Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism.

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here, we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily ("8HUM") can circumvent these problems. The pharmacokinetics, metabolite profiles, and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani, and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery.

Photobiomodulation in promoting increased Skin Flap Viability: a systematic review of animal studies.

Necrosis is common in skin flap surgeries. Photobiomodulation, a noninvasive and effective technique, holds the potential to enhance microcirculation and neovascularization. As such, it has emerged as a viable approach for mitigating the occurrence of skin flap necrosis. The aim of this systematic review was to examine the scientific literature considering the use of photobiomodulation to increase skin-flap viability. The preferred reporting items for systematic reviews and meta-analyses (PRISMA), was used to conducted systematic literature search in the databases PubMed, SCOPUS, Elsevier and, Scielo on June 2023. Included studies investigated skin-flap necrosis employing PBMT irradiation as a treatment and, at least one quantitative measure of skin-flap necrosis in any animal model. Twenty-five studies were selected from 54 original articles that addressed PBMT with low-level laser (LLL) or light-emitting diode (LED) in agreement with the qualifying requirements. Laser parameters varied markedly across studies. In the selected studies, the low-level laser in the visible red spectrum was the most frequently utilized PBMT, although the LED PBMT showed a similar improvement in skin-flap necrosis. Ninety percent of the studies assessing the outcomes of the effects of PBMT reported smaller areas of necrosis in skin flap. Studies have consistently demonstrated the ability of PBMT to improve skin flap viability in animal models. Evidence suggests that PBMT, through enhancing angiogenesis, vascular density, mast cells, and VEGF, is an effective therapy for decrease necrotic tissue in skin flap surgery.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Experience and professional training in the School Health Program.

OBJECTIVE: To analyze the association between experience and professional training in the School Health Program. METHOD: Descriptive, inferential, quantitative and normative study. The data were collected from May to July 2017 through a questionnaire based in the School Health Program, with the participation of professionals from the Family Health Strategy. RESULTS: 105 professionals participated in the study. The average time working in the Family Health Strategy and in the School Health Program is 12.1 and 7.2 years, respectively. 94.3% of the professionals feel qualified to perform the activities of the School Health Program, although only 30.5% have participated in training. There is statistical association between experience and professional training. CONCLUSION: The professionals who conduct activities in the School Health Program undergo few training processes, but feel qualified to carry out the activities proposed.

Effects of periodontal treatment on exacerbation frequency and lung function in patients with chronic periodontitis: study protocol of a 1-year randomized controlled trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has been associated with periodontal disease (PD), and periodontal treatment (PT) has been connected to reduction of lung disease exacerbations. Bronchiectasis has many clinical similarities with COPD but, although it is also a chronic lung disease, to date it has not been studied with relation to PD. The aim of this study is to evaluate whether PT associated with photodynamic therapy (PDT) reduces the number of exacerbations, improves pulmonary function, periodontal clinical parameters and quality of life after 1 year of periodontal treatment follow-up. METHODS: Bronchiectasis patients will undergo medical anamnesis and periodontal examination. Participants with periodontitis will be divided into two groups and PT will be performed as G1 control group (n = 32) - OHO (oral hygiene orientation) + supragingival treatment + simulation of using photodynamic therapy (PDT); G2 experimental (n = 32) - scaling and root planing + PDT + OHO. Lung function will be assessed both at baseline and after 1 year by spirometry, exacerbation history will be analyzed through clinical records monitoring. Three instruments for quality of life assessment will also be applied - Saint George's Respiratory Questionnaire and Impact Profile Analysis Oral health (OHIP-14). It is expected that periodontal treatment can improve the analyzed parameters after 1 year. DISCUSSION: Although only one study evaluates exacerbation in COPD after 1 year of PT, bronchiectasis has not been studied in the dentistry field to date. TRIAL REGISTRATION: NCT02514226. Version #1. This study protocol receives grant from FAPESP (São Paulo Research Foundation) #2015/20535-1. First received: July 22, 2015, 1st version. This protocol has been approved by the Research Ethics Committee of Nove de Julho University.

Analogues of Marine Guanidine Alkaloids Are in Vitro Effective against Trypanosoma cruzi and Selectively Eliminate Leishmania (L.) infantum Intracellular Amastigotes.

