1、喜树碱类衍生物抗HIV构效关系与作用机制研究2、HIV/AIDS患者疱疹病毒感染状况及性病患者的HIV感染状况分析

1、喜树碱类衍生物抗HIV构效关系与作用机制研究 喜树碱为传统的抗肿瘤药物。本研究对经过化学结构修饰的喜树碱类衍生物进行抗HIV活性及作用机制的研究，并初步探讨了其抗HIV构效关系。 我们对喜树碱类衍生物A系列化合物A1(喜树碱)、A2(10-羟基喜树碱)及A3(7-羟基喜树碱)进行了抗HIV活性检测。化合物A1和A3有较好的抗HIV-1和抗HIV-2活性，化合物A2没有显示抗HIV活性。表明化合物A1的C-10位上-OH基团修饰可能会降低抗HIV活性，化合物A1的C-7位上-CH2OH基团修饰和C-20位-CH3缺失可能会提高其抗HIV活性。对化合物A3和A1的抗HIV机制研究发现：二者对整合酶有一定的结合活性，对慢性感染H9/HIV-1ⅢB 和Jurkat/HIV-1ⅢB细胞中病毒复制没有抑制活性、不能阻断H9/HIV-1ⅢB与正常细胞间的融合，对重组的HIV-1蛋白酶和逆转录酶没有抑制活性。化合物A1和A3不具有选择性杀伤HIV-1ⅢB慢性感染的H9和Jurkat细胞系的作用。进一步进行化合物A3诱导 H9和H9/HIV-1ⅢB、Jurkat和Jurkat/HIV-1ⅢB的凋亡实验显示，化合物A3诱导感染HIV-1ⅢB和未感染病毒细胞的凋亡没有选择性。据此我们初步认为化合物A3和A1的抗HIV作用可能与抑制整合酶活性有关，该化合物可能还作用于其它靶点。 喜树碱类衍生物B系列中化合物B1为20(S)-O - [-O-( 1'-氧基-2',2',6',6'-四甲基哌啶-4'-丁二酸)]-20-喜树碱酯，化合物B2为20(S)-O - [-N-( 1'-氧基-2',2',6',6'-四甲基-1',2',5',6'-四氢吡啶酰胺)-4'-丙氨酸)]-20-喜树碱酯)。我们对化合物B1和B2进行了抗HIV活性检测。结果显示：化合物B2有较好的抗HIV-1和抗HIV-21、喜树碱类衍生物抗HIV构效关系与作用机制研究 喜树碱为传统的抗肿瘤药物。本研究对经过化学结构修饰的喜树碱类衍生物进行抗HIV活性及作用机制的研究，并初步探讨了其抗HIV构效关系。 我们对喜树碱类衍生物A系列化合物A1(喜树碱)、A2(10-羟基喜树碱)及A3(7-羟基喜树碱)进行了抗HIV活性检测。化合物A1和A3有较好的抗HIV-1和抗HIV-2活性，化合物A2没有显示抗HIV活性。表明化合物A1的C-10位上-OH基团修饰可能会降低抗HIV活性，化合物A1的C-7位上-CH2OH基团修饰和C-20位-CH3缺失可能会提高其抗HIV活性。对化合物A3和A1的抗HIV机制研究发现：二者对整合酶有一定的结合活性，对慢性感染H9/HIV-1ⅢB 和Jurkat/HIV-1ⅢB细胞中病毒复制没有抑制活性、不能阻断H9/HIV-1ⅢB与正常细胞间的融合，对重组的HIV-1蛋白酶和逆转录酶没有抑制活性。化合物A1和A3不具有选择性杀伤HIV-1ⅢB慢性感染的H9和Jurkat细胞系的作用。进一步进行化合物A3诱导 H9和H9/HIV-1ⅢB、Jurkat和Jurkat/HIV-1ⅢB的凋亡实验显示，化合物A3诱导感染HIV-1ⅢB和未感染病毒细胞的凋亡没有选择性。据此我们初步认为化合物A3和A1的抗HIV作用可能与抑制整合酶活性有关，该化合物可能还作用于其它靶点。 喜树碱类衍生物B系列中化合物B1为20(S)-O - [-O-( 1'-氧基-2',2',6',6'-四甲基哌啶-4'-丁二酸)]-20-喜树碱酯，化合物B2为20(S)-O - [-N-( 1'-氧基-2',2',6',6'-四甲基-1',2',5',6'-四氢吡啶酰胺)-4'-丙氨酸)]-20-喜树碱酯)。我们对化合物B1和B2进行了抗HIV活性检测。结果显示：化合物B2有较好的抗HIV-1和抗HIV-2活性，而化合物B1的抗HIV活性差。表明化合物B1的C-4’位-CH2被-NH取代，同时C-3’位-CH3修饰可能会提高其抗HIV活性。对化合物B2的抗HIV机制研究发现，化合物B2对慢性感染H9/HIV-1ⅢB细胞中病毒复制没有抑制活性、不能阻断H9/HIV-1ⅢB与正常细胞间的融合，对HIV-1蛋白酶、重组的HIV-1逆转录酶及整合酶没有抑制活性。化合物B2不具有选择性杀伤HIV-1ⅢB慢性感染的H9细胞系的作用。化合物B2抗HIV的作用机制还需进一步研究。 2、HIV/AIDS患者疱疹病毒感染状况及性病患者的HIV感染状况分析 疱疹病毒是AIDS患者合并感染的常见病原体。引起人类疾病的8种疱疹病毒与HIV感染及AIDS进展、机会性感染、恶性肿瘤密切相关。为了解HIV/AIDS患者人类8型疱疹病毒感染状况，我们检测了30例AIDS患者、40例HIV携带者及70例正常对照的液标本中8型疱疹病毒感染状况。采用ELISA法检测单纯疱疹病毒1型(HSV-1)、单纯疱疹病毒2型(HSV-2)、水痘-带状疱疹病毒(VZV)和巨细胞病毒(CMV)；采用PCR法检测EB病毒(EBV)、疱疹病毒6型(HHV-6)、疱疹病毒7型(HHV-7)及疱疹病毒8型(HHV-8)。结果显示，HIV/AIDS患者中HSV-1、HSV-2、VZV、CMV、HHV-6、HHV-8 阳性率均高于健康体检者，其中AIDS患者VZV感染率与HIV携带者有显著性差异；在AIDS患者中多种疱疹病毒共感染普遍存在，必须重视HIV/AIDS患者合并疱疹病毒感染的防治。 性病可促进HIV的传播，了解性病患者的HIV感染状况及临床特征具有重要的意义。在自愿接受HIV咨询检测的基础上，对临床确诊的412例性病患者进行HIV-1/2抗体检测，并对其临床特征进行分析研究。结果显示412例性病患者的HIV检出率为2.9%。性病患者中检出HIV阳性率依次为：尖锐湿疣(6.2%)、生殖器疱疹(4.2%)、梅毒(3.4%)、淋病(1.5%)及非淋菌性尿道炎(1.0%)。83.3%合并感染HIV的性病患者存在多性伴，商业性行为普遍存在，安全套使用率极低现象。感染HIV的尖锐湿疣及生殖器疱疹患者以频繁复发为突出表现，1例合并感染HIV的梅毒患者半年即进展为神经梅毒。性病患者是HIV感染的重要高危人群，危险性行为是其感染HIV和其它性病的主要原因，应该加强性病患者的HIV检测。对临床上频繁复发的尖锐湿疣及生殖器疱疹患者、快速进展的梅毒患者应高度怀疑合并HIV感染的可能。

Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery

Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality. (C) 2011 Elsevier Ltd. All rights reserved.

NF-κB is a critical mediator of BRCA1-induced chemoresistance

BRCA1 mediates resistance to apoptosis in response to DNA-damaging agents, causing BRCA1 wild-type tumours to be significantly more resistant to DNA damage than their mutant counterparts. In this study, we demonstrate that following treatment with the DNA-damaging agents, etoposide or camptothecin, BRCA1 is required for the activation of nuclear factor-?B (NF-?B), and that BRCA1 and NF-?B cooperate to regulate the expression of the NF-?B antiapoptotic targets BCL2 and XIAP. We show that BRCA1 and the NF-?B subunit p65/RelA associate constitutively, whereas the p50 NF-?B subunit associates with BRCA1 only upon DNA damage treatment. Consistent with this BRCA1 and p65 are present constitutively on the promoters of BCL2 and XIAP, whereas p50 is recruited to these promoters only in damage treated cells. Importantly, we demonstrate that the recruitment of p50 onto the promoters of BCL2 and XIAP is dependent upon BRCA1, but independent of its NF-?B partner subunit p65. The functional relevance of NF-?B activation by BRCA1 in response to etoposide and camptothecin is demonstrated by the significantly reduced survival of BRCA1 wild-type cells upon NF-?B inhibition. This study identifies a novel BRCA1-p50 complex, and demonstrates for the first time that NF-?B is required for BRCA1-mediated resistance to DNA damage. It reveals a functional interdependence between BRCA1 and NF-?B, further elucidating the role played by NF-?B in mediating cellular resistance of BRCA1 wild-type tumours to DNA-damaging agents.

