff base, showed the highest increase in the molecular weight and net negative charge, whereas glucose modification caused a small increase in the molecular weight even incubation for after 4 weeks. N^ε‐(carboxymethyl)lysine (CML), N^ε‐(carboxyethyl)lysine (CEL), and imidazolone increased in modified HSA in correlation with their lysine and arginine modification, whereas high amounts of GA‐pyridine was detected in HSA modified with glycolaldehyde. Furthermore, the binding ability of HSA to warfarin and ketoprofen was more effectively decreased by methylglyoxal modification than the other aldehydes. These results indicated that changes in the physicochemical properties and the formation of AGE structures are highly dependent on the aldehydes. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1614–1625, 2010     

The Effect and Mechanism of Oxysophoridine on Central Nervous System

Objectives To study the central pharmacological effects of oxysophoride and its basic mechanism.     

The Effect of Electric Properties of Liposomes on SOD-like Activity ofCopper Salicylate

The SOD-like activities of copper salicylate encapsulated in neutral positively and negativelycharged liposomes were studied with cytochrome c method. The results showed that the SOD-like activity of coppersalicylate encapsulated in neutral liposomes was enhanced over 3 times more than that of copper salicylate in aqueoussolution. However, the activity decreased for copper salicylate encapsulated in positively and negatively chargedliposomes. It was also found that at experimental concentrations there was no regularity for the change of activity ofcopper salicylate encapsulated in negatively charged liposomes, and copper salicylate encapsulated in liposomes withmore negative charges could promote the dismutation of superoxide anion free radicals, however, so could coppersalicylate encapsulated in liposomes with fewer negative charges only in lower concentrations.     

The Effect of Childhood Supervisory Neglect on Emerging Adults’ Drinking

This study investigated the effect of childhood supervisory neglect on emerging adults’ drinking. Child supervisory neglect is the most common form of child maltreatment in the United States, but few studies explore supervisory neglect separate from other forms of maltreatment among emerging adults, 18–25 years old. The study sample included (n = 11,117) emerging adults, 18–25 years old who participated in Waves I and III of the National Longitudinal Study of Adolescent Health (Add Health). We conducted separate analyses for male and female emerging adults, because they have different rates of alcohol consumption and alcohol risk behaviors. Our study used latent class analysis to understand how patterns of alcohol risk behaviors clustered together. For males, we found the following four classes: (1) multiple-risk drinkers, (2) moderate-risk drinkers, (3) binge-drinkers, and (4) low-risk drinkers or abstainers. For females, we found the following three classes: (1) multiple-risk drinkers, (2) moderate-risk drinkers, and (3) low-risk drinkers or abstainers. For both males and females, supervisory neglect increased the odds of membership in the multiple-risk drinkers’ class compared to the low-risk drinkers or abstainers’ class. Single males who did not live with their parents, and who were white had increased odds of being in the multiple-risk drinkers. For females, being more educated, or in a serious romantic relationship increased the odds of membership in the multiple-risk drinkers’ class. Practitioners should ask about histories of supervisory neglect among emerging adults who engage in alcohol risk behaviors.     

The Effects of Eupatilin (Stillen?) on Motility of Human Lower Gastrointestinal Tracts

Gastrointestinal motility consists of phasic slow-wave contractions and the migrating motor complex (MMC). Eupatilin (Stillen®) has been widely used to treat gastritis and peptic ulcers, and various cytokines and neuropeptides are thought to be involved, which can affect gastrointestinal motility. We performed a study to identify the effects of eupatilin on lower gastrointestinal motility with electromechanical recordings of smooth muscles in the human ileum and colon. Ileum and colon samples were obtained from patients undergoing bowel resection. The tissues were immediately stored in oxygenated Krebs-Ringer''s bicarbonate solution, and conventional microelectrode recordings from muscle cells and tension recordings from muscle strips and ileal or colonic segments were performed. Eupatilin was perfused into the tissue chamber, and changes in membrane potentials and contractions were measured. Hyperpolarization of resting membrane potential (RMP) was observed after administration of eupatilin. The amplitude, AUC, and frequency of tension recordings from circular and longitudinal smooth muscle strips and bowel segments of the ileum and colon were significantly decreased after admission of eupatilin. Eupatilin elicited dose-dependent decreases during segmental tension recordings. In conclusion, eupatilin (Stillen®) showed inhibitory effects on the human ileum and colon. We propose that this drug may be useful for treating diseases that increase bowel motility, but further studies are necessary.     