Synthetic analogues of marine sponge guanidine alkaloids showed in vitro antiparasitic activity against Leishmania (L.) infantum and Trypanosoma cruzi. Guanidines 10 and 11 presented the highest selectivity index when tested against Leishmania. The antiparasitic activity of 10 and 11 was investigated in host cells and in parasites. Both compounds induced depolarization of mitochondrial membrane potential, upregulation of reactive oxygen species levels, and increased plasma membrane permeability in Leishmania parasites. Immunomodulatory assays suggested an NO-independent effect of guanidines 10 and 11 on macrophages. The same compounds also promoted anti-inflammatory activity in L. (L.) infantum-infected macrophages cocultived with splenocytes, reducing the production of cytokines MCP-1 and IFN-γ. Guanidines 10 and 11 affect the bioenergetic metabolism of Leishmania, with selective elimination of parasites via a host-independent mechanism.

Anti-parasitic Guanidine and Pyrimidine Alkaloids from the Marine Sponge Monanchora arbuscula.

HPLC-UV-ELSD-MS-guided fractionation of the anti-parasitic extract obtained from the marine sponge Monanchora arbuscula, collected off the southeastern coast of Brazil, led to the isolation of a series of guanidine and pyrimidine alkaloids. The pyrimidines monalidine A (1) and arbusculidine A (7), as well as the guanidine alkaloids batzellamide A (8) and hemibatzelladines 9-11, represent new minor constituents that were identified by analysis of spectroscopic data. The total synthesis of monalidine A confirmed its structure. Arbusculidine A (7), related to the ptilocaulin/mirabilin/netamine family of tricyclic guanidine alkaloids, is the first in this family to possess a benzene ring. Batzellamide A (8) and hemibatzelladines 9-11 represent new carbon skeletons that are related to the batzelladines. Evaluation of the anti-parasitic activity of the major known metabolites, batzelladines D (12), F (13), L (14), and nor-L (15), as well as of synthetic monalidine A (1), against Trypanosoma cruzi and Leishmania infantum is also reported, along with a detailed investigation of parasite cell-death pathways promoted by batzelladine L (14) and norbatzelladine L (15).

Alchornedine, a new anti-trypanosomal guanidine alkaloid from Alchornea glandulosa.

Bioactivity-guided fractionation of the MeOH extract from the leaves of Alchornea glandulosa afforded a new guanidine alkaloid named alchornedine, as well as two other inactive derivatives (pteroginine and pteroginidine). The structure of alchornedine, which shows a very rare ring system, was elucidated based on NMR, IR, and MS spectral analyses. This compound displayed antiprotozoal activity against Trypanosoma cruzi (Y strain). By using the MTT assay, the trypomastigotes showed an IC50 value of 93 µg/mL (443 µM), a similar effectiveness to the standard drug benznidazole. Alchornedine also showed activity against the intracellular amastigotes, with an IC50 value of 27 µg/mL (129 µM). Using benznidazole as a standard drug, this guanidine alkaloid was approximately 3-fold more effective against the intracellular form of T. cruzi. The mammalian cytotoxicity of alchornedine was verified against NCTC cells and demonstrated an IC50 of 50 µg/mL (237 µM), but this compound demonstrated a selective elimination of parasites inside macrophages without affecting the morphology of the host cells. Alchornedine was effective against both clinical forms of T. cruzi and could be used as a scaffold for future drug design studies against American trypanosomiasis.

Conjugation to 4-aminoquinoline improves the anti-trypanosomal activity of Deferiprone-type iron chelators.

Iron is an essential growth component in all living organisms and plays a central role in numerous biochemical processes due to its redox potential and high affinity for oxygen. The use of iron chelators has been suggested as a novel therapeutic approach towards parasitic infections, such as malaria, sleeping sickness and leishmaniasis. Known iron chelating agents such as Deferoxamine and the 3-hydroxypyridin-4-one (HPO) Deferiprone possess anti-parasitic activity but suffer from mammalian toxicity, relatively modest potency, and/or poor oral availability. In this study, we have developed novel derivatives of Deferiprone with increased anti-parasitic activity and reduced cytotoxicity against human cell lines. Of particular interest are several new derivatives in which the HPO scaffold has been conjugated, via a linker, to the 4-aminoquinoline ring system present in the known anti-malaria drug Chloroquine. We report the inhibitory activity of these novel analogues against four parasitic protozoa, Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum, and, for direct comparison, against human cells lines. We also present data, which support the hypothesis that iron starvation is the major cause of growth inhibition of these new Deferiprone-Chloroquine conjugates in T. brucei.

