Assessment of analytical techniques for characterization of crystalline clopidogrel forms in patent applications

The aim of this study was to evaluate two important aspects of patent applications of crystalline forms of drugs: (i) the physicochemical characterization of the crystalline forms; and (ii) the procedure for preparing crystals of the blockbuster drug clopidogrel. To this end, searches were conducted using online patent databases. The results showed that: (i) the majority of patent applications for clopidogrel crystalline forms failed to comply with proposed Brazilian Patent Office guidelines. This was primarily due to insufficient number of analytical techniques evaluating the crystalline phase. In addition, some patent applications lacked assessment of chemical/crystallography purity; (ii) use of more than two analytical techniques is important; and (iii) the crystallization procedure for clopidogrel bisulfate form II were irreproducible based on the procedure given in the patent application.

Este trabalho tem como objetivo avaliar dois aspectos importantes em um pedido de patente de formas cristalinas de fármacos: (i) caracterização físico-química das formas cristalinas e (ii)o procedimento de preparo da forma II do fármaco clopidogrel, um blockbuster de vendas. Realizaram-se buscas em bancos de dados patentários on line. Os resultados mostraram que (i) a maioria dos pedidos de patente de formas cristalinas do clopidogrel não se adequam com proposta do INPI devido ao número insuficiente de técnicas analíticas utilizadas na caracterização da fase cristalina. Ainda, em alguns pedidos de patente não há a presença da avaliação da pureza química/cristalográfica; (ii) a importância de se utilizar mais de duas técnicas de avaliação e (iii) que não foi possível a reprodução da cristalização com o procedimento apresentado no pedido de patente.

Proteomics analysis of water insoluble-urea soluble crystallins from normal and dexamethasone exposed lens.

PURPOSE: The aim of this study was to identify glucocorticoid induced cataracts (GIC)-specific modified water insoluble-urea soluble (WI-US) crystallins and related changes after rat lens were exposed to dexamethasone (Dex). METHODS: We separated WI-US lens proteins by two-dimensional electrophoresis (2-DE). The crystallins were then analyzed with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Protein levels and morphological changes of αA- and αB-crystallins were also determined. Electronic microscope of lens and native-page analysis of crystallins were further determined. RESULTS: Measured masses, isoelectric points (pIs), and amino acid sequences of all detected crystallins matched previously-reported data. Analysis by 2-DE indicated that αA- and αB-crystallin increased when the lens was viewed under 1 µM and 10 µM Dex, which was identical with the results of western-blot, immuno histochemistry or fluorescence; ßB2- and ßA3-crystallin increased when lens was viewed under 1 µM Dex and 100 µM Dex. ßA1-, ßA4-, and ßB1-crystallins decreased under 0.1-100 µM Dex. Electronic microscope figures showed the condition of the lens center gradually worsened and cracked between fiber cells that became larger under 1-100 µM Dex. Moreover, αA-crystallins were associated with increased phosphorylation (PI decreased).The newly protein spots: ßA2-, ßA3-, ßB1-, and γs-crystallin appeared under 0.1-100 µM Dex. Native-page showed α-crystallin increased when the lens was exposed to 1 µM Dex; however, ß-crystallin did not decrease under 0.1-100 µM Dex. The percentage of α-crystallin gradually decreased, however ß-crystallin gradually increased, perhaps because the emergence of newly appeared ß-crystallin under Dex. CONCLUSIONS: Our results showed multiple WI-US crystallins may be more vulnerable to glucocorticoid stress because of diminished important roles, which will in turn provide a mechanism for GIC from a proteomics perspective.

Crystallins of the beta/gamma-superfamily mimic the effects of lens injury and promote axon regeneration.

