Comparison of Copaxone® and Synthon's therapeutically equivalent glatiramer acetate.

Glatiramer acetate is indicated for the treatment of patients with relapsing forms of multiple sclerosis (RMS). In 2016, an alternative to the originator product was approved in the EU through the hybrid procedure regulatory pathway. This paper reviews the scientifically rigorous and multifaceted program undertaken to demonstrate the equivalence of this glatiramer acetate follow-on product (GTR) and the reference product Copaxone®, which resulted in the EU approval of GTR 20 mg/mL and 40 mg/mL. Establishing therapeutic equivalence for non-biological complex drugs is not trivial and requires a complex and multidisciplinary effort. Ultimately, there is not a single test or study that establishes therapeutic equivalence of two heterogeneous products. Instead, it requires a good understanding of the synthesis process together with a full set of data that includes comparative physicochemical testing, nonclinical in vitro and in vivo studies, and a comparative clinical study to allow for a valid conclusion that two products are therapeutically equivalent. The detailed understanding of glatiramer's synthesis process and its impact on the characteristics of glatiramer, combined with the results of a scientifically rigorous and multifaceted physicochemical and biological characterization program, and the clinical data from the 794-patient Phase III GATE study, demonstrate that GTR and Copaxone are therapeutically equivalent. The data further demonstrate that Synthon's manufacturing process consistently yields drug substance of the same quality as Copaxone and that switching from Copaxone to GTR is safe and well-tolerated.

Tissue- and time-dependent estrogen receptor activation in estrogen reporter mice.

With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.

Identification of beta-endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio).

Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.

alpha-MSH acetylation in the pituitary gland of the sea bream (Sparus aurata L.) in response to different backgrounds, confinement and air exposure.

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.

The stress response of the gilthead sea bream (Sparus aurata L.) to air exposure and confinement.

We investigated short-term effects (up to 24 h) of air exposure and confinement, and long-term effects (up to 11 days) of confinement, to elucidate signalling pathways in the stress response of gilthead sea bream Sparus aurata L. Plasma glucose and lactate were taken as indicators of sympathetic activation, and alpha-melanocyte stimulating hormone (alpha-MSH), adrenocorticotrophic hormone (ACTH) and cortisol as indicators of activation of the brain-pituitary-interrenal (BPI) axis. Air exposure for 3 min resulted, within 30 min, in an increase in plasma concentrations of cortisol, alpha-MSH, glucose, lactate, osmolality and plasma Na, Cl and Mg. Plasma ACTH and beta-endorphin and plasma K, Ca and P did not change. We conclude that air exposure mainly activates the brain-sympathetic-chromaffin cell (BSC) axis. In fish confined at a density of 70 kg/m(3) (compared with 4 kg/m(3) in controls), cortisol, ACTH and alpha-MSH increased within 1 h, indicating activation of the BPI axis. Plasma glucose, Na, Cl and Mg increased with an 8 h delay compared with the response to air exposure. No changes in plasma lactate, osmolality, K, Ca and P were observed. Long-term confinement induced a biphasic cortisol response with peaks at 1 h and at 2 and 3 days. A gradual increase in plasma beta-endorphin concentrations peaked at 7 days; the concentration of alpha-MSH increased rapidly within 1 h and then declined to control values 4 days after the onset of confinement. No changes in ACTH were detected. Our data provide evidence that a stressor-specific activation of the BSC and BPI axes may occur in Sparus aurata.

Cloning and expression of two proopiomelanocortin mRNAs in the common carp (Cyprinus carpio L.).

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.

Differential expression of two pro-opiomelanocortin mRNAs during temperature stress in common carp (Cyprinus carpio L.).

Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and beta-endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laser-induced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be strain-dependent and influenced by temperature. In strain E20xR8, the ratio had altered in favour of POMC-I from 1:3.2 (POMC-I:POMC-II) in fish adapted to 24 degreesC to 1:1.2 in fish adapted to a decrease of 9 degreesC in ambient temperature. A rapid drop in temperature from 24 to 15 degreesC decreased the POMC mRNA ratio at the expense of POMC-I from 1:1.9 in the control fish (strain E4xR3R8) to 1:4.2 3 h after the temperature drop of 9 degreesC. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.