Reflex strategy to ensure accurate total testosterone results from consumer initiated, self-collected capillary samples.

BACKGROUND: Self-collected capillary samples are convenient for direct access testing (DAT), but exogenous testosterone use may cause falsely elevated total testosterone (TT) results. We designed a quality assurance workflow to differentiate between accurate or erroneous supraphysiological TT concentrations. METHODS: Clinical samples with TT > 1500 ng/dL were reflexed to luteinizing hormone (LH) and follicle stimulating hormone (FSH) and screened for exogenous testosterone use. Samples (n = 120) with normal TT were reflexed to LH/FSH as a control. RESULTS: A total of 8572 TT samples were evaluated, of which 533 (6.2 %) had TT > 1500 ng/dL and were reflexed. Of these, 441 (82.7 %) had significantly decreased LH/FSH (<0.85/<0.7mIU/mL, respectively), 72 (13.5 %) had normal or borderline normal LH/FSH, and 20 (3.8 %) had insufficient plasma volume. In patients with TT > 1500 ng/dL, injectable exogenous testosterone use was most commonly accompanied by significantly decreased LH/FSH, while topical testosterone use was most commonly accompanied by detectable LH/FSH. Control samples were almost all (99.2 %) within or above the LH/FSH reference intervals. Unique patients ordered 351 TT tests where at least one TT result was > 1500 ng/dL. Based on TT and LH/FSH results, we hypothesized that patients were intermittently or consistently overusing exogenous testosterone, resolved elevated TT with recollection, or repeatedly contaminated their sample. CONCLUSION: Self-collected capillary specimens are acceptable for TT testing. A quality assurance reflex to LH/FSH can determine the validity of supraphysiological TT results in a consumer initiated/DAT population.

Frenotomy with breastfeeding support versus breastfeeding support alone for infants with tongue-tie and breastfeeding difficulties: the FROSTTIE RCT.

Background: Tongue-tie can be diagnosed in 3-11% of babies, with some studies reporting almost universal breastfeeding difficulties, and others reporting very few feeding difficulties that relate to the tongue-tie itself, instead noting that incorrect positioning and attachment are the primary reasons behind the observed breastfeeding difficulties and not the tongue-tie itself. The only existing trials of frenotomy are small and underpowered and/or include only very short-term or subjective outcomes. Objective: To investigate whether frenotomy is clinically and cost-effective to promote continuation of breastfeeding at 3 months in infants with breastfeeding difficulties diagnosed with tongue-tie. Design: A multicentre, unblinded, randomised, parallel group controlled trial. Setting: Twelve infant feeding services in the UK. Participants: Infants aged up to 10 weeks referred to an infant feeding service (by a parent, midwife or other breastfeeding support service) with breastfeeding difficulties and judged to have tongue-tie. Interventions: Infants were randomly allocated to frenotomy with standard breastfeeding support or standard breastfeeding support without frenotomy. Main outcome measures: Primary outcome was any breastmilk feeding at 3 months according to maternal self-report. Secondary outcomes included mother's pain, exclusive breastmilk feeding, exclusive direct breastfeeding, frenotomy, adverse events, maternal anxiety and depression, maternal and infant NHS health-care resource use, cost-effectiveness, and any breastmilk feeding at 6 months of age. Results: Between March 2019 and November 2020, 169 infants were randomised, 80 to the frenotomy with breastfeeding support arm and 89 to the breastfeeding support arm from a planned sample size of 870 infants. The trial was stopped in the context of the COVID-19 pandemic due to withdrawal of breastfeeding support services, slow recruitment and crossover between arms. In the frenotomy with breastfeeding support arm 74/80 infants (93%) received their allocated intervention, compared to 23/89 (26%) in the breastfeeding support arm. Primary outcome data were available for 163/169 infants (96%). There was no evidence of a difference between the arms in the rate of breastmilk feeding at 3 months, which was high in both groups (67/76, 88% vs. 75/87, 86%; adjusted risk ratio 1.02, 95% confidence interval 0.90 to 1.16). Adverse events were reported for three infants after surgery [bleeding (n = 1), salivary duct damage (n = 1), accidental cut to the tongue and salivary duct damage (n = 1)]. Cost-effectiveness could not be determined with the information available. Limitations: The statistical power of the analysis was extremely limited due to not achieving the target sample size and the high proportion of infants in the breastfeeding support arm who underwent frenotomy. Conclusions: This trial does not provide sufficient information to assess whether frenotomy in addition to breastfeeding support improves breastfeeding rates in infants diagnosed with tongue-tie. Future work: There is a clear lack of equipoise in the UK concerning the use of frenotomy, however, the effectiveness and cost-effectiveness of the procedure still need to be established. Other study designs will need to be considered to address this objective. Trial registration: This trial is registered as ISRCTN 10268851. Funding: This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment Programme (project number 16/143/01) and will be published in full in Health Technology Assessment; Vol. 27, No. 11. See the NIHR Journals Library website for further project information. The funder had no role in study design or data collection, analysis and interpretation. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

