Safety and efficacy of N-acetylcysteine in hospitalized patients with HIV-associated tuberculosis: An open-label, randomized, phase II trial (RIPENACTB Study)

Despite the availability of effective antimicrobials, tuberculosis (TB) is still a serious health threat. Mortality is even higher in people living with HIV who are diagnosed with TB. New therapies are needed to shorten the time required to cure TB and decrease fatality rates in this population. N-acetylcysteine (NAC) is a glutathione precursor and has shown recently in experimental setting to present in vitro and in vivo anti-mycobacterial activity. We test the hypothesis that NAC is safe, well tolerated and secondarily efficacious as adjunctive anti-TB therapy in hospitalized individuals with HIV-associated TB. Patients were enrolled sequentially in a tertiary care center, in the Brazilian Amazon. We performed a randomized, parallel group, single-center, open study trial of two arms, in hospitalized patients over 18 years of age, with microbiologically confirmed pulmonary TB in HIV: one with rifampicin, isoniazid, pyrazinamide and ethambutol at standard doses (Control Group), and a second in which NAC 600 mg bid for eight weeks was added (NAC Group). A total of 21 and 18 patients were enrolled to the Control Group and NAC Group, respectively. Adverse event rates were similar in the two arms. Our findings suggest that in the more critical population of hospitalized patients with HIV-associated TB, the use of NAC was not unsafe, despite the low sample size, and a potential impact on faster negative cultures needs to be further explored in larger studies.

(68.7%), followed by Manacapuru, Tabatinga, Itacoatiara and Tefé. There were 1,929 cases reported in indigenous peoples, a majority in communities that are a predominantly indigenous population, such as São Gabriel da Cachoeira, Itamarati and Santa Isabel do Rio Negro (14).

Tuberculosis and HIV
After the onset of Acquired Immunodeficiency Syndrome (AIDS), there was a worldwide increase in the number of TB cases. HIV is the main predisposing factor for the development of TB. The risk of latent form progression is 20 to 37 times higher in HIVpositive (18). In 2014, of the 9.6 million who developed TB worldwide, 1.2 million (12%) had the virus. Africa accounted for 74% of cases. In some parts of the continent, more than 50% were co-infected. Globally, in 2014, 51% of TB patients had a documented HIV test (13). In Brazil, of the 67,966 new cases in 2014, 62.7% were tested for HIV, resulting in 10.4% of TB / HIV co-infection. The highest percentages were in Porto Alegre (28%), Curitiba (22%), Florianópolis (20.4%) and Manaus (20%) (18). For example, it is a test for tuberculosis treatment (2011) aimed at all patients with tuberculosis, poisoning, cold and bad breath (19).
A study conducted in Manaus, Amazonas, Brazil, was a leading cause of death (28%) among 129 autopsied AIDS patients (20). Janneke et al. (2010) reviewed studies that autopsied the HIV-positive series in sub-Saharan Africa over the past two decades, totaling 593 adult autopsies. Infectious diseases were the main post mortem diagnosis, being a more common TB ranging from 20-54% (21). In 2013, 360,000 co-infected people died worldwide, 50% women, making TB one of the leading causes of death in women with reproductive age HIV (12). In 2014, it is estimated that there were 385,000 deaths, 14% of children (13). Therefore, TB remains one of the leading causes of death in people with AIDS, especially in underdeveloped or socio-economic developing countries.