Efficient Drug Delivery and Induction of Apoptosis in Colorectal Tumors Using a Death Receptor 5-Targeted Nanomedicine

Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.Molecular Therapy (2014); doi:10.1038/mt.2014.137.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

Pharmacogenomics and metastatic colorectal cancer:current knowledge and perspectives

The pharmacogenomics field is crucial for optimizing the selection of which chemotherapy regimen to use according to the patient's genomic profile. Indeed, the individual's inherited genome accounts for a large proportion of the variation in his or her response to chemotherapeutic agents both in terms of efficiency and toxicity. Patients with metastatic disease are more likely to receive different lines of chemotherapy with variable efficacy and experience some related complications. It is therefore critical to tailor the best therapeutic arsenal to improve the efficacy and avoid as much as possible related complications that are susceptible to interrupt the treatment. The pharmacogenomics approach investigates for each drug the implicated metabolic pathway and the potential personal variations in gene function. The aim of this review is to present a clear overview of the most accurate polymorphisms that have been identified as related to drug response in patients with mCRC.

Mécanismes d'activation de la voie lysosomale durant l'apoptose chimio-induite

L’apoptose est une forme de mort cellulaire essentielle au développement et au maintien de l’homéostase chez les animaux multicellulaires. La machinerie apoptotiq ue requiert la participation des caspases, des protéases conservées dans l’évolution et celle des organelles cytoplasmiques. Les lysosomes subissent des ruptures partielles, labilisation de la membrane lysosomale (LML), qui entraînent l’activation des cathepsines dans le cytoplasme de cellules cancéreuses humaines en apoptose induite par la camptothecin (CPT), incluant les histiocytes humains U-937. Ces modifications lysosomales se manifestent tôt durant l’activation de l’apoptose, concomitamment avec la perméabilisation de la mitochondrie et l’activation des caspases. Une étude protéomique quantitative et comparative a permis d’identifier des changements précoces dans l’expression/localisation de protéines lysosomales de cellules U-937 en apoptose. Lors de deux expériences indépendantes, sur plus de 538 protéines lysosomales identifiées et quantifiées grâce au marquage isobarique iTRAQ et LC-ESIMS/ MS, 18 protéines augmentent et 9 diminuent dans les lysosomes purifiés de cellules en cours d’apoptose comparativement aux cellules contrôles. Les candidats validés par immuno-buvardage et microscopie confocale incluent le stérol-4-alpha-carboxylate 3- déhydrogénase, le prosaposin et la protéine kinase C delta (PKC-d). Des expériences fonctionnelles ont démontrées que la translocation de PKC-d aux lysosomes est requise pour la LML puisque la réduction de son expression par ARN interférents ou l’inhibition de son activité à l’aide du rottlerin empêche la LML lors de l’apoptose induite par la CPT. La translocation de PKC-d aux lysosomes conduit à la phosphorylation et l’activation de la sphingomyelinase acide lysosomale (ASM), et à l’accroissement subséquent du contenu en céramide (CER) à la membrane lysosomale. Cette accumulation de CER endogène aux lysosomes est un évènement critique pour la LML induite par la CPT car l’inhibition de l’activité de PKC-d ou de ASM diminue la formation de CER et la LML.Ces résultats révèlent un nouveau mécanisme par lequel la PKC-d active l’ASM qui conduit à son tour à l’accumulation de CER à la membrane lysosomale et déclenche la LML et l’activation de la voie lysosomale de l’apoptose induite par la CPT. En somme, ce mécanisme confirme l’importance du métabolisme des sphingolipides dans l’activation de la voie lysosomale de l’apoptose.