Many mothers and babies experience difficulties in establishing breastfeeding. In some babies it is thought that their difficulties may be linked to a condition called tongue-tie, in which a piece of skin tightly joins the middle part of the underside of the tongue to the base of the baby's mouth. This can be treated by an operation to divide the tight part/skin in the middle of the underneath of the tongue. We planned to carry out a trial of 870 babies to find out whether an operation together with breastfeeding support helps more mothers and babies with tongue-tie to continue breastfeeding until the baby is 3 months old compared to breastfeeding support on its own and whether the costs were different between the two groups of mothers and babies. We were only able to recruit 169 babies as the trial was stopped because of slow recruitment, changes to services in the COVID-19 pandemic and a high proportion of the babies in the breastfeeding support group going on to have an operation. There were no differences in the rate of breastfeeding at 3 months between the babies in the group who had an operation straightaway and those in the group that had breastfeeding support alone, or had an operation later. More than four in every five babies in both groups were still breastmilk feeding at 3 months. Three babies who had an operation, around 1 in 50 babies, had a complication of the operation (bleeding, scarring or a cut to the tube that makes saliva). Because of the small size of the study, we cannot say whether an operation to divide a tongue-tie along with breastfeeding support helps babies with tongue-tie and breastfeeding difficulties or has different costs. We will need to try different types of research to answer the question.

Embryonal tumor with multilayered rosettes: Post-treatment maturation and implications for future therapy.

BACKGROUND: Embryonal tumor with multilayered rosettes (ETMR) is a deadly grade IV pediatric brain tumor. Despite an intensive multimodal treatment approach that includes surgical resection, high-dose chemotherapy, and radiotherapy, the progression-free survival at 5 years is less than 30%. CASE: We report a case of long-term survival in a 5-month old female with a large mass in the posterior fossa, diagnosed as ETMR, which subsequently underwent treatment-induced maturation. Prior to chemotherapy, histopathology revealed an abundance of highly proliferative, undifferentiated cells and multilayered rosette structures. Conversely, post-treatment histopathology revealed cell populations that differentiated into neuronal and ganglionic phenotypes. At 5-year follow-up, the patient remains progression-free. CONCLUSION: This finding contributes to the few reports to date of post-treatment differentiation/maturation of ETMR cell populations, with an implication for less cytotoxic therapeutic interventions aimed at differentiation.

Mass spectrometry imaging discriminates glioblastoma tumor cell subpopulations and different microvascular formations based on their lipid profiles.

Glioblastoma is a prevalent malignant brain tumor and despite clinical intervention, tumor recurrence is frequent and usually fatal. Genomic investigations have provided a greater understanding of molecular heterogeneity in glioblastoma, yet there are still no curative treatments, and the prognosis has remained unchanged. The aggressive nature of glioblastoma is attributed to the heterogeneity in tumor cell subpopulations and aberrant microvascular proliferation. Ganglioside-directed immunotherapy and membrane lipid therapy have shown efficacy in the treatment of glioblastoma. To truly harness these novel therapeutics and develop a regimen that improves clinical outcome, a greater understanding of the altered lipidomic profiles within the glioblastoma tumor microenvironment is urgently needed. In this work, high resolution mass spectrometry imaging was utilized to investigate lipid heterogeneity in human glioblastoma samples. Data presented offers the first insight into the histology-specific accumulation of lipids involved in cell metabolism and signaling. Cardiolipins, phosphatidylinositol, ceramide-1-phosphate, and gangliosides, including the glioblastoma stem cell marker, GD3, were shown to differentially accumulate in tumor and endothelial cell subpopulations. Conversely, a reduction in sphingomyelins and sulfatides were detected in tumor cell regions. Cellular accumulation for each lipid class was dependent upon their fatty acid residue composition, highlighting the importance of understanding lipid structure-function relationships. Discriminating ions were identified and correlated to histopathology and Ki67 proliferation index. These results identified multiple lipids within the glioblastoma microenvironment that warrant further investigation for the development of predictive biomarkers and lipid-based therapeutics.