Pathophysiology of tuberculosis
After infection of infectious particles containing living bacilli, these are passed along the trachea, bronchi, bronchioles and reach the alveoli. Along the airways, a respiratory mucosa triggers the first line of defense. The alveoli are formed by type I and II epithelial cells, macrophages, neutrophils and dendritic cells (22). Macrophages are important phagocytes in an initial (or innate) immune response to Mtb. Bacillus phagocytosis involves the participation of different types of receptors and data in intracellular environment, a sequence of events are triggered in the final phase of the process of creating a microorganism. Among these events, we can highlight: the fusion of phagosome with lysosomoma enables the proteolytic pH enzymes to act on the microorganism; production of antimicrobial chemicals such as hydrogen peroxide (reactive oxygen intermediates) and nitric oxide (nitrogen reactive intermediates), toxic metals such as iron, zinc and copper; and cell death by apoptosis (23). The response is immune to the microbiological capacity of phagocytes and Mtb virulence (24).
The dentitic cells, after bacillus phagocytosis, migrate at the site to closer lymph nodes, present Mtb antigenic peptides to CD4 + T lymphocytes, through the expression of the class II complex histocompatibility receptor (MHC) (25). This is a cellular version that represents a late (or acquired) response option that causes lymphocytes to migrate from lymph node tissue to the lung parenchyma, helping phagocytes to present (26).
Macropagyos are antigen presenting strains, but this process occurs without pulmonary alveolus. The control of Mtb, mainly the success of this interaction. The CD4 + T lymphocyte deficit in AIDS shows a cell present in tuberculosis control. Proteins are required for the production of cytokines, which are a function of modulating the action of immune cells. They influence naive CD4 + T lymphocytes to differentiate into different strains. An interleukin (IL) 12 produced by phagocytes induces a production and production of Interferon Gamma (IFN-γ), which is a major cytokine involved in the Mtb immune response. Other cells may produce IFN-γ, but these lymphocytes are the main producers. In contrast, IL-4-induced lineage inhibits IFN-γ production and consequently phagocyte activation (24). When the Th1 immune response predominates the formation of granulomas occurs, which are cell-fighting in the content of spreading the bacilli, which is not wirelessly eradicating them, which features a latent TB (27). If immune surveillance fails, the disease can be reactivated and disseminated. The bacilli tend to concentrate in the center of the often necrotic granuloma and contain macrophages surrounded by fibroblasts, neutrophils, dendritic cells and lymphocytes. Necrosis is called caseosa, from the Latin caseum, which means cheese (28). Although granuloma is intended to contain the spread of the bacillus, some studies suggest that the caseous necrosis environment may be propagated to a multiplication of Mtb (29).
Interference with host defense mechanisms is crucial for the survival of Mtb in the intracellular environment. Some people are able to adapt to the response by escaping host strategies (30). For example, an inhibition of fusion with lysosome represents one of the main mechanisms of bacillus escape from lysosomal hydrolases (31). This block allows the stability of intracellular results and prevents the presentation of antigens necessary for lymphocyte activation (32). Mtb can also counteract a production of reactive oxygen intermediates (33). Other of these mechanisms, one of the late response evocation tactics is the stimulation of IL-10 production by CD4 + T lymphocytes, tending to a Th2-type response, where an IFN-γ secretion is inhibited, favoring Mtb ( 34).
The different manifestations of TB reflect the balance between Mtb and host defense networks (24). In 90% of infected people Mtb is contained in the latent form (35). The spread and elimination of the bacilli is not, first of all sufficient, a lymphatic spread to the satellite lymph node, from which there may be a hematogenous spread, causing extrapulmonary forms of TB. A more common clinical manifestation is lung disease.
Classical complaints may be dry or sputum, haemoptysis, fever, night sweats, asthenia and weight loss. Extrapulmonary tuberculosis can occur in any case and follow-up is local (36).

The role of glutathione and N-acetylcysteine
Glutathione is a tripeptide synthesized by the media from cysteine sequential chain to acid glutamic, followed by addition of glycine (γ-glutamylcysteinylglycine). The cysteine sulfhydryl group (-SH) is the active componente. This tripeptide is present in most cells of the human organism and occupies the functions of functions, among them the maintenance of the cellular pathway. One of the mechanisms used to measure intracellular mortality of microorganisms is the production of oxidants such as reactive oxygen and nitrogen intermediates. Also important for this process is a simultaneous synthesis of antioxidants, a process of protection such as host cells against the effects of this bactericidal mechanism. Glutathione is one of the most important antioxidants produced by the host. The imbalance between oxidants and antioxidants is called oxidative stress, leading to decreased levels of glutathione and its precursors (38,39). Its action on system modulation has also been launched, particularly on lymphocytes. Low levels of glutathione may inhibit as cytokines in a Th1 response and induce apoptosis of CD4 + T lymphocytes (40,41).
Unlike some antioxidants, they can also be reverted to a synthesis of glutathione (39).
Also, the administration of glutathione is not aerogenized due to its bioavailability and its limited ability to cross a phospholipid bilayer of cells. Similarly, a cysteine undergoes rapid oxidation to its disulfide, cystine, which has poor solubility. N-acetylcysteine (NAC) first made its report during the 1960s, when it proved to be an effective mucolytic agent in individuals with cystic fibrosis. Subsequently, a new role in investigating its therapeutic potential in acetaminophen poisoning. Cleavage of the acetyl group makes cysteine available for later adoption in glutathione synthesis (Figure 1 Reduced glutathione levels are also measured in AIDS patients (50). Guerra et al. (2011) demonstrated that HIV-negative levels have CD4 + T lymphocyte glutathione levels when compared to healthy control, and these levels are restored after in vivo supplementation with NAC, favoring cytokine production in a Th1 response. We also observed that glutathione depletion in lymphocytes correlated with one of the levels of tumor necrosis factor (TNF) and free radicals. TNF is a proinflammatory cytokine associated with the progression of HIV and the maintenance of inflammatory damage (50). The reactive energies of oxidized oxygen are involved in the activation of nuclear factor-kB (NF-kB), which controls gene transcription for HIV replication (51). The hypothesis of its response is Th1 immune and has a direct antimicrobial action against HIV, which justifies this study. This proposal has become more possible when you built the effect of hepatoprotectant on mycobacteriostats.

