The Effect of Benzyl Alcohol on Recombinant Human Interferon-γ

Purpose. The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN- and benzyl alcohol concentrations on the dynamics of the interaction in liquid formulations were also examined. Methods. The effect of benzyl alcohol on the secondary and tertiary structure of rhIFN- in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic light scattering was employed to detect aggregate formation due to the interaction of benzyl alcohol with rhIFN-. Results. The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN- formulations induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remained unaltered. There were gradual decreases in ellipticity with time throughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high molecular weight aggregates as measured by dynamic light scattering. Loss in near-UV ellipticity was accelerated at lower protein concentration and by increasing buffer or benzyl alcohol concentration. It was also faster in succinate than in acetate buffer. Formulation ionic strength did not affect the CD spectral changes in both the near- and far-UV regions. Conclusions. Interaction between benzyl alcohol and rhIFN- is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significant role in determining the extent of the interaction and consequently the stability of the product.     

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.     

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.     

Inhibitory Effect of Zinc on PEPT1-Mediated Transport of Glycylsarcosine and β-Lactam Antibiotics in Human Intestinal Cell Line Caco-2

Purpose. The aim of this study was to examine the effects of zinc on the intestinal peptide transporters (PEPT1 and basolateral peptide transporter) and to elucidate the mechanism of the interactions. Methods. Caco-2 cells were pretreated with zinc, and the uptake studies were carried out. Results. Zinc treatment resulted in the inhibition of [¹⁴C]glycylsarcosine (Gly-Sar) uptake via PEPT1 in a concentration-dependent manner, whereas it showed moderate inhibitory effect on the basolateral peptide transporter. Zinc also inhibited the uptake of oral -lactam antibiotics such as ceftibuten and cephradine by PEPT1. Kinetic analysis showed that zinc treatment increased K _m values without affecting V _max values of the [¹⁴C]Gly-Sar uptake. The inhibition of [¹⁴C]Gly-Sar uptake induced by zinc was observed in the presence of an H⁺ gradient but not in the absence of an H⁺ gradient. Conclusions. These results indicate that zinc is a competitive inhibitor of PEPT1. Zinc inhibited the PEPT1 function, possibly by interacting with histidine residues of PEPT1 that are part of an H⁺-binding site. These findings would provide important information for clinical, physiologic, and biochemical aspects of peptide transporters.     

Effects of Angiotensin II AT1–Receptor Blockade on High Fat Diet–Induced Vascular Oxidative Stress and Endothelial Dysfunction in Dahl Salt-Sensitive Rats

We examined the effects of angiotensin II AT₁–receptor blockade with olmesartan on high fat (HF) diet–induced vascular oxidative stress and endothelial dysfunction in normal salt (NS) diet–fed Dahl salt-sensitive (DSS) rats. Treatment with NS + HF diet (32% crude fat, 0.3% NaCl) for 20 weeks significantly increased blood pressure in DSS rats. NS + HF diet–fed DSS rats also showed higher plasma levels of thiobarbituric acid–reactive substances, aortic superoxide production, and mRNA levels of p22^phox and gp91^phox in aortic tissues than NS diet–fed DSS rats. Furthermore, acetylcholine-induced vasorelaxation of aorta from NS + HF diet–fed DSS rats was significantly reduced. In NS + HF diet–fed DSS rats, treatment with olmesartan medoxomil (10 mg/kg per day, p.o.) and hydralazine (25 mg/kg per day, p.o.) similarly decreased blood pressure. However, in these animals, only olmesartan normalized plasma levels of thiobarbituric acid–reactive substances, vascular superoxide in aortic tissues, and acetylcholine-induced vasorelaxation. These data indicate that HF diet–induced hypertension is associated with vascular oxidative stress and endothelial dysfunction in NS diet–treated DSS rats. Inhibition of angiotensin II AT₁ receptors may elicit beneficial effects on HF-induced hypertension and vascular injury in subjects that have genetically enhanced sodium-sensitive blood pressure.     