Spatial quantitation of antibiotics in bone tissue compartments by laser-capture microdissection coupled with UHPLC-tandem mass spectrometry.

Bones are the site of multiple diseases requiring chemotherapy, including cancer, arthritis, osteoporosis and infections. Yet limited methodologies are available to investigate the spatial distribution and quantitation of small molecule drugs in bone compartments, due to the difficulty of sectioning undecalcified bones and the interference of decalcification methods with spatially resolved drug quantitation. To measure drug concentrations in distinct anatomical bone regions, we have developed a workflow that enables spatial quantitation of thin undecalcified bone sections by laser-capture microdissection coupled to HPLC/tandem mass spectrometry, and spatial mapping on adjacent sections by mass spectrometry imaging. The adhesive film and staining methods were optimized to facilitate histology staining on the same sections used for mass spectrometry image acquisition, revealing drug accumulation in the underlying bone tissue architecture, for the first time. Absolute spatial concentrations of rifampicin, bedaquiline, doxycycline, vancomycin and several of their active metabolites are shown for both small rodent bones and larger rabbit bones that more closely resemble human bone density. Overlaid MALDI mass spectrometry images of drugs and histology staining enabled the generation of semi-quantitative data from regions of interest within anatomical bone compartments. These data correlated with absolute drug concentrations determined by HPLC-MS/MS in laser-capture microdissection samples. Collectively, these techniques enable semi- and fully quantitative drug distribution investigations within bone tissue compartments for the first time. Our workflow can be translated to image and quantify not only drugs but also biomarkers of disease to investigate drug penetration as well as mechanisms underlying bone disorders.

Circulating Exosome Cargoes Contain Functionally Diverse Cancer Biomarkers: From Biogenesis and Function to Purification and Potential Translational Utility.

Although diagnostic and therapeutic treatments of cancer have tremendously improved over the past two decades, the indolent nature of its symptoms has made early detection challenging. Thus, inter-disciplinary (genomic, transcriptomic, proteomic, and lipidomic) research efforts have been focused on the non-invasive identification of unique "silver bullet" cancer biomarkers for the design of ultra-sensitive molecular diagnostic assays. Circulating tumor biomarkers, such as CTCs and ctDNAs, which are released by tumors in the circulation, have already demonstrated their clinical utility for the non-invasive detection of certain solid tumors. Considering that exosomes are actively produced by all cells, including tumor cells, and can be found in the circulation, they have been extensively assessed for their potential as a source of circulating cell-specific biomarkers. Exosomes are particularly appealing because they represent a stable and encapsulated reservoir of active biological compounds that may be useful for the non-invasive detection of cancer. T biogenesis of these extracellular vesicles is profoundly altered during carcinogenesis, but because they harbor unique or uniquely combined surface proteins, cancer biomarker studies have been focused on their purification from biofluids, for the analysis of their RNA, DNA, protein, and lipid cargoes. In this review, we evaluate the biogenesis of normal and cancer exosomes, provide extensive information on the state of the art, the current purification methods, and the technologies employed for genomic, transcriptomic, proteomic, and lipidomic evaluation of their cargoes. Our thorough examination of the literature highlights the current limitations and promising future of exosomes as a liquid biopsy for the identification of circulating tumor biomarkers.

Decreasing the Lower Limit of Quantitation for Urine Albumin Improves Clinical Utility.