Les protéines Staufen et leurs rôles dans la régulation posttranscriptionnelle de l’expression des gènes, la réponse aux dommages à l’ADN et le cycle cellulaire

Les différents mécanismes de régulation posttranscriptionnelle de l’expression des gènes sont de plus en plus reconnus comme des processus essentiels dans divers phénomènes physiologiques importants, comme la prolifération cellulaire et la réponse aux dommages à l’ADN. Deux des protéines impliquées dans ce type de régulation sont Staufen1 (Stau1) et Staufen2 (Stau2). Elles sont des protéines de liaison à l’ARN double brin qui contribuent au transport de l’ARN messager (ARNm), au contrôle de la traduction, à l’épissage alternatif et sont responsables de la dégradation de certains ARNm spécifiques. Les protéines Staufen peuvent en effet s’associer à des ARNm bien précis, d’autant plus que, majoritairement, Stau1 et Stau2 ne se retrouvent pas en complexe avec les mêmes cibles. De nombreuses évidences récentes montrent l’implication de divers mécanismes de régulation posttranscriptionnelle dans la réponse aux dommages à l’ADN, plusieurs protéines de liaison à l’ARN y participant d’ailleurs. De façon importante, cette réponse dicte un ou plusieurs destin(s) à la cellule qui doit réagir à la suite de dommages à l’intégrité de son ADN: réparation de l’ADN, arrêt de la prolifération cellulaire, apoptose. Nous avons donc fait l’hypothèse que l’expression de Stau1 et/ou de Stau2 pourrait être affectée en réponse à un stress génotoxique, ce qui pourrait avoir comme conséquence de moduler l’expression et/ou la stabilité de leurs ARNm cibles. De même, notre laboratoire a récemment observé que l’expression de Stau1 varie pendant le cycle cellulaire, celle-ci étant plus élevée jusqu’au début de la mitose (prométaphase), puis elle diminue alors que les cellules complètent leur division. Par conséquent, nous avons fait l’hypothèse que Stau1 pourrait lier des ARNm de façon différentielle dans des cellules bloquées en prométaphase et dans des cellules asynchrones. D’un côté, en employant la camptothécine (CPT), une drogue causant des dommages à l’ADN, pour traiter des cellules de la lignée de cancer colorectal HCT116, nous avons observé que seule l’expression de Stau2 est réduite de façon considérable, tant au niveau de la protéine que de l’ARNm. L’utilisation d’autres agents cytotoxiques a permis de confirmer cette observation initiale. De plus, nous avons constaté que l’expression de Stau2 est touchée même dans des conditions n’engendrant pas une réponse apoptotique, ce qui suggère que cette déplétion de Stau2 est possiblement importante pour la mise en place d’une réponse appropriée aux dommages à l’ADN. D’ailleurs, la surexpression de Stau2 conjointement avec le traitement à la CPT entraîne un retard dans l’induction de l’apoptose dans les cellules HCT116. Nous avons aussi montré que la diminution de l’expression de Stau2 est due à une régulation de sa transcription en réponse au stress génotoxique, ce pourquoi une région minimale du promoteur putatif de Stau2 est nécessaire. Également, nous avons identifié que le facteur de transcription E2F1, couramment impliqué dans la réponse aux dommages à l’ADN, peut contrôler l’expression de Stau2. Ainsi, E2F1 permet une augmentation de l’expression de Stau2 dans des cellules non traitées, mais cette hausse est abolie dans des cellules traitées à la CPT, ce qui suggère que la CPT pourrait agir en inhibant l’activation transcriptionnelle de Stau2 par E2F1. Enfin, nous avons observé que certains ARNm associés à Stau2, et codant pour des protéines impliquées dans la réponse aux dommages à l’ADN et l’apoptose, sont exprimés différemment dans des cellules traitées à la CPT et des cellules non traitées. D’un autre côté, nous avons identifié les ARNm associés à Stau1 lors de la prométaphase, alors que l’expression de Stau1 est à son niveau le plus élevé pendant le cycle cellulaire, grâce à une étude à grande échelle de micropuces d’ADN dans des cellules HEK293T. Nous avons par la suite confirmé l’association entre Stau1 et certains ARNm d’intérêts, donc codant pour des protéines impliquées dans la régulation de la prolifération cellulaire et/ou le déroulement de la mitose. Une comparaison de la liaison de ces ARNm à Stau1 dans des cellules bloquées en prométaphase par rapport à des cellules asynchrones nous a permis de constater une association préférentielle dans les cellules en prométaphase. Ceci suggère une augmentation potentielle de la régulation de ces ARNm par Stau1 à ce moment du cycle cellulaire. Les données présentées dans cette thèse indiquent vraisemblablement que la régulation posttranscriptionnelle de l’expression génique contrôlée par les protéines Staufen se fait en partie grâce à la modulation de l’expression de Stau1 et de Stau2 en fonction des conditions cellulaires. Nous envisageons alors que cette variation de l’expression des protéines Staufen ait des conséquences sur des sous-ensembles d’ARNm auxquels elles sont liées et que de cette façon, elles jouent un rôle pour réguler des processus physiologiques essentiels comme la réponse aux dommages à l’ADN et la progression dans le cycle cellulaire.

