Novel approaches for culturing hepatocytes for drug testing applications

Dissertation presented to obtain the Ph.D degree in Biochemisry, Biotechnology

Investigation and Control of a Large Outbreak of Multi-Drug Resistant Tuberculosis at a Central Lisbon Hospital

An increase in the number of new cases of tuberculosis (TB) combined with poor clinical outcome was identified among HIV-infected injecting drug users attending a large HIV unit in central Lisbon. A retrospective epidemiological and laboratory study was conducted to review all newly diagnosed cases of TB from 1995 to 1996 in the HIV unit. Results showed that from 1995 to 1996, 63% (109/173) of the Mycobacterium tuberculosis isolates from HIV-infected patients were resistant to one or more anti-tuberculosis drugs; 89% (95) of these were multidrug-resistant, i.e., resistant to at least isoniazid and rifampicin. Eighty percent of the multidrug-resistant strains (MDR) available for restriction fragment length polymorphism (RFLP) DNA fingerprinting clustered into one of two large clusters. Epidemiological data support the conclusion that the transmission of MDR-TB occurred among HIV-infected injecting drug users exposed to infectious TB cases on open wards in the HIV unit. Improved infection control measures on the HIV unit and the use of empirical therapy with six drugs once patients were suspected to have TB, reduced the incidence of MDR-TB from 42% of TB cases in 1996 to 11% in 1999.

Drug-Induced Anaphylaxis Survey in Portuguese Allergy Departments

Background and Objective: Drug-induced anaphylaxis is an unpredictable and potentially fatal adverse drug reaction. The aim of this study was to identify the causes of drug-induced anaphylaxis in Portugal. Methods: During a 4-year period a nationwide notification system for anaphylaxis was implemented, with voluntary reporting by allergists. Data on 313 patients with drug anaphylaxis were received and reviewed. Statistical analysis included distribution tests and multiple logistic regression analysis to investigate significance, regression coefficients, and marginal effects. Results: The mean (SD) age of the patients was 43.8 (17.4) years, and 8.3% were younger than 18 years. The female to male ratio was 2:1. The main culprits were nonsteroidal anti-inflammatory drugs (NSAIDs) (47.9% of cases), antibiotics (35.5%), and anesthetic agents (6.1%). There was a predominance of mucocutaneous symptoms (92.2%), followed by respiratory symptoms (80.4%) and cardiovascular symptoms (49.0%). Patients with NSAID-induced anaphylaxis showed a tendency towards respiratory and mucocutaneous manifestations. We found no significant associations between age, sex, or atopy and type of drug. Anaphylaxis recurrence was observed in 25.6% of cases, and the risk was higher when NSAIDs were involved. Conclusions: NSAIDs were the most common cause of anaphylaxis in this study and were also associated with a higher rate of recurrence. We stress the need for better therapeutic management and prevention of recurring episodes of drug-induced anaphylaxis.

Differences in Nevirapine Biotransformation as a Factor for its Sex-Dependent Dimorphic Profile of Adverse Drug Reactions

OBJECTIVES: Nevirapine is widely used for the treatment of HIV-1 infection; however, its chronic use has been associated with severe liver and skin toxicity. Women are at increased risk for these toxic events, but the reasons for the sex-related differences are unclear. Disparities in the biotransformation of nevirapine and the generation of toxic metabolites between men and women might be the underlying cause. The present work aimed to explore sex differences in nevirapine biotransformation as a potential factor in nevirapine-induced toxicity. METHODS: All included subjects were adults who had been receiving 400 mg of nevirapine once daily for at least 1 month. Blood samples were collected and the levels of nevirapine and its phase I metabolites were quantified by HPLC. Anthropometric and clinical data, and nevirapine metabolite profiles, were assessed for sex-related differences. RESULTS: A total of 52 patients were included (63% were men). Body weight was lower in women (P = 0.028) and female sex was associated with higher alkaline phosphatase (P = 0.036) and lactate dehydrogenase (P = 0.037) levels. The plasma concentrations of nevirapine (P = 0.030) and the metabolite 3-hydroxy-nevirapine (P = 0.035), as well as the proportions of the metabolites 12-hydroxy-nevirapine (P = 0.037) and 3-hydroxy-nevirapine (P = 0.001), were higher in women, when adjusted for body weight. CONCLUSIONS: There was a sex-dependent variation in nevirapine biotransformation, particularly in the generation of the 12-hydroxy-nevirapine and 3-hydroxy-nevirapine metabolites. These data are consistent with the sex-dependent formation of toxic reactive metabolites, which may contribute to the sex-dependent dimorphic profile of nevirapine toxicity.