BACKGROUND: Urine albumin-to-creatinine ratio (uACR) is a screening assay for chronic kidney disease (CKD). A value of >30 mg/g is flagged abnormal, but lower ratios have prognostic implications. Thus, to maximize diagnostic utility, urine albumin (uAlb) should be measurable to 3 mg/L to match the lowest creatinine concentration generally utilized (10 mg/dL). Most uAlb assays have lower limits of quantitation (LLOQs) 2- to 4-fold higher. We sought to determine the performance characteristics of a commonly used uAlb assay at 3 mg/L and to evaluate the clinical screening impact of reducing the LLOQ. METHODS: Urine was serially diluted to assess uAlb linearity and precision for concentrations near the claimed LLOQ (12 mg/L). Samples (n = 30) with uAlb <12 mg/L were compared between laboratories. Sequential samples (n = 1239) were evaluated for clinical impact of reducing the measuring range to 3 mg/L. RESULTS: The assay was linear to 1.6 mg/L. Interday precision at 3.7 mg/L and 4.3 mg/L was 7.7% and 8.6%, respectively. Minimal bias was observed between labs (y = 1.091x - 0.75; average bias = -0.13 mg/L). Clinical validation demonstrated 501 of 1239 samples (40.4%) had uAlb <12 mg/L. Using 11.9 mg/L as the numerator for samples with uAlb <12 mg/dL and urine creatinine >10 mg/L, 107 of 499 (21.4%) would have a ratio flagged abnormal at >30 mg/g. Using the numeric value for these samples to 3 mg/L reduced alarm to <1%. CONCLUSIONS: A uAlb LLOQ of 3 mg/L improves screening utility of uACR by simplifying reporting and clinical interpretation when uAlb is low and provides clinical information for prognostic tools developed for people at risk of CKD.

Drug concentration at the site of disease in children with pulmonary tuberculosis.

BACKGROUND: Current TB treatment for children is not optimized to provide adequate drug levels in TB lesions. Dose optimization of first-line antituberculosis drugs to increase exposure at the site of disease could facilitate more optimal treatment and future treatment-shortening strategies across the disease spectrum in children with pulmonary TB. OBJECTIVES: To determine the concentrations of first-line antituberculosis drugs at the site of disease in children with intrathoracic TB. METHODS: We quantified drug concentrations in tissue samples from 13 children, median age 8.6 months, with complicated forms of pulmonary TB requiring bronchoscopy or transthoracic surgical lymph node decompression in a tertiary hospital in Cape Town, South Africa. Pharmacokinetic models were used to describe drug penetration characteristics and to simulate concentration profiles for bronchoalveolar lavage, homogenized lymph nodes, and cellular and necrotic lymph node lesions. RESULTS: Isoniazid, rifampicin and pyrazinamide showed lower penetration in most lymph node areas compared with plasma, while ethambutol accumulated in tissue. None of the drugs studied was able to reach target concentration in necrotic lesions. CONCLUSIONS: Despite similar penetration characteristics compared with adults, low plasma exposures in children led to low site of disease exposures for all drugs except for isoniazid.

Alterations of human skin microbiome and expansion of antimicrobial resistance after systemic antibiotics.

Although systemic antibiotics are critical in controlling infections and reducing morbidity and mortality, overuse of antibiotics is presumed to contribute to negative repercussions such as selection of antimicrobial-resistant organisms and collateral damage to commensal microbes. In a prospective, randomized study of four clinically relevant antibiotic regimens [doxycycline (20 mg or 100 mg), cephalexin, or trimethoprim/sulfamethoxazole], we investigated microbial alterations on skin after administration of systemic antibiotics to healthy human volunteers. Samples from different skin and oral sites, as well as stool, were collected before, during, and up to 1 year after antibiotic use, and shotgun metagenomic sequencing was performed. Taxonomic analysis showed that subjects receiving doxycycline 100 mg and trimethoprim/sulfamethoxazole (TMP/SMX) exhibited greater changes to their skin microbial communities, as compared to those receiving other regimens or untreated controls. Oral and stool microbiota also demonstrated fluctuations after antibiotics. Bacterial culturing in combination with whole-genome sequencing revealed specific emergence, expansion, and persistence of antibiotic-resistant staphylococci harboring tetK or tetL and dfrC or dfrG genes in all subjects who received doxycycline 100 mg or TMP/SMX, respectively. Last, analysis of metagenomic data revealed an increase of genes involved in gene mobilization, indicating stress responses of microbes to antibiotics. Collectively, these findings demonstrate direct, long-lasting effects of antibiotics on skin microbial communities, highlighting the skin microbiome as a site for the development and persistence of antibiotic resistance and the risks of overprescribing.

