Physicochemical and Biological Examination of Two Glatiramer Acetate Products.

Herein we compared 40 mg/mL lots of the active ingredient, glatiramer acetate, manufactured by Mylan/Natco to the active ingredient, glatiramer acetate in Copaxone (Teva Pharmaceuticals, Ltd., Netanya Israel) using physicochemical (PCC) methods and biological assays. No differences were seen between the Mylan/Natco and Teva lots with some low resolution release PCC assays (amino acid analysis, molecular weight distribution, interaction with Coomassie Brilliant Blue G-250). Changes in polydispersity between Mylan/Natco and Copaxone lots were found using size exclusion chromatography and the high resolution PCC method, known as Viscotek, and suggestive of a disparity in the homogeneity of mixture, with a shift towards high molecular weight polypeptides. Using RPLC-2D MALLS, 5 out of 8 Mylan/Natco lots fell outside the Copaxone range, containing a high molecular weight and high hydrophobicity subpopulation of polypeptides not found in Copaxone lots. Cation exchange chromatography showed differences in the surface charge distribution between the Copaxone and Mylan/Natco lots. The Mylan/Natco lots were found to be within Copaxone specifications for the EAE model, monoclonal and polyclonal binding assays and the in vitro cytotoxicity assay, however higher IL-2 secretion was shown for three Mylan/Natco lots in a potency assay. These observations provide data to inform the ongoing scientific discussion about the comparability of glatiramer acetate in Copaxone and follow-on products.

Modafinil and the risk of cardiovascular events: Findings from three US claims databases.

PURPOSE: This study examined the potential risk of cardiovascular (CV) events associated with modafinil and the consistency of the risk estimates across databases. METHODS: A retrospective, inception cohort design of patients who initiated treatment with modafinil between 2006 and 2008 was used in three US health care claims databases. Modafinil users were matched with nonusers. Patients were further divided into two cohorts of obstructive sleep apnea (OSA) and non-OSA (NOSA) cohorts. Endpoints of interest, including myocardial infarction (MI), stroke, CV hospitalizations, and all-cause death, were assessed using incidence rates and Cox proportional hazard ratios (HRs), adjusted for potential confounding factors. RESULTS: The cohorts included a total of 175 524 patients in MarketScan CM; 77 266-in IMS LifeLink; and 8174-in MarketScan Medicaid. No increased risk for MI in the OSA and NOSA cohorts was observed across all three databases. The risks of CV hospitalization in the OSA and NOSA cohorts were not different between the modafinil users and nonusers, except for IMS LifeLink database where the HR was lower than one in the modafinil users compared with the nonusers (HR, 0.69; 95% confidence interval [CI], 0.54 to 0.87). For OSA patients with prior stroke, an adjusted HR of 1.96 (95% CI, 1.02 to 3.76) was observed for stroke among modafinil users compared with nonusers. Among the NOSA, the HRs for all-cause death in the OSA were inconsistent across databases. CONCLUSIONS: Except for few CV outcomes, applying one common protocol generated consistent risk estimates of CV events following modafinil use across cohorts and databases.

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model.

Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.

Lipid peroxidation in the presence of albumin, inhibitory and prooxidative effects.

Oxidative modifications of LDL are involved in atherogenesis. Previously we have developed a simple assay to evaluate the susceptibility of lipids to copper-induced peroxidation in the relatively natural milieu of unfractionated serum in the presence of excess citrate. Based on our previous results we have proposed that the inducer of peroxidation in our optimized assay is a copper-citrate complex. Recent investigations indicate that under certain conditions a copper-albumin complex may induce peroxidation of ascorbate. Two different complexes may be formed in albumin-containing systems (e.g. serum) namely 1:1 and 2:1 copper-albumin complexes. The aim of the present work was to evaluate the possibility that at least one of these complexes may be responsible for the induction of peroxidation of lipids in lipidic systems containing copper and albumin, including our optimized assay. Towards this end, we have investigated the dependence of copper-induced peroxidation on the concentration of added albumin in lipidic systems in the absence and presence of citrate. In all the systems investigated in this study (PLPC liposomes, LDL, HDL and mixtures of HDL and LDL) we found that at low concentrations of free copper (e.g. in the presence of excess citrate) the 2:1 copper-albumin complex is redox-active and that this complex is the major contributor to the initiation of lipid peroxidation in these systems and in our optimized assay. The possible relevance of the induction of peroxidation in vivo by the latter complex has yet to be studied.

Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants.

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.

Copper-induced peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants and its dependence on the oxidative stress.

In an attempt to deepen our understanding of the mechanisms responsible for lipoprotein peroxidation, we have studied the kinetics of copper-induced peroxidation of the polyunsaturated fatty acid residues in model membranes (small, unilamellar liposomes) composed of palmitoyllinoleoylphosphatidylcholine (PLPC). Liposomes were prepared by sonication and exposed to CuCl(2) in the absence or presence of naturally occurring reductants (ascorbic acid (AA) and/or alpha-tocopherol (Toc)) and/or a Cu(I) chelator (bathocuproinedisulfonic acid (BC) or neocuproine (NC)). The resultant oxidation process was monitored by recording the time-dependence of the absorbance at several wavelengths. The observed results reveal that copper-induced peroxidation of PLPC is very slow even at relatively high copper concentrations, but occurs rapidly in the presence of ascorbate, even at sub-micromolar copper concentrations. When added from an ethanolic solution, tocopherol had similar pro-oxidative effects, whereas when introduced into the liposomes by co-sonication tocopherol exhibited a marked antioxidative effect. Under the latter conditions, ascorbate inhibited peroxidation of the tocopherol-containing bilayers possibly by regeneration of tocopherol. Similarly, both ascorbate and tocopherol exhibit antioxidative potency when the PLPC liposomes are exposed to the high oxidative stress imposed by chelated copper, which is more redox-active than free copper. The biological significance of these results has yet to be evaluated.