Acute coronary syndrome associated with continuous 5-fluorouracil infusion in a patient with metastatic colorectal cancer-a case report with a discussion on this clinical dilemma

Introduction: 5-Fluorouracil (5-FU) is considered to be the backbone of colorectal cancer (CRC) systemic therapy since the great majority of recommended regimens include its administration. A clinical picture consisting of chest pain, sometimes cardiac enzyme elevation, electrocardiogram abnormalities consistent with myocardial ischemia, and normal coronary angiogram associated with 5-FU administration have been infrequently reported. The clinical dilemma is: Which chemotherapy regimen should we use in CRC patients with a previous acute coronary syndrome (ACS) associated with 5-FU? Case Report: We describe the case of a 55-year-old otherwise healthy woman with metastatic colon adenocarcinoma who presented an ACS probably secondary to arterial vasospasm while receiving continuous intravenous 5-FU infusion (mFOLFOX6 regimen). After the ACS, the patient was treated with raltitrexate plus oxaliplatin (TOMOX) and subsequently with irinotecan plus cetuximab with no other cardiac event. Conclusion: The risk of cardiotoxicity associated with 5-FU is low but real. The probable mechanism is arterial vasospasm, as suggested by our case report. Both the use of the TOMOX regimen and irinotecan plus cetuximab seems to be safe regimens to be considered in this clinical scenario. © 2009 Humana Press Inc.

Recent applications of analytical techniques for quantitative pharmaceutical analysis: A review

The intension of this paper was to review and discuss some of the current quantitative analytical procedures which are used for quality control of pharmaceutical products. The selected papers were organized according to the analytical technique employed. Several techniques like ultraviolet/visible spectrophotometry, fluorimetry, titrimetry, electroanalytical techniques, chromatographic methods (thin-layer chromatography, gas chromatography and high-performance liquid chromatography), capillary electrophoresis and vibrational spectroscopies are the main techniques that have been used for the quantitative analysis of pharmaceutical compounds. In conclusion, although simple techniques such as UV/VIS spectrophotometry and TLC are still extensively employed, HPLC is the most popular instrumental technique used for the analysis of pharmaceuticals. Besides, a review of recent works in the area of pharmaceutical analysis showed a trend in the application of techniques increasingly rapid such as ultra performance liquid chromatography and the use of sensitive and specific detectors as mass spectrometers.

High concentrations of glucose reduce the oxidative metabolism of dog neutrophils in vitro

Background: Dogs are commonly affected by hyperglycemic conditions. Hyperglycemia compromises the immune response and favors bacterial infections; however, reports on the effects of glucose on neutrophil oxidative metabolism and apoptosis are conflicting in humans and rare in dogs. Considering the many complex factors that affect neutrophil oxidative metabolism in vivo, we investigated in vitro the specific effect of high concentrations of glucose on superoxide production and apoptosis rate in neutrophils from healthy dogs.Results: The capacity of the neutrophils to reduce tetrazolium nitroblue decreased significantly in the higher concentration of glucose (15.13 ± 9.73% (8 mmol/L) versus 8.93 ± 5.71% (16 mmol/L)). However, there were no changes in tetrazolium nitroblue reduction at different glucose concentrations when the neutrophils were first activated with phorbol myristate acetate. High concentrations of glucose did not affect the viability and apoptosis rate of canine neutrophils either with or without prior camptothecin stimulation. This study provides the first evidence that high concentrations of glucose inhibit the oxidative metabolism of canine neutrophils in vitro in a manner similar to that which occurs in humans, and that the decrease in superoxide production did not increase the apoptosis rate.Conclusions: A high concentration of glucose reduces the oxidative metabolism of canine neutrophils in vitro. It is likely that glucose at high concentrations rapidly affects membrane receptors responsible for the activation of NADPH oxidase in neutrophils; therefore, the nonspecific immune response can be compromised in dogs with acute and chronic hyperglycemic conditions. © 2013 Bosco et al.; licensee BioMed Central Ltd.

Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease

Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production.

Estresse oxidativo, produção de superóxido e a apoptose de neutrófilos de cães com insuficiência renal crônica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação in vitro do P-cresol sobre o metabolismo oxidativo e apoptose dos neutrófilos de cães

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)