Adult retinal ganglion cells (RGCs) can survive axotomy and regrow lengthy axons when exposed to lens injury (LI). The neuroprotective and axon-growth-promoting effects of LI have been attributed to an infiltration of activated macrophages into the inner eye and recently also to astrocyte-derived CNTF. The present work reveals that certain purified lens proteins (crystallins) cause the effects of LI. Intravitreal injections of beta- or gamma-crystallins, but not of alpha-crystallin, strongly enhanced axon regeneration from retinal explants in culture, within peripheral nerve grafts or the crushed optic nerve. Deposition of the effective crystallins within the vitreous body was also associated with an influx of circulating macrophages and an activation of retinal astrocytes, Müller cells, and resident microglia. Furthermore beta-crystallin induced CNTF expression in retinal astrocytes and activation of CNTF's major downstream signaling pathway (JAK/STAT3) when intravitreally injected or added to the culture medium ex vivo. Consistently, in culture the addition of beta- and gamma-crystallins to the medium also increased axon regeneration from retinal explants. These results demonstrate that crystallins of the beta/gamma-superfamily are the lens-derived activators of cascades, which lead to axonal regeneration and suggest that their effects might be mediated by astrocyte-derived CNTF.

Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin.

We have previously identified a gelsolin-like protein (C/L-gelsolin) as a corneal crystallin in zebrafish. Here we show by phylogenetic analysis that there are at least six genes encoding gelsolin-like proteins based on their gelsolin domains in zebrafish: gsna and gsnb group with the vertebrate gelsolin gene, scina and scinb group with the scinderin (adseverin) gene, and scinla (C/L-gelsolin) and scinlb are novel scinderin-like genes. RT-PCR showed that scinla, scinlb, and gsnb are preferentially expressed in the adult cornea whereas gsna is expressed to a similar extent in cornea, lens, brain, and heart; scina and scinb expression were detectable only in whole zebrafish and not in these adult tissues. Quantitative RT-PCR and 2-dimensional polyacrylamide gel electrophoresis followed by MALDI/TOF mass spectroscopy confirmed high expression of beta-actin and scinla, moderate expression of scinlb, and very low expression of gsna and gsnb in the cornea. Finally, transgenic zebrafish carrying a green fluorescent protein reporter transgene driven by a 4 kb scinla promoter fragment showed expression in the cornea, snout, dorsal fin, and tail fin of 3-day-old zebrafish larvae. Our data suggest that scinla and scinlb are diverged paralogs of the vertebrate scinderin gene and show that scinla encodes the zebrafish corneal crystallin previously called C/L-gelsolin.

Modulation of alpha and beta crystallin expression in rat retinas with ocular hypertension-induced ganglion cell degeneration.

The expression of alpha (alphaA and alphaB) and beta (betaA1/A3, betaA2, betaA4, and betaB2) crystallin genes were analyzed at the mRNA and protein levels in rat retinas with ocular hypertension-induced ganglion cell death. An animal model with progressive loss of retinal ganglion cells (RGC) was generated by elevation of intraocular pressure (IOP). The estimated RGC loss was approximately 8% and 20% at 2 and 5 weeks post IOP elevation, respectively. mRNA and protein quantification showed that alpha and beta crystallin genes were downregulated at both transcriptional (alphaA, alphaB, betaA1/A3, betaA4, and betaB2 approximately 50% and betaA2~40%) and protein (alphaA~50%, alphaB~63%, betaA1/A3~70%, and betaB2~38%) levels 2 weeks after IOP elevation. In experimental retinas 5 weeks after IOP elevation, the levels of crystallin mRNAs were higher than at 2 weeks and were comparable to that of control retinas. However, the levels of the corresponding proteins were still lower (alphaA, alphaB, and betaB2 approximately 37% and betaA1/A3~70%) than in control retinas. Furthermore, we found that the expression of these genes in the retina is predominantly localized to the cells in the GCL and to a lesser degree in the INL and ONL. Colocalization of the crystallin-positive and Fluorogold retrogradely labeled cells indicated that the cells expressing alpha and beta crystallins in the GCL are RGCs. In summary, we showed that alpha and beta crystallins are expressed in the retina predominantly by RGCs and that their expression is affected by ocular hypertension.