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi.

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25-0.68 µM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 µM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50=34 µM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX(®) Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.

[Effectiveness in the diagnosis of tuberculosis in Foz do Iguaçu, the triple-border area of Brazil, Paraguay and Argentina]. / Efetividade no diagnóstico da tuberculose em Foz do Iguaçu, tríplice fronteira Brasil, Paraguai e Argentina.

This study sought to assess the effectiveness of health services in the diagnosis of tuberculosis in Foz do Iguaçu-PR, the triple border region of Brazil, Paraguay, and Argentina. In this epidemiologic, cross-sectional study, 101 persons with tuberculosis were interviewed in 2009 by using an instrument based on the Primary Care Assessment Tool . The analysis was based on proportions and respective 95% confidence intervals (95%) and means. Emergency units (37%) and primary health care units (26%) were the most sought units. Access to medical consultation on the same day reached 70%, but tuberculosis was suspected in less than 47% of patients; bacilloscopy was conducted in 50% of patients. We conclude that although these services provide rapid care, they do not determine the true diagnosis and lead the patient to seek specialized services. Specialty services are more effective in establishing the correct diagnosis. In the triple border region, seeking care at a primary health care unit led to extra time and more returns to the hospital for a tuberculosis diagnosis.

RES-Seq-a barcoded library of drug-resistant Leishmania donovani allowing rapid assessment of cross-resistance and relative fitness.

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. New therapeutic options with diverse mechanisms of actions (MoAs) are required to consolidate progress toward control of this disease and combat drug resistance. Here, we describe the development of a scalable resistance library screen (RES-Seq) as a tool to facilitate the identification and prioritization of anti-leishmanial compounds acting via novel MoA. We have amassed a large collection of Leishmania donovani cell lines resistant to frontline drugs and compounds in the VL pipeline, with resistance-conferring mutations fully characterized. New phenotypic hits screened against this highly curated panel of resistant lines can determine cross-resistance and potentially shared MoA. The ability to efficiently identify compounds acting via previously established MoA is vital to maintain diversity within drug development portfolios. To expedite screening, short identifier DNA barcodes were introduced into resistant clones enabling pooling and simultaneous screening of multiple cell lines. Illumina sequencing of barcodes enables the growth kinetics and relative fitness of multiple cell lines under compound selection to be tracked. Optimal conditions allowing discrimination of resistant and sensitive clones were established (3× and 10× EC50 for 3 days) and applied to screening of a complex library with VL preclinical and clinical drug candidates. RES-Seq is set to play an important role in ensuring that anti-leishmanial compounds exploiting diverse mechanisms of action are developed, ultimately providing options for future drug combination strategies.IMPORTANCEVisceral leishmaniasis (VL) remains the third largest parasitic killer worldwide, responsible for 20,000-30,000 deaths each year. Control and ultimate elimination of VL will require a range of therapeutic options with diverse mechanisms of action to combat drug resistance. One approach to ensure that compounds in development exploit diverse mechanisms of action is to screen them against highly curated cell lines resistant to drugs already in the VL pipeline. The identification of cross-resistant cell lines indicates that test compounds are likely acting via previously established mechanisms. Current cross-resistance screens are limited by the requirement to profile individual resistant cell lines one at a time. Here, we introduce unique DNA barcodes into multiple resistant cell lines to facilitate parallel profiling. Utilizing the power of Illumina sequencing, growth kinetics and relative fitness under compound selection can be monitored revolutionizing our ability to identify and prioritize compounds acting via novel mechanisms.

The role of invariant surface glycoprotein 75 in xenobiotic acquisition by African trypanosomes.

The surface proteins of parasitic protozoa mediate functions essential to survival within a host, including nutrient accumulation, environmental sensing and immune evasion. Several receptors involved in nutrient uptake and defence from the innate immune response have been described in African trypanosomes and, together with antigenic variation, contribute towards persistence within vertebrate hosts. Significantly, a superfamily of invariant surface glycoproteins (ISGs) populates the trypanosome surface, one of which, ISG75, is implicated in uptake of the century-old drug suramin. By CRISPR/Cas9 knockout and biophysical analysis, we show here that ISG75 directly binds suramin and mediates uptake of additional naphthol-related compounds, making ISG75 a conduit for entry of at least one structural class of trypanocidal compounds. However, ISG75 null cells present only modest attenuation of suramin sensitivity, have unaltered viability in vivo and in vitro and no alteration to suramin-invoked proteome responses. While ISG75 is demonstrated as a valid suramin cell entry pathway, we suggest the presence of additional mechanisms for suramin accumulation, further demonstrating the complexity of trypanosomatid drug interactions and potential for evolution of resistance.

