Begoña Calvo

HLA-DRB1 and MICA in Autoimmunity

Abstract: Autoimmune disorders such as type 1 diabetes (T1DM), celiac disease (CD), and Addisons disease (ADD) develop in individuals with genetic susceptibility that are exposed to environmental triggering factors not completely defined. Patients with an autoimmune disease (and their relatives) are at increased risk of developing another disorder, and this might be caused by a common genetic origin of autoimmunity; for example, HLA class II region in 6p21 shows a very strong association with most diseases. The aim of this study was to determine whether shared susceptibility markers extend from the central (DRB1) through the telomeric (MICA) HLA region. We analyzed three independent sets of families with one autoimmune disease, T1DM, CD, or ADD, and genotyped them for HLA‐DRB1 and for the exon 5 GCT polymorphism of MICA. For HLA‐DRB1, allele DRB1*0301 was the only one associated with risk for all three diseases; in the case of MICA, allele A9 was found to be the common protective allele. Haplotype analysis shows that haplotype A5.1‐DRB1*0301 confers risk to autoimmunity. Our results show that there are common risk and protection alleles in both loci, suggesting a core of genetic association with autoimmunity (HLA‐DRB1*0301 risk; A9 protection) that could be modulated by other alleles/loci or environmental factors toward one or another disease. Some alleles are part of conserved haplotypes (A5.1‐DR3, A5.1‐DR2), whereas others seem to have independent effect (A9) and support the idea of two independent loci in this region.

Production of BCG alginate-PLL microcapsules by emulsification/internal gelation

A biocompatible emulsification method for microencapsulation of live cells and enzymes within a calcium alginate matrix applied to Bacillus Calmette-Guérin (BCG) has been developed. Small-diameter alginate beads (microcapsules) were formed via internal gelation of an alginate solution emulsified within vegetable oil. Five different oils (sesame, sweet almond, perhydrosqualene, camomile and jojoba) were used. The rheological analysis of the oils showed a Newtonian behaviour, with viscosities = 30.0, 37.7, 51.2, 59.3 and 67.1 mPa.s for perhydrosqualene, jojoba, camomile, sesame and sweet almond oil respectively. The particle size of the microcapsules obtained ranged from 30.3 microns for the microcapsules prepared with sweet almond oil to 57.0 microns for those made with perhydrosqualene. The mean particle diameter obtained was found to be dependent on the viscosity of the oil employed, according to the equation: phi (micron) = 76.6-0.628 eta (mPa.s) (r2 = 0.943). The encapsulated BCG was identified by the Difco TB stain set K, followed by observation under optical microscopy. Freeze-drying of the microcapsules was carried out to ensure their stability during storage. Two batches of microcapsules (those prepared with sesame and jojoba oil) and four types of cryoprotectors (glucose, trehalose, mannitol and sorbitol), at three concentration levels (5, 10 and 20% w/v) were studied. The parameters evaluated were particle size, physical appearance, reconstitution of lyophilizates and microscopical evaluation. For both batches of microcapsules the best results were obtained with trehalose 5%, showing particle sizes of 42.1 microns in the case of the microcapsules prepared with sesame oil, and of 45.3 microns for those prepared with jojoba.

Biosimilars: pharmacovigilance and risk management†

Biosimilars cannot be authorised based on the same requirements that apply to generic medicines. Despite the fact that the biosimilar and reference drug can show similar efficacy, the biosimilar may exhibit different safety profile in terms of nature, seriousness or incidence of adverse reactions. However, the data from pre‐authorisation clinical studies normally are insufficient to identify all potential differences. Therefore, clinical safety of similar biological medicinal products must be monitored closely on an ongoing basis during the post‐approval phase including continued risk–benefit assessment.

