Étude des biais sur la dose parentérale d'un nouveau médicament lors d'un essai clinique avec profil cinétique

Introduction et objectif: Lors d'essais cliniques, le pharmacien est responsable de la préparation et de la dispensation des médicaments à évaluer. Un article récent a toutefois montré que les aspects pharmaceutiques liés au contrôle de la dose administrée in fine étaient souvent mal contrôlés. Il peut exister une différence entre la dose nominale fournie par le certificat d'analyse du fabricant et la dose réellement administrée au sujet, biais qui se reporte en cascade sur l'estimation des paramètres pharmacocinétiques (PK), comme la clairance ou le volume de distribution. Ce travail visait à évaluer les biais entachant la quantité de médicament réellement injectée (iv/sc) aux volontaires d'un essai clinique étudiant la PK et la relation dose-réponse d'un nouveau produit biotechnologique. Méthode: La dose de médicament administrée lors de l'essai clinique (D) a été calculée de la manière suivante: D = C * V - pertes. La concentration du produit (C; titre nominal du fabricant) a été vérifiée par immuno-essai. Le volume de médicament injecté (V) a été déterminé pour chaque injection par pesée (n=72), en utilisant la masse de la seringue avant et après injection et la densité du produit. Enfin, une analyse in vitro a permis d'évaluer les pertes liées à l'adsorption du produit dans les lignes de perfusion et de choisir le dispositif adéquat in vivo. Résultats: La concentration du médicament s'est révélée proche du titre nominal (96 ± 7%), et a été utilisée comme référence. Le volume injecté était quant à lui entaché d'un biais systématique par rapport à la valeur théorique correspondant à 0.03 mL pour la dose minimale (i.e. 75% du volume à injecter à cette dose). Une analyse complémentaire a montré que cela s'expliquait par une réaspiration partielle de la solution médica-menteuse avant le retrait de la seringue après injection sc, due à l'élasticité du piston. En iv, le biais était par contre provoqué par une réaspiration du soluté de perfusion co-administré. Enfin, la mesure des quantités de médicament récupérées après injection dans le dispositif de perfusion a démontré des pertes minimales par adsorption. Discussion-conclusion: Cette étude confirme l'existence de biais inversement corrélés au volume et à la concentration du médicament administré, pouvant provoquer des erreurs importantes sur les paramètres PK. Ce problème est négligé ou insuffisamment considéré dans les protocoles de Phase I et nécessiterait une planification rigoureuse. Les procédures opératoires devraient attirer l'attention sur ce point crucial.

Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates.

UNLABELLED: We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4(+) T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8(+) T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine. IMPORTANCE: The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.

Hepatitis C virus infection among Brazilian hemophiliacs: a virological, clinical and epidemiological study

We determined and analyzed risk factors of hepatitis C virus (HCV)-infected Brazilian hemophiliacs according to their virological, clinical and epidemiological characteristics. A cross-sectional and retrospective study of 469 hemophiliacs was carried out at a Brazilian blood center starting in October 1997. The prevalence of HCV infection, HCV genotypes and factors associated with HCV RNA detection was determined. The seroprevalence of anti-HCV antibodies (ELISA-3.0) was 44.6% (209/469). Virological, clinical and epidemiological assessments were completed for 162 positive patients. There were seven (4.3%) anti-HCV seroconversions between October 1992 and October 1997. During the same period, 40.8% of the positive anti-HCV hemophiliacs had abnormal alanine transaminase (ALT) levels. Plasma HCV RNA was detected by nested-RT-PCR in 116 patients (71.6%). RFLP analysis showed the following genotype distribution: HCV-1 in 98 hemophiliacs (84.5%), HCV-3 in ten (8.6%), HCV-4 in three (2.6%), HCV-2 in one (0.9%), and not typeable in four cases (3.4%). Univariate analysis indicated that older age (P = 0.017) and abnormal ALT levels (P = 0.010) were associated with HCV viremia, while the presence of inhibitor antibodies (P = 0.024) and HBsAg (P = 0.007) represented a protective factor against the presence of HCV RNA. These findings may contribute to a better understanding of the relationship between HCV infection and hemophilia.

Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/ß-actin-mRNA ratio (mean ± SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 ± 0.15; N = 5; P < 0.05) compared to non-responders (0.35 ± 0.17; N = 12) and controls (0.30 ± 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

Validation of the Social Phobia and Anxiety Inventory for Children (SPAI-C) in a sample of Brazilian children

The purpose of the present study was to examine the factor structure and psychometric properties of the Social Phobia and Anxiety Inventory for Children (SPAI-C), an instrument developed in the United States and applied to a sample of Brazilian schoolchildren. The process included the translation of the original material from English into Portuguese by two bilingual psychiatrists and a back translation by a bilingual physician. Both the front and back translations were revised by a bilingual child psychiatrist. The study was performed using a cross-sectional design and the Portuguese version of the SPAI-C was applied to a sample of 1954 children enrolled in 3rd to 8th grade attending 2 private and 11 public schools. Eighty-one subjects were excluded due to an incomplete questionnaire and 2 children refused to participate. The final sample consisted of 1871 children, 938 girls (50.1%) and 933 boys (49.8%), ranging in age from 9 to 14 years. The majority of the students were Caucasian (89.0%) and the remainder were African-Brazilian (11.0%). The Pearson product-moment correlation showed that the two-week test-retest reliability coefficient was r = 0.780 and Cronbach's alpha was 0.946. The factor structure was almost similar to that reported in previous studies. The results regarding the internal consistency, the test-retest reliability and the factor structure were similar to the findings obtained in studies performed on English speaking children. The present study showed that the Portuguese language version of SPAI-C is a reliable and valid measure of social anxiety for Brazilian children.

Persons who served more than six months in the War of 1812 : letter from the Secretary of War, transmitting in compliance with a resolution of the House calling for a statement of the number of officers, non-commissioned officers, privates, &c., who served for a period of six months and upwards in the War of 1812.--

Caption title.

Les Antonins : - ans de J.C., 69-180

UANL

Búsqueda de selección en el polimorfismo 677C>T (c.665C>T) del gen de la metilentetrahidrofolato reductasa (MTHFR) en una población Colombiana

Se realizó un estudio genético – poblacional en dos grupos etarios de población colombiana con la finalidad de evaluar las diferencias genéticas relacionadas con el polimorfismo MTHFR 677CT en busca de eventos genéticos que soporten la persistencia de este polimorfismo en la especie humana debido que este ha sido asociado con múltiples enfermedades. De esta manera se genotipificaron los individuos, se analizaron los genotipos, frecuencias alélicas y se realizaron diferentes pruebas genéticas-poblacionales. Contrario a lo observado en poblaciones Colombianas revisadas se identificó la ausencia del Equilibrio Hardy-Weinberg en el grupo de los niños y estructuras poblacionales entre los adultos lo que sugiere diferentes historias demográficas y culturales entre estos dos grupos poblacionales al tiempo, lo que soporta la hipótesis de un evento de selección sobre el polimorfismo en nuestra población. De igual manera nuestros datos fueron analizados junto con estudios previos a nivel nacional y mundial lo cual sustenta que el posible evento selectivo es debido a que el aporte de ácido fólico se ha incrementado durante las últimas dos décadas como consecuencia de las campañas de fortificación de las harinas y suplementación a las embarazadas con ácido fólico, por lo tanto aquí se propone un modelo de selección que se ajusta a los datos encontrados en este trabajo se establece una relación entre los patrones nutricionales de la especie humana a través de la historia que explica las diferencias en frecuencias de este polimorfismo a nivel espacial y temporal.  

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.