The Relaxing Effect of ��-Defensin 1 on the Adrenergic Responses of Rat Bladder

Defensins, cysteine-rich cationic polypeptides released from neutrophils, are known to have powerful antimicrobial properties. In this study, we sacrificed 30 rats to investigate the effects of α-defensin 1 on detrusor muscle contractions in isolated rat bladder. From the experiments we found relaxing effects of α-defensin 1 on the contractions induced by phenylephrine (PE) but not by bethanechol (BCh) in the detrusor smooth muscles. To determine the mechanisms of the effects of α-defensin 1, the changes of effects on PE-induced contraction by α-defensin 1 pretreatment were observed after pretreatment of Rho kinase inhibitor (Y-27632), protein kinase C (PKC) inhibitor (Calphostin C), potent activator of PKC (PDBu; phorbol 12,13-dibutyrate), and NF-κB inhibitors (PDTC; pyrrolidinedithiocarbamate and sulfasalazine). The contractile responses of PE (10^-9~10^-4 M) were significantly decreased in some concentrations of α-defensin 1 (5×10^-9 and 5×10^-8 M). When strips were pretreated with NF-κB inhibitors (PDTC and sulfasalazine; 10^-7~10^-6 M), the relaxing responses by α-defensin 1 pretreatment were disappeared. The present study demonstrated that α-defensin 1 has relaxing effects on the contractions of rat detrusor muscles, through NF-κB pathway. Further studies in vivo are required to clarify whether α-defensin 1 might be clinically related with bladder dysfunction by inflammation process.     

The Effects of Tumour Necrosis Factor α on Mediator Release from Human Lung

Summary: Although tamour necrosis factor α (TNFα) may be involved in the pathology of asthma, little is known about its role in mediator release from inflammatory cells in human lung. We investigated whether TNFα induced histamine release from mast cells in human chopped lung tissue and whether it modulated antigen-induced release of histamine and leukotrienes C₄/D₄/E₄ from passively sensitized lung tissue. Spontaneous histamine release in the presence of 1 nM TNFα for up to 4 h at 37°C was not significantly different from spontaneous histamine release alone (6.1±1.3% and 6.1±1.5% of total tissue histamine at 4 h respectively; n=3). Lung tissue was passively sensitized to the house dust mite Dermatophagoides pteronyssinus by incubating it in serum from an atopic volunteer donor for 3 h at 37°C. Treatment of the sensitized lung tissue with 1 nM TNFα for 60 min prior to challenge with a low concentration (1.8 AU) of D. pteronyssinus caused a significant increase in the amount of histamine release induced by the antigen from 0.2±0.6% to 1.9±1.0% of total tissue histamine (P=0.045, n=6). The release of leukotriene C₄/D₄/E₄ induced by the same concentration of antigen was not significantly changed by the TNFα treatment (39.5±9.1 and 55.6±17.7 pg/100 μl supernatant sample respectively; n=6). These results suggest that TNFα may be involved in potentiation of histamine release in allergic asthma, particularly in the presence of low antigen concentrations.     

Non-Polarized Secretion of Mouse Interferon-β from Gene-Transferred Human Intestinal Caco-2 Cells

Purpose. The intestinal epithelium is considered to be a feasible target for somatic gene therapy. To this end, Caco-2 cells derived from human colon carcinoma were transfected with a mouse interferon- (IFN-) expression vector and several stable sublines were established; this hetero-specific cytokine allows unexpected cellular effects to be avoided. Using the highest mouse IFN--producing sublines, the mode of IFN secretion was examined. Methods. The secretion polarity of mouse IFN- in its gene-transduced Caco-2 sublines was studied in a bicameral culture system in which the chambers were separated by microporous filters. Results. Mouse IFN- was secreted to the same extent from both apical and basolateral surfaces of the transduced cells regardless of cell aging. Conclusions. These results suggest that in the intestinal epithelium exogenous gene products such as IFNs can be delivered to both the luminal and blood sides in vivo. Thus, the intestinal epithelium may be suitable for systemic and local delivery of therapeutic proteins by gene transfer.     

Effect of ω-conotoxin GVIA on release of 3H-γ-aminobutyric acid from slices of rat neostriatum

Summary -Conotoxin GVIA (-CT) diminished the potassium-induced in vitro release of ³H--aminobutyric acid (³H-GABA) from slices of rat neostriatum in a manner which depended on the concentration of potassium. -CT (0.1 nmol/l) decreased the release of ³H-GABA induced by 25 mmol/l K⁺ from 11.6% to 6.1% of tissue content, ie. by 48%, while it did not affect the release of ³H-GABA caused by 20 mmol/l K⁺, which was 4.8% of tissue content. However, in the presence of a polyclonal antiserum or cysteamine (600 mol/l), both of which diminish the effects of endogenous somatostatin, 0.1–10 nmol/l -CT decreased the release of ³H-GABA induced by 20 mmoles/l K⁺ by 40%. It is concluded that -CT did not only inhibit GABA-neurones, but had an additional inhibitory effect on somatostatin neurones which are known to depress the release of ³H-GABA. It is further concluded that neuronal interactions, which are possible in brain slice preparations, may impede the interpretation of effects of drugs, especially if agents are used which affect basic mechanisms of transmitter release and thus the release of various transmitters from neurones. Send offprint requests to D. K. Meyer at the above address