On-Slide Heat Sterilization Enables Mass Spectrometry Imaging of Tissue Infected with High-Threat Pathogens Outside of Biocontainment: A Study Directed at Mycobacterium tuberculosis.

Mass spectrometry imaging investigations of tissues infected with agents that require high-security biocontainment, such as Mycobacterium tuberculosis, have been limited due to incompatible sterilization techniques. Here we describe an on-slide heat sterilization method that enables mass spectrometry imaging investigations of pharmaceuticals, lipids, and metabolites in infected tissue samples outside of biocontainment. An evaluation of different temperatures and incubation times determined that 100 °C for 1 h was essential to sterilize 5 times the bacterial burden observed in tuberculosis (TB) cavity sections. Laser-capture microdissection combined with liquid chromatography with tandem mass spectrometry quantitation, in addition to mass spectrometry imaging, showed that no degradation was observed following the on-slide heat sterilization protocol for a variety of drug classes covering a range of physicochemical properties. Utilizing the tissue mimetic model, we demonstrated that the detection of lipid and metabolite ions was not impacted by heat sterilization and that, for several metabolites, the on-slide heat sterilization method improved the sensitivity when compared to control samples. An application of the on-slide heat sterilization to M. tuberculosis infected tissue enabled the first detection and spatial distribution of lipids indicative of a lysosomal storage disease phenotype within TB granuloma macrophages, in addition to the differential distribution of metabolites central to the fatty acid oxidation pathway. These initial investigations detected a pronounced heterogeneity within the cellular regions and necrotic cores of individual TB granulomas and across different evolving granulomas. This study provides the framework for mass spectrometry imaging investigations of high-threat pathogens outside of biocontainment.

In Vitro Miniaturized Tuberculosis Spheroid Model.

Tuberculosis (TB) is a public health concern that impacts 10 million people around the world. Current in vitro models are low throughput and/or lack caseation, which impairs drug effectiveness in humans. Here, we report the generation of THP-1 human monocyte/macrophage spheroids housing mycobacteria (TB spheroids). These TB spheroids have a central core of dead cells co-localized with mycobacteria and are hypoxic. TB spheroids exhibit higher levels of pro-inflammatory factor TNFα and growth factors G-CSF and VEGF when compared to non-infected control. TB spheroids show high levels of lipid deposition, characterized by MALDI mass spectrometry imaging. TB spheroids infected with strains of differential virulence, Mycobacterium tuberculosis (Mtb) HN878 and CDC1551 vary in response to Isoniazid and Rifampicin. Finally, we adapt the spheroid model to form peripheral blood mononuclear cells (PBMCs) and lung fibroblasts (NHLF) 3D co-cultures. These results pave the way for the development of new strategies for disease modeling and therapeutic discovery.

Comparative Analysis of Pharmacodynamics in the C3HeB/FeJ Mouse Tuberculosis Model for DprE1 Inhibitors TBA-7371, PBTZ169, and OPC-167832.

Multiple drug discovery initiatives for tuberculosis are currently ongoing to identify and develop new potent drugs with novel targets in order to shorten treatment duration. One of the drug classes with a new mode of action is DprE1 inhibitors targeting an essential process in cell wall synthesis of Mycobacterium tuberculosis. In this investigation, three DprE1 inhibitors currently in clinical trials, TBA-7371, PBTZ169, and OPC-167832, were evaluated side-by-side as single agents in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon tuberculosis infection. The goal was to confirm the efficacy of the DprE1 inhibitors in a mouse tuberculosis model with advanced pulmonary pathology and perform comprehensive analysis of plasma, lung, and lesion-centric drug levels to establish pharmacokinetic-pharmacodynamic (PK-PD) parameters predicting efficacy at the site of infection. Results showed significant efficacy for all three DprE1 inhibitors in the C3HeB/FeJ mouse model after 2 months of treatment. Superior efficacy was observed for OPC-167832 even at low-dose levels, which can be attributed to its low MIC, favorable distribution, and sustained retention above the MIC throughout the dosing interval in caseous necrotic lesions, where the majority of bacteria reside in C3HeB/FeJ mice. These results support further progression of the three drug candidates through clinical development for tuberculosis treatment.