Structure–Activity Relationships (SAR) Research of Thiourea Derivatives as Dual Inhibitors Targeting both HIV-1 Capsid and Human Cyclophilin A

HIV-1 capsid (CA) and human cyclophilin A (CypA) play important roles in HIV-1 assembly and disassembly processes, which are critical in HIV-1 replication. Based on the discovery of thiourea derivatives targeting both of the two proteins and indicating effective inhibitory activities in our group, we designed and synthesized a new class of thiourea derivatives. Their abilities to bind to capsid and cyclophilin A were determined by ultraviolet spectroscopic analysis, fluorescence binding affinity, and PPIase inhibition assay. Furthermore, the newly synthesized compounds were tested for their antiviral activities and cytotoxicities using CEM cells. According to the biological evaluation and subsequent molecular docking analyses, we studied the structure–activity relationships of thiourea derivatives. Three optimal compounds (K17, K24, K25) based on the achieved structure–activity relationships would be the basis for future optimization.     

Solid-State Stability of Human Insulin I. Mechanism and the Effect of Water on the Kinetics of Degradation in Lyophiles from pH 2–5 Solutions

Purpose. Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range. Methods. Solutions of 1.0 mg/mL insulin were adjusted to pH 2–5 using HC1, freeze-dried, then exposed to various relative humidities at 35°C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance. Results. Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH (pH) yielding, at any given water content, solid-state pH-rate profiles parallel to the solution pH-rate profile. This pH dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of 4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3–5 and at 35°C involves rate-limiting intramolecular nucleophilic attack of the Asn_A21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., Phe_B1, and Gly_Al) of another insulin molecule to generate either deamidated insulin (Asp_A21) or an amide-linked covalent dimer (e.g., [Asp_A21-Phe_B1] or [Asp_A21-Gly_A1]), respectively. The rate of insulin degradation in lyophilized powders at 35°C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20–50%) required to induce a glass transition. Conclusions. The decomposition of human insulin in lyophilized powders between pH 3–5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.     

Effect of compression speeds on the compaction properties of a 1:1 paracetamol–microcrystalline cellulose mixture prepared by single compression and by combinations of pre-compression and main-compression

A 1:1 blend of paracetamol and microcrystalline cellulose was compacted at different compression speeds by single compression or combinations of pre- and main-compression. The tensile strengths of the tablets decreased from 0.74±0.01 to 0.44±0.05 MPa as the compression speed was increased from 78 to 390 mm/s when a single compression pressure of 80 MPa was used to compress the tablets. When combinations of pre- and main-compression of 320 and 240 MPa were used to compress the tablets, tensile strengths decreased from 3.12±0.67 MPa at a compression speed of 78 mm/s to 1.24±0.36 MPa when the compression speed was 390 mm/s. The energies of compression and the ratio of elastic to plastic energies increased with increase in compression speed. This was because the material was becoming more elastic and more energy was required for the elastic expansion leading to a reduction in the energy available for plastic deformation and bond formation which resulted in a decrease in tensile strengths. Pre-compression played a major role at high compression speeds. The tensile strengths of tablets (1.2±0.08 MPa) compressed with a pre-compression of 160 MPa followed by a main-compression of 80 MPa (compression speed of 390 mm/s) were similar to the tensile strengths of tablets (1.1±0.10 MPa) compressed using a single compression of 320 MPa at the same compression speed of 390 mm/s. Thus, combinations of lower pressures can be employed to compress the material to the same tensile strength as a high single compression.     

Concentration-Dependent Effect of Naringin on Intestinal Absorption of β<Subscript>1</Subscript>-Adrenoceptor Antagonist Talinolol Mediated by P-Glycoprotein and Organic Anion Transporting Polypeptide (Oatp)

Purpose  The purpose of this study is to clarify the impact of P-gp and Oatp on intestinal absorption of the β₁-adrenoceptor antagonist talinolol. Methods  P-gp-mediated transport was measured in LLC-PK1/MDR1 cells. Oatp-mediated uptake was evaluated with Xenopus oocytes expressing Oatp1a5. Rat intestinal permeability was measured by the in situ closed loop method. In vivo absorption was pharmacokinetically assessed by measuring plasma concentration after oral administration in rats. Results  In LLC-PK1/MDR1 cells, the permeability of talinolol was markedly higher in the secretory direction than in the absorptive one. The uptake of talinolol by Xenopus oocytes expressing Oatp1a5 was significantly increased compared with that by water-injected oocytes. Naringin inhibited talinolol uptake by Oatp1a5 (IC ₅₀ = 12.7 μM). The reported IC ₅₀ value of naringin for P-gp-mediated transport of talinolol is approximately 2,000 μM. Rat intestinal permeability of talinolol was significantly decreased in the presence of 200 μM naringin, but was significantly increased by 2,000 μM naringin. Similar results were obtained in in vivo absorption studies in rats. Conclusion  The absorption behavior of talinolol can be explained by the involvement of both P-gp and Oatp, based on characterization of talinolol transport by Oatp1a5 and P-gp, and the effects of naringin.     