General objective
To evaluate the effects of NAC in individuals coinfected with HIV;

Specific objectives
• Evaluate the safety, tolerance and effectiveness of adjuvant therapy in the treatment of tuberculosis; • Observe smear and culture conversion rates for mycobacteria in the first 8 weeks of NAC use;

Study Design
RIPENACTB is a phase II, randomized, controlled, open, single-group, parallel clinical trial.

Study site
The study will be conducted at the Tropical Medicine Foundation, in the city of Manaus, capital of the state of Amazonas. FMT-HVD is an institution of the Government of the State of Amazonas, created in 1974, which has as national and international reference the medical assistance, teaching and research in infectious and parasitic diseases. The complex is divided into inpatient unit, outpatient unit, clinical analysis laboratories (including TB laboratory, security level III) and research management. -Consent signed and dated prior to initial assessment. There is therefore a reason for withdrawal statement. The same should happen in the two FMT-HVD specialized outpatient clinics to continue the treatment of TB and HIV.

Via tuberculosis laboratory
The FMT-HVD tuberculosis laboratory will be oriented to conduct a series of studies on the positivity of some test for TB, regardless of the biological material analyzed. The physician should investigate the patient's procedure and obtain information necessary to analyze a possibility of inclusion in the study.

Via intensive care unit visits, wards and emergency care
The study team will make daily visits to these groups, along with the care team, for eligibility issues.

Eligible Individuals
If the candidate is eligible, he/she will be invited to participate in the RIPENACTB study through the informed consent form, as determined by the researcher. Unable to understand, IC will be offered to the nearest family member. To be accepted, a proof collection of the RIPENACTB protocol should be performed as needed. There will be an attempt to match a study collection with a technical assistance collection, with the intention of reducing the number of punishments. In the presence of central venous access, a medical record will be aspirated from this device. The results of the RIPENACTB protocol are not D0, occurrences obtained, are valid if they obey the following tolerance intervals: In case of absence, a tolerance of seven days will be given for rescheduling, otherwise it will be considered as impossible to follow up. Babies are advised to come up with a medical indication. Upon completion of TB and FDA treatment, they will be referred to the FMT-HVD HIV Outpatient Clinic for follow-up.

Collection of sputum or tracheal aspirate
The material will be delivered to the FMT-HVD TB lab by the study nurse. As the basins must be subjected to bacilloscopy, Xpert MTB / RIF® (diagnostic only) and culture for continuous liquid mycobacteria. Should be processed within one hour and should be oriented to data processing and reported for the following cases: not identified or misidentified; present leakage; or inappropriate pool. The antimicrobial susceptibility test will be done on sample D0, which is being observed Mtb. NAC is a drug approved by the United States Food and Drug (FDA) and ANVISA, category B in pregnancy. When administered orally it is easily absorbed by the intestinal mucosa. In the liver, it is converted to cysteine to form glutathione, which is then secreted into the circulation. Its half life is 5.6 hours and approximately 30% is excreted by the kidneys. Nausea, vomiting and heartburn may occur, but side effects are uncommon (55). Oral administration of NAC at doses up to 8000 mg / day did not lead to clinically significant adverse reactions (56). Interaction with tuberculostatics is not known.