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates.

The beta and gamma crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of gamma-crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of gammaN-crystallin, an evolutionary bridge between the beta and gamma families. In all species examined, gammaN-crystallins have a hybrid gene structure, half beta and half gamma, and thus appear to be the 'missing link' between the beta and gamma crystallin lineages. Overall, there are four major classes of gamma-crystallin: the terrestrial group (including mammalian gammaA-F); the aquatic group (the fish gammaM-crystallins); the gammaS group; and the novel gammaN group. Like the evolutionarily ancient beta-crystallins (but unlike the terrestrial gammaA-F and aquatic gammaM groups), both the gammaS and gammaN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, gammaN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the gamma-crystallins, also in the retina. Although well conserved throughout vertebrate evolution, gammaN in primates has apparently undergone major changes and possible loss of functional expression.

Characterization of gammaS-crystallin isoforms from a catfish: evolutionary comparison of various gamma-, gammaS-, and beta-crystallins.

gammaS-Crystallin from catfish eye lenses, formerly designated betas-crystallin in mammalian lenses, is structurally characterized in this study by cDNA cloning and sequencing. To facilitate sequence characterization of gammaS-crystallin with structural properties lying between beta- and gamma-crystallins, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from catfish eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain nucleotide segments encoding multiple gammaS-crystallin isoforms. Sequencing several positive clones revealed that at least two distinct isoforms exist in the gammaS-crystallin class of this teleostean fish, similar to the authentic gamma-crystallin family characterized previously in species of the piscine class. Comparison of protein sequences encoded by two representative catfish gammaS1 and gammaS2 cDNAs with the published sequences of beta-, gamma-, and gammaS-crystallins from shark, carp, bullfrog, bovine, and human lenses indicates that there is about 20-50% sequence homology between catfish gammaS-crystallins and various members of the related beta/gamma-crystallin superfamily from different evolutionary classes, with a higher sequence similarity being found between catfish gammaS- and mammalian gamma-crystallins than between catfish gammaS- and bovine or carp gammaS-crystallins. Phylogenetic trees constructed on the basis of the nucleotide and protein sequence divergence among various beta-, gamma-, and gammaS-crystallins corroborate the closer relatedness of catfish gammaS- to authentic gamma-crystallin than to bovine and carp gammaS-crystallins. The results suggest that evolution of catfish gammaS-crystallins follows a different path from that of bovine and carp gammaS-crystallins and may represent a more ancient offshoot from the ancestral gamma/gammaS coding gene than carp and bovine gammaS-crystallins.

Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice.

gamma-Crystallins are major structural components of the lens fiber cells in amphibians and mammals. Many dominant inherited cataracts in humans and mice have been shown to map within the gamma-crystallin gene cluster. Several transcription factors, including PAX6 and SOX proteins, have been suggested as candidates for crystallin gene regulation. Here we show that the targeted deletion of Sox1 in mice causes microphthalmia and cataract. Mutant lens fiber cells fail to elongate, probably as a result of an almost complete absence of gamma-crystallins. It appears that the direct interaction of the SOX1 protein with a promoter element conserved in all gamma-crystallin genes is responsible for their expression.

Rho B-crystallin, an aldose reductase-like lens protein in the gecko Lepidodactylus lugubris.

The ocular lenses of the diurno-nocturnal gecko Lepidodactylus lugubris contain a monomeric 38-kDa protein at a level of 20 to 22% of the total water-soluble protein. Amino acid sequences of peptides from this protein are most similar--up to 72% identity--to mammalian aldose reductase, an NADPH-dependent reductase which normally occurs at house-keeping levels in the eye lens, and which is involved in the development of diabetic cataract. The sequences show 56% identity with rho-crystallin from lenses of the frog genus Rana. It is concluded that different genes from the same superfamily of NADPH-dependent reductases have been recruited to become highly expressed as lens proteins in at least two different evolutionary lineages. To reflect the relationship with frog rho-crystallin, the gecko lens protein is designated as rho B-crystallin. As for frog rho-crystallin, no enzymatic activity could be established for rho B-crystallin in the gecko lens. Up to now, rho B-crystallin has not been detected in lenses of other reptiles or amphibians.