Effects of Synthetic Sphingosine-1-Phosphate Analogs on Cytosolic Phospholipase A2α–Independent Release of Arachidonic Acid and Cell Toxicity in L929 Fibrosarcoma Cells: the Structure–Activity Relationship

Sphingolipid metabolites including ceramide, sphingosine, and their phosphorylated products [sphingosine-1-phosphate (S1P) and ceramide-1-phosphate] regulate cell functions including arachidonic acid (AA) metabolism and cell death. The development of analogs of S1P may be useful for regulating these mediator-induced cellular responses. We synthesized new analogs of S1P and examined their effects on the release of AA and cell death in L929 mouse fibrosarcoma cells. Among the analogs tested, several compounds including DMB-mC11S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3’-undecyl)phenylpropyl phosphate] and DMB-mC9S [dimethyl (2S,3R)-2-tert-butoxycarbonylamino-3-hydroxy-3-(3’-nonyl)phenylpropyl phosphate] released AA within 1 h and caused cell death 6 h after treatment. The release of AA was observed in C12 cells [a L929 variant lacking a type α cytosolic phospholipase A₂ (cPLA₂α)] and L929-cPLAα–siRNA cells (L929 cells treated with small interference RNA for cPLA₂α). Treatment with pharmacological inhibitors of secretory and Ca²⁺-independent PLA₂s decreased the DMB-mC11S–induced release of AA. The effect of the S1P analogs tested on the release of AA was comparable to that on cell death in L929 cells, and a high correlation coefficient was observed. Two analogs lacking a butoxycarbonyl moiety [DMAc-mC11S (dimethyl (2S,3R)-2-acetamino-3-hydroxy-3-(3’-undecyl)phenylpropyl phosphate] and DMAm-mC11S [dimethyl (2S,3R)-2-amino-3-hydroxy-3-(3’-undecyl)phenylpropyl phosphate)] had inhibitory effects on the release of AA and cell toxicity induced by DMB-mC11S. Synthetic phosphorylated lipid analogs may be useful for studying PLA₂ activity and its toxicity in cells.[Supplementary Fig. 1: available only at http://dx.doi.org/10.1254/jphs.08284FP]     

Effect of the endogenous κ opioid agonist dynorphin A(1–17) on cocaine-evoked increases in striatal dopamine levels and cocaine-induced place preference in C57BL/6J mice

Rationale Effects of synthetic kappa opioid receptor agonists on cocaine-induced reward have been studied extensively in rats but relatively few studies have used the endogenous kappa agonist dynorphin A(1–17).Objectives Three studies were conducted to examine the effect of the natural sequence dynorphin on cocaine-induced increases in dopamine, on the formation of conditioned place preference and on increases in locomotor activity in C57BL/6 J mice.Methods After implantation of guide cannulae into the caudate putamen, mice were allowed 4–5 days to recover from surgery. In the first study, dynorphin A (0, 1, 2, 4.4 nmol) was infused into the caudate putamen and dopamine levels were measured by in vivo microdialysis in that brain region. Then, the effect of dynorphin A (4.4 nmol) on increases in dopamine levels induced by 15 mg/kg cocaine i.p. was also measured with in vivo microdialysis. The third experiment examined the effect of dynorphin A (4.4 nmol) on conditioned place preference and locomotion induced by 15 mg/kg cocaine.Results Dynorphin A significantly decreased basal dopamine levels in a dose-dependent manner by more than 60% at the highest dose, and this effect was completely blocked by pre-injection of the kappa-opioid receptor antagonist nor-BNI (10 mg/kg). The highest dose of dynorphin (4.4 nmol) blocked increases in dopamine levels, the formation of conditioned place preference and attenuated locomotion induced by 15 mg/kg cocaine.Conclusion The blockade of the cocaine-induced rise in striatal dopamine may contribute to both dynorphins ability to prevent the development of cocaine-induced conditioned place preference and to attenuate the increase in locomotor activity.