Medication storage and handling
Study drugs will be stored in a safe place and under appropriate physical conditions. Access will be limited to the researcher and authorized staff. The release will be made during medical consultations registered in the source file "idoctor".

Duration of treatment
The use of NAC and the RIPE scheme will be for two months (intensive phase). The use of rifampicin and isoniazid (maintenance phase) cannot be pre-established because, although this phase lasts an average of four months, it can be extended as directed by a physician.

Dosage and administration
The RIPE regimen will be administered orally or nasogastric tube via the 4 in 1 tablet, with the dose adjusted for weight once a day and preferably for fasting. NAC will be administered orally or via a nasally sachet-shaped effervescent sachet at a dose of 1200 mg (two 600 mg sachets) to be diluted in 200 ml of water and administered once daily.

Treatment group
The examples will be assigned to treatment according to a randomization table.

Medication Accounting
The researcher should document in the medical records the amount of medicines dispensed, administered and administered by a case, returned. Accounting records should be updated until treatment ends.

Treatment adherence
They will be charged for medical appointments such as dispensing medication packages and first-time telephone operators once a week, during working hours, or mandatory SMS or WhatsApp® messages to emphasize adherence to treatment.

Concomitant medications and non-drug therapies
All concomitant medications known during the study should be done in a medical record. There is no indication for concomitant use of any medications.

Treatment after study termination
Not much planned extension time. Upon completion of TB treatment, doctors will be revoked at the FMT-HVD HIV / AIDS outpatient clinic.

Overdose treatment of the investigational drug
An overdose for this study will be calculated as any dose higher than planned. So far there is no one specific to the NAC. Tax on the adverse effect due to overdose of the NAC, the object must be managed with supportive measures and may be hospitalized in PESCLIN, receiving the care of the researcher. After hospital discharge, the main responsible is to return to the tuberculosis treatment routine, in addition to being able to return to treatment. In the presence of a four-or four-year adverse or laboratory problem, researcher discontinuity will be judged by the researcher, as the adverse event must be reported by an external control committee within 24 hours. The chance of being recorded in the patient's medical record must be related to TB treatment.

Primary work
A safety, tolerability and action of the NAC.

Secondary Outcomes
The converse to conversion of bacilloscopy and the culture in the weeks.
Monitor drug hepatotoxicity to tuberculostatics.
To evaluate the effect of NAC on viral load and CD4 + cell count.

Sample Size and Randomization
To not fifty evidence should be planned to be randomized (twenty five in each group), using the table of the program Excel®.

Statistical Analysis
Data will be entered in an Excel® 8.0 spreadsheet and statistical analysis will be performed using the Stata 11.0 statistical program. For a descriptive analysis: 1) means and standard deviation are calculated for continuous variables with normal distribution; 2) medians and interquartile ranges (IQI, 25-75% percentile) for continuous asymmetric distribution variables; 3) proportions for categorical variables. In the bivariate exploratory investigation the chi-square hypothesis test or Fisher's exact test will be applied to the variable variables and the continuous distributed variables will be analyzed with the Student test in the media communication, with or without adjustment of unequal variances. Continuous variables Asymmetry distribution or variables are analyzed using the nonparametric Mann-Whitney or Kruskal-Wallis test to compare the medians. The measure used to calculate the difference between risk variables (RR) and explanatory variables (covariates of interest). Multivariate will be detected by logistic regression model, with derivation of odds ratio (OR or odds ratio). Confusion will be analyzed through non-automatic editing of variables back and forth one by one (backwards, step by step). It will be a protocol data analysis (PP) and an intention-to-treat analysis (ITT).
Confidence interval of 95% (95% CI) and importance values will be considered when less than 0.05. They will be a preliminary investigation when reaching the follow-up of half of the patients to be randomized.

Ethical Considerations
This study will be submitted to the FMT-HVD Research Ethics Committee (CEP), preserving the rights of the research subjects.

Financing
A NAC will be purchased by by INTERFAM. Reference therapy was no longer flat because it is being advocated as first-line TB treatment by the Ministry of Health.

Comments on the feasibility and implementation of the proposal
For the feasibility and execution of the first study shift, data selection is available for the study; financing for a purchase of consumables; eligible patients, as they are a national and international referral center for infectious and parasitic diseases; and partnership with researchers in the area.

Conflicts of Interest
There is no conflict of interest in the development of this study.