[Study on soluble proteins in human fetal lens].

Soluble proteins from corter of human fetal clear lenses were separated by column chromatography with Sepharyl S-300 (superfine) and determined by disc-PAGE and SDS-PAGE. The results documented from 18W, 21W (2 cases) and 24W fetal lenses suggested that a negative correlation tended to be presented between the age of fetus and fraction of alpha, beta 1 crystallins (r = -0.9542; r = -0.9674) and a positive correlation between the age of fetus and fraction of beta 2, gamma crystallins (r = 0.9666; r = 0.9970). There was no change in the alpha, beta 1, beta 2 crystallins on disc-PAGE. Among the fetuses of different ages, the blurred band of gamma crystallin seen in 18W and 21W fetal lenses was thickened in the 24W fetal lens. The SDS-PAGE showed that there was no significant difference among the polypeptides in each of alpha, beta 1, beta 2, gamma crystallins in the studied fetal lenses of different age groups. It is presumed that the normal lens is proportionally composed of four fractions of crystallin. This is hypothetically contributed to the maintenance of normal structure of the lens, why the proper proportion of four fractions of crystallins is depended either on proteins synthesis or on protein molecular assembly is not yet clear.

Drosocrystallin, a major 52 kDa glycoprotein of the Drosophila melanogaster corneal lens. Purification, biochemical characterization, and subcellular localization.

We have identified a 52 kDa protein, which is a potent substrate for cholera toxin-dependent ADP-ribosylation, in the compound eye preparation of the fruit fly, Drosophila melanogaster. We find that the 52 kDa protein is a glycoprotein and a Ca2+ binder bearing a high content of leucine, serine and glycine. By microsequencing we determined its 13 N-terminal sequence, AYL*PIDLNQLAK, with the asterisk representing an ambiguous signal. In order to study further the 52 kDa protein we have raised a polyclonal antibody against a synthetic oligopeptide representing the N-terminal 13 residues of the 52 kDa protein. By immunogold labelling with the antibody, the epitopes were localized at the EM level to the laminated corneal lens. The number of the gold particles per microns2 in the electron-dense layer of the corneal lens was 2.5 times higher than that of the electron-lucent layer. The pattern of the 52 kDa protein distribution in the corneal lens suggests that the 52 kDa protein is the major protein component that participates in the pattern formation of the alternate refractive indices of the D. melanogaster corneal lens. An X-ray dispersion analysis in situ revealed that the laminated corneal lens contained a higher concentration of Ca2+, supporting the hypothesis that the 52 kDa protein binds Ca2+ in vivo. To the best of our knowledge, this is the first report that identifies the protein entity of an arthropod corneal lens. We propose to designate this 52 kDa protein drosocrystallin.

Isolation and characterization of cDNAs encoding beta A2- and beta A4-crystallins: heterologous interactions in the predicted beta A4-beta B2 heterodimer.