Development and validation of an instrument for the evaluation of HIV care in Primary Health Care.

OBJECTIVE: To develop and validate an instrument to evaluate the decentralization process of care for People Living with HIV in Primary Health Care. METHOD: Methodological study, developed in four stages: elaboration of the logical model based on the triad Structure-Process-Outcomes; development of the instrument; content validation by expert judges and technical reviewers; and semantic validation. Online questionnaires were used, and the Kappa index was used for analysis. RESULTS: The instrument with 68 items and 8 factors was submitted to validation by expert judges who recommended the exclusion of 3 items and the alteration of 2 factors. In the validation by technical reviewers, 2 items were excluded and 6 factors were highlighted; the agreement index was ≥0.75. In the semantic validation, 87.3% of the judges answered "totally agree" for the items presented. CONCLUSION: The instrument is validated for its content, has 63 items and has the potential to assess the care provided for people living with HIV in Primary Health Care.

Structure-Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.

Intersections between rural women's resilience and quality of life: a mixed-methods study. / Interseções entre resiliência e qualidade de vida em mulheres rurais: estudo de métodos mistos.

OBJECTIVE: to analyze the intersections between rural women's quality of life and resilience. METHOD: convergent mixed methods design in which a cross-sectional quantitative study is triangulated with a qualitative study guided by Oral History. Data were collected concomitantly, using a socio-demographic form, Resilience Scale, Medical Outcomes Study 36-Item Short-Form Health Survey, and open-ended interviews. The analysis was based on descriptive and inferential statistics and inductive thematic analysis, which was integrated later. RESULTS: an association was found between the social aspects domain of quality of life and a moderate level of resilience related to the characteristics of life in rural areas. The integration of results enabled verifying that these two constructs (which mutually influence each other) are mediated by protective factors, resilience developed by the rural women, such as spirituality and the formation of social support, enchantment, and a feeling of belonging to their context. CONCLUSION: by developing protective factors, rural women develop a resilient behavior that favors their quality of life. Identifying these factors enables the development of psychosocial interventions to promote rural women's health.

The use of phenomenological frameworks in Brazilian nursing research at the stricto sensu level: an overview.

OBJECTIVE: To characterize the stricto sensu scientific production of Brazilian nursing that resorts to phenomenological theoretical frameworks. METHOD: Descriptive, exploratory, and document study carried out using the CAPES catalog of theses and dissertations from January to April 2022. RESULT: The sample included 600 dissertations and theses, with a predominance of the Heideggerian framework, followed by Schütz and Merleau-Ponty. Topics discussed included nursing care in women's health, mental health, pediatric and adolescent nursing, oncology nursing, obstetric nursing, Primary Health Care, as well as family and nursing education. CONCLUSION: Phenomenology was found to be a powerful reference for the unveiling of the phenomena of interest to the field of nursing, contributing to the construction of knowledge about the being who cares, the being who is cared for, and the care itself.

Elements that influenced immediate mother-neonate contact during the golden hour.

OBJECTIVE: To characterize the elements that influenced the immediate mother-neonate contact during the golden hour. METHOD: A cross-sectional observational study with a quantitative approach. A total of 105 parturient women hospitalized in two maternity hospitals with usual risk were observed. The instrument was based on Brazilian National Normal Childbirth Care Guidelines and World Health Organization good obstetric practices, totaling 36 questions. The analysis took place in a descriptive way using the Chi-Square Test for proportion comparison. RESULTS: Of the parturient women, 2.8% (n = 3) experienced the golden hour, and 82.9% (n = 87), immediate contact between 1 and 5 minutes. In 85.7% (n = 90) of the group, there were no causes that contraindicated immediate contact. For 48.0% (n = 49) of participants, contact was re-established by the nursing staff within 31-60 minutes. CONCLUSION: Immediate contact during the golden hour had low hospital care compliance. Neonatal procedures that can be postponed predominated as influencing elements of the golden hour. The assistance observed in the birth rooms investigated reflects the need to reduce interventions in labor and birth.