Lesion Penetration and Activity Limit the Utility of Second-Line Injectable Agents in Pulmonary Tuberculosis.

Amikacin and kanamycin are second-line injectables used in the treatment of multidrug-resistant tuberculosis (MDR-TB) based on the clinical utility of streptomycin, another aminoglycoside and first-line anti-TB drug. While streptomycin was tested as a single agent in the first controlled TB clinical trial, introduction of amikacin and kanamycin into MDR-TB regimens was not preceded by randomized controlled trials. A recent large retrospective meta-analysis revealed that compared with regimens without any injectable drug, amikacin provided modest benefits, and kanamycin was associated with worse outcomes. Although their long-term use can cause irreversible ototoxicity, they remain part of MDR-TB regimens because they have a role in preventing emergence of resistance to other drugs. To quantify the contribution of amikacin and kanamycin to second-line regimens, we applied two-dimensional matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging in large lung lesions, quantified drug exposure in lung and in lesions of rabbits with active TB, and measured the concentrations required to kill or inhibit growth of the resident bacterial populations. Using these metrics, we applied site-of-action pharmacokinetic and pharmacodynamic (PK-PD) concepts and simulated drug coverage in patients' lung lesions. The results provide a pharmacological explanation for the limited clinical utility of both agents and reveal better PK-PD lesion coverage for amikacin than kanamycin, consistent with retrospective data of contribution to treatment success. Together with recent mechanistic studies dissecting antibacterial activity from aminoglycoside ototoxicity, the limited but rapid penetration of streptomycin, amikacin, and kanamycin to the sites of TB disease supports the development of analogs with improved efficacy and tolerability.

Pharmacokinetics and Target Attainment of SQ109 in Plasma and Human-Like Tuberculosis Lesions in Rabbits.

SQ109 is a novel well-tolerated drug candidate in clinical development for the treatment of drug-resistant tuberculosis (TB). It is the only inhibitor of the MmpL3 mycolic acid transporter in clinical development. No SQ109-resistant mutant has been directly isolated thus far in vitro, in mice, or in patients, which is tentatively attributed to its multiple targets. It is considered a potential replacement for poorly tolerated components of multidrug-resistant TB regimens. To prioritize SQ109-containing combinations with the best potential for cure and treatment shortening, one must understand its contribution against different bacterial populations in pulmonary lesions. Here, we have characterized the pharmacokinetics of SQ109 in the rabbit model of active TB and its penetration at the sites of disease-lung tissue, cellular and necrotic lesions, and caseum. A two-compartment model with first-order absorption and elimination described the plasma pharmacokinetics. At the human-equivalent dose, parameter estimates fell within the ranges published for preclinical species. Tissue concentrations were modeled using an "effect" compartment, showing high accumulation in lung and cellular lesion areas with penetration coefficients in excess of 1,000 and lower passive diffusion in caseum after 7 daily doses. These results, together with the hydrophobic nature and high nonspecific caseum binding of SQ109, suggest that multiweek dosing would be required to reach steady state in caseum and poorly vascularized compartments, similar to bedaquiline. Linking lesion pharmacokinetics to SQ109 potency in assays against replicating, nonreplicating, and intracellular M. tuberculosis showed SQ109 concentrations markedly above pharmacokinetic-pharmacodynamic targets in lung and cellular lesions throughout the dosing interval.

Visualizing the dynamics of tuberculosis pathology using molecular imaging.

Nearly 140 years after Robert Koch discovered Mycobacterium tuberculosis, tuberculosis (TB) remains a global threat and a deadly human pathogen. M. tuberculosis is notable for complex host-pathogen interactions that lead to poorly understood disease states ranging from latent infection to active disease. Additionally, multiple pathologies with a distinct local milieu (bacterial burden, antibiotic exposure, and host response) can coexist simultaneously within the same subject and change independently over time. Current tools cannot optimally measure these distinct pathologies or the spatiotemporal changes. Next-generation molecular imaging affords unparalleled opportunities to visualize infection by providing holistic, 3D spatial