Development of chitosan-based microparticles for pulmonary drug delivery

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Nevirapine in an animal model of pre-diabetes: study of drug pharmacokinetic and its effects on fasting glycemia and insulin resistance

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Risky behavior regarding drug use and HIV infection: an Internet questionnaire coupled with short education texts for Portuguese speakers

Surveys of risky behavior relating to HIV/AIDS are generally made for groups at risk of infection, for which HIV/AIDS prevalence is usually expected to be higher than in the general population. Therefore, an educational homepage in Portuguese was created on the Internet to inform/ask internauts regarding knowledge and behavior. The internauts were classified as adolescents (13 to 25 years) and adults (>25 years). The number of STDs was reported as 1. 8 ± 2. 6 infections (range: 1 to 20 infections); 43% used condoms during sexual intercourse. Alcohol consumption was reported by 63% and illicit drug use by 32% (marijuana 24% and inhalants 15%). Among the adolescents, 31% did not classified alcohol as a drug. The adults more frequently reported homosexuality, anal intercourse and STDs, although the adolescents also presented high rates of risky behavior. These results show the need to reach out to internauts through better control strategies. Different types of strategies must be encouraged, in order to reach people that use this means of communication and entertainment.

Insights into the epidemiology of drug-resistant Streptococcus pneumoniae and closely-related streptococci in the era of conjugate vaccines

In Portugal, the introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) has led to significant changes in the population structure of Streptococcus pneumoniae. However, the levels of antimicrobial resistance have not decreased and have been a matter of concern. (...)

Antituberculosis drug-induced hepatotoxicity: a comparison between patients with and without human immunodeficiency virus seropositivity

INTRODUCTION: The prevalence and risk factors for rifampin, isoniazid and pyrazinamide hepatotoxicity were evaluated in HIV-infected subjects and controls. METHODS: Patients with tuberculosis (30 HIV positive and 132 HIV negative), aged between 18 and 80 years-old, admitted to hospital in Brazil, from 2005 to 2007, were selected for this investigation. Three definitions of hepatotoxicity were used: I) a 3-fold increase in the lower limit of normal for alanine-aminotransferase (ALT); II) a 3-fold increase in the upper limit of normal (ULN) for ALT, and III) a 3-fold increase in the ULN for ALT plus a 2-fold increase in the ULN of total bilirubin. RESULTS: In groups with and without HIV infection the frequency of hepatotoxicity I was 77% and 46%, respectively (p < 0.01). Using hepatotoxicity II and III definitions no difference was observed in the occurrence of antituberculosis drug-induced hepatitis. Of the 17 patients with hepatotoxicity by definition III, 3 presented no side effects and treatment was well tolerated. In 8 (36.4%) out of 22, symptoms emerged and treatment was suspended. Alcohol abuse was related to hepatotoxicity only for definition I. CONCLUSIONS: Depending on the definition of drug-induced hepatitis, HIV infection may or may not be associated with hepatotoxicity. The impact that minor alterations in the definition had on the results was impressive. No death was related to drug-induced hepatotoxicity. The emergence of new symptoms after initiating antituberculosis therapy could not be attributed to hepatotoxicity in over one third of the cases.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120); 5% (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

The patent expiry as a major inflection point in a branded Rx drug's life and strategies to counter generic competition - exemplified at Pfizer's blockbuster drug Lipitor in the USA

A large number of expensive, but highly profitable branded prescription drugs will go off-patent in the USA between 2011 and 2015. Their revenues are crucial to fund the immense costs associated with the development of an innovative drug. The rising cost pressure on pharmaceutical stakeholders has increased the demand for more affordable medications, as provided by the branded drug's generic counterpart. Yet, research based incumbents are moving beyond the traditional late lifecycle strategies and deploy more aggressive tactics in order to protect their brands, as seen with Pfizer's Lipitor!. It is doubtful, whether these efforts will help the blockbuster business model to resist current market conditions.

Adverse reactions following mass drug administration with diethylcarbamazine in lymphatic filariasis endemic areas in the Northeast of Brazil

INTRODUCTION: The Global Programme to Eliminate Lymphatic Filariasis was launched with the goal of eliminating this disease via the annual mass drug administration (MDA) of a single dose of antifilarial drugs. Adverse drug reactions following MDA are a major factor of poor treatment adherence in several countries. This study assessed the occurrence of adverse drug reactions (ADRs) following the first round of mass treatment in two communities treated with different dosages of diethylcarbamazine (DEC) in the City of Recife, Brazil. METHODS: Population-based cross-sectional surveys were conducted in a random sample of the population living in both communities (Areas I and II). The dose of DEC recommended by the WHO (6mg/kg) was calculated based on the individual's weight-for-age. In Area II, weight differences between the genders were also considered when determining dosage. Data were obtained through interviews conducted in the first 12 to 48h and on the 5th day after MDA during household visits. RESULTS: A total of 487 and 365 individuals were interviewed in Areas I and II, respectively. The prevalence of ADRs in Area I (23.6; 95%CI: 19.1-29.5) was higher than in Area II (16.2; 95%CI:11.9-21.5)(p=0.0078). The prevalence of ADRs among females was higher than in males in Area I (p=0.0021). In Area II, no significant difference between the genders was observed (p=0.1840). Age was not associated with ADRs in either area. CONCLUSIONS: Adjusting MDA dosage schedules according to weight-for-age and sex may be may contribute to reduce the occurrence of adverse drug reactions in the population.

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.