Except for the two acidic chains, beta A2 and beta A4, the primary structures of all bovine beta-crystallins have previously been elucidated, either by direct protein sequencing or prediction from cDNA sequencing. Both beta A2 and beta A4 were found to be synthesized in half-year-old calf lenses and are therefore likely to be present in a cDNA bovine library constructed from mRNA isolated from lenses of that age. A large number of cDNA clones was screened with all available crystallin, actin, vimentin and lens membrane protein MP26 probes and finally with a randomly primed mRNA probe. Clones positive for the latter, but negative for known lens proteins, were isolated and sequenced. beta A2, comprising 197 aa, and beta A4, comprising 209 aa, were identified. Both proteins have a conserved two-domain structure and an N-terminal extension which is variable. A three-dimensional model of the structure of beta A4 was made based on the coordinates of one subunit from the beta B2 dimer which has recently been solved using x-ray diffraction techniques. The resulting heterodimer structure, together with the compiled bovine beta-crystallin sequences, was used to indicate those regions of the sequences which distinguish acidic from basic beta-crystallins with a view to defining structural features necessary for subunit recognition in beta-crystallin aggregates. With the aid of the present data, the complete evolutionary tree of the bovine beta-crystallin family has been constructed, which confirms the early separation of the genes encoding the three acidic and the three basic beta-crystallins.

Independent regulation of two coexpressed delta-crystallin genes in chick lens and nonlens tissues.

It is known that delta-crystallin is super-abundant in the early chick lens, but it is found at lower levels in certain other tissues. Ninety-nine percent of the lens delta-crystallin poly(A)+ RNA is from the delta 1-crystallin gene. We report here that the delta 1- and delta 2-crystallin genes are both transcribed in the chick lens and retina throughout embryonic development and that both RNAs are found in embryo adenohypophysis and epiphysis and in day-old posthatch chick tibiofemoral chondrocytes and striated muscle. delta 1-crystallin RNA is more abundant in lens tissues, while delta 2-crystallin RNA is more abundant in all nonlens tissues. However, delta 1-crystallin RNA is processed more efficiently than delta 2-crystallin RNA in all early embryonic tissues examined. A comparison of lens epithelium and fibers established that levels of delta 2-crystallin RNA are the same but those of delta 1-crystallin RNA are over 100-fold higher in fibers compared to epithelial cells. The evidence implies independent regulation both of transcription and of post-transcriptional events for these two genes.

Interaction of a lens cell transcription factor with the proximal domain of the mouse gamma F-crystallin promoter.

The elements regulating lens-specific expression of the mouse gamma F-crystallin gene were examined. Here we show that mouse gamma F-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different gamma-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse gamma F-crystallin gene.

Direct assignment of the human beta B2 and beta B3 crystallin genes to 22q11.2----q12: markers for neurofibromatosis 2.

We have isolated a human probe specific for the beta B3 crystallin gene (CRYB3) and hybridized it to Southern blots of human X rodent cell hybrids with known human chromosomal constitution. In this way we could directly assign CRYB3 to chromosome 22. Cell hybrids with translocation chromosomes containing distinct portions of chromosomes 22 were used to regionally localize the gene to 22q11.2----q12. Owing to its known close proximity to the beta B3 crystallin gene, the beta B2-1 crystallin gene (CRYB2-1) also maps in this region. A second beta B2 crystallin gene, beta B2-2 (CRYB2-2), not linked to the CRYB2-1/CRYB3 cluster, could be localized in the same region. This implies that the three known beta B crystallin genes are all within 22q11.2----q12. This small region contains D22S1, the only marker that shows no recombination with neurofibromatosis 2. Therefore, the beta B crystallin genes on chromosome 22 might be markers for this disease. Two DNA fragments revealing useful polymorphisms associated with the beta B crystallin genes were identified. One detects a two-system MspI restriction fragment length polymorphism specific for the CRYB2-1/CRYB3 cluster. The other detects an informative PstI polymorphism that is in linkage equilibrium with the MspI polymorphism.

[Antibodies against alpha, beta, and gamma crystallins in human serum].

Serum antibodies against alpha, beta, and gamma crystallins in 79 normal subjects and 38 patients with senile cataract were determined by ELISA, with the result that the presence of the antibodies was related to age, not to sex, and the rate of presence in patients with senile cataract did not differ from that of controls. The relationship between the presence of the antibodies and senile cataract was discussed.

Evolution of eye lens crystallins: the stress connection.

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.