Conserved extended haplotypes discriminate HLA-DR3-homozygous Basque patients with type 1 diabetes mellitus and celiac disease

The major susceptibility locus for type 1 diabetes mellitus (T1D) maps to the human lymphocyte antigen (HLA) class II region in the major histocompatibility complex on chromosome 6p21. In southern European populations, like the Basques, the greatest risk to T1D is associated with DR3 homo- and heterozygosity and is comparable to that of DR3/DR4, the highest risk genotype in northern European populations. Celiac disease (CD) is another DR3-associated autoimmune disorder showing certain overlap with T1D that has been explained by the involvement of common genetic determinants, a situation more frequent in DR3-rich populations, like the Basques. As both T1D- and CD-associated HLA alleles are part of conserved extended haplotypes (CEH), we compared DR3-homozygous T1D and CD patients to determine whether CEHs were equally distributed between both disorders or there was a differential contribution of different haplotypes. We observed a very pronounced distribution bias (P<10−5) of the two major DR3 CEHs, with DR3-B18 predominating in T1D and DR3-B8 in CD. Additionally, high-density single nucleotide polymorphism (SNP) analysis of the complete CEH [A*30-B*18-MICA*4-F1C30-DRB1*0301-DQB1*0201-DPB1*0202] revealed extraordinary conservation throughout the 4.9 Mbp analyzed supporting the existence of additional diabetogenic variants (other than HLA-DRB1*0301-DQB1*0201), conserved within the DR3-B18 CEH (but not in other DR3 haplotypes) that could explain its enhanced diabetogenicity.

EU’s New Pharmacovigilance Legislation: Considerations for Biosimilars

Biosimilars are biological medicines, the active substances of which are highly similar to those of biologics that have already been authorized. As for any other medicine, the applicant of the biosimilar marketing authorization must submit a risk-management plan (RMP)/pharmacovigilance plan. The pharmacovigilance plan should take into account risks identified during product development, the potential risks and how those risks will be addressed after authorization of the product.Recently, new European Pharmacovigilance legislation has been implemented, ensuring proper risk management through the recording of suspected adverse drug reactions and data collection from all stakeholders. The new regulation entails a reduction of the administrative burden on companies and regulatory agencies, as obligations of the responsible parties are clearly established and duplication of effort avoided.This article analyzes the new European Pharmacovigilance System requirements, with special focus on those medicines requiring additional monitoring, such as biosimilars, which are priorities for pharmacovigilance. Further, it provides the new obligations to marketing authorization holders, such as the continuous benefit–risk assessment.

Determination of salbutamol enantiomers by high-performance capillary electrophoresis and its application to dissolution assays

Capillary zone electrophoresis was successfully applied to the chiral separation of salbutamol after addition of a suitable cyclodextrin chiral selector to the electrophoresis buffer. Parameters important in achieving enantiomeric separation are cyclodextrin type, mobile phase pH and applied field strength. In our study, salbutamol enantiomeric separation was obtained with the following conditions: heptakis (2,6-di-O-methyl)-beta-cyclodextrin in 40 mM Tris (pH 2.5) and at 15 kV, obtaining a 3.09 resolution with migration times of 13.74 min for (R)-salbutamol and 13.98 min for (S)-salbutamol. Linearity, limit of quantitation, precision and accuracy were established using this method. The calibration curve was linear in a range of 1-40 micrograms ml-1 of racemic salbutamol (0.5-20 micrograms ml-1 of each enantiomer). This method was applied to evaluate the enantioselective release of salbutamol and taking into account the hypothesis that one enantiomer of a chiral drug would be released faster than the other from a pharmaceutical dosage form containing a racemic drug and a chiral excipient. For this purpose, matrix tablets formed by chiral excipients such as hydroxypropylmethylcellulose (HPMC) were considered. The release of the enantiomers of salbutamol from the formulations containing HPMC was found to be equivalent, with constant dissolution values (K) of 1.187 +/- 0.223% min-n for (R)-salbutamol and 1.076 +/- 0.268% min-n for (S)-salbutamol.

Contribution of MIC-A polymorphism to type 1 diabetes mellitus in Basques.

Abstract: The maximum genetic susceptibility to type 1 diabetes (T1DM) in Basques is conferred by extended HLA haplotype F1C30‐DR3‐DQ2‐DPB1*0202. Due to the strong linkage disequilibrium within the haplotype, it is difficult to determine which individual allele shows the strongest association with T1DM. Recent studies of the MIC‐A gene have shown an HLA‐independent association of allele A5 with T1DM and A5.1 with Addisons disease. In order to test for association of the MIC‐A exon 5 polymorphism with T1DM and to further characterize risk and protection haplotypes in Basques, we typed 70 Basque families with T1DM for MIC‐A exon 5 polymorphism using fluorescent PCR and electrophoresis on an ABI sequencing machine. When analyzed individually, allele A4 was associated with disease [OR = 2.93 (1.58‐5.5)], while the presence of A9 conferred protection from T1DM [OR = 0.27 (0.08‐0.74)]. In the context of HLA haplotypes, allele A4 was found to be associated to the F1C30‐DR3‐DQ2‐DPB*0202 risk haplotype, both for T1DM and AFBAC alleles (Pc= 0.0003). Allele A5.1 was strongly associated with protective haplotype SC31‐DRB1*1501‐DQB1*0602, present only in AFBAC alleles, but also with risk haplotype SC01‐DR3‐DQ2. In conclusion, polymorphisms in exon 5 of the MIC‐A gene are associated with genetic susceptibility/protection to T1DM, but in the context of susceptibility HLA haplotypes. Nevertheless, the protective effect of A9 allele seems independent from HLA, since it does not appear to be associated with any particular extended haplotype.

Interspecies Scaling of Cimetidine–Theophylline Pharmacokinetic Interaction: Interspecies Scaling in Pharmacokinetic Interactions

The aim of the present study was the use of an interspecies scaling approach to predict drug interactions during preclinical drug disposition studies. Theophylline and cimetidine were selected because of their documented interaction. The literature was searched for pharmacokinetic data of intravenously administered theophylline alone and in the presence of cimetidine in humans, dogs and rats. Further, we determined the theophylline-cimetidine drug interaction in rabbits. Application of allometric equations to the pharmacokinetic parameters and the conversion of chronological time into pharmacokinetic time allowed us to obtain the complex Dedrick plot for theophylline when administered alone or in combination with cimetidine. A superimposable kinetic profile was obtained for the plasma levels of theophylline in all species studied, both with and without cimetidine. From the terminal phase of the curves it is possible to calculate the elimination half-life: 2.69 apolisychrons for theophylline when it is administered alone and 3.86 apolisychrons when it is administered in combination with cimetidine. This 43% increase in t1/2 is similar to the increase in the elimination half-life of theophylline in humans when it is administered after pretreatment with cimetidine. These results show that an interspecies scaling approach may be useful to predict the effect of interactions in humans from the results obtained in preclinical research with new drugs.

Biosimilars approval process.

For similar biological medicinal products, the so-called biosimilars, clinical trials are required rather than just the bioequivalence studies required to support the registration of a generic small molecule drug product. The EU Directive 2001/83/EC, as amended, stated that where a biological medicinal product which is similar to a reference biological product, does not meet the conditions in the definition of generic medicinal products the results of appropriate pre-clinical tests or clinical trials relating to these conditions must be provided. The challenge is to determine the exact nature of the non-clinical and clinical programme required to gain regulatory approval. The applicant is encouraged to provide a detailed description of the strategy used to demonstrate the biosimilar and the reference product have similar profiles in terms of quality, safety and efficacy. The extent to which comparability can be proven will have quite an impact on how many non-clinical and clinical studies the biosimilar applicant will be required to conduct. The dossier submitted by the applicant to the EMEA should cover all aspects of the comparability assessment and must include data on possible unwanted immune reactions to the therapeutic protein. Post-marketing pharmacovigilance plans are also expected to be included in the biosimilar dossier.

Release of salbutamol sulfate enantiomers from hydroxypropylmethylcellulose matrices

The in vitro dissolution profiles of nine sustained-release formulations of (±)-salbutamol were determined. The formulations contained chiral pharmaceutical excipients, such as hydroxypropylmethylcellulose (HPMC) and heptakis (2,6-di-O-methyl)-β-cyclodextrin (DMCD). The influence of the degree of ionization was studied in six formulations that were buffered at different pH values. The quantitative determination of salbutamol enantiomers released by the matrices was carried out using a stereospecific capillary electrophoresis method. The release of salbutamol enantiomers from all formulations was found to be non-enantioselective. Likewise, the addition of buffering agents to the formulation did not provide an enantioselective release of salbutamol enantiomers from the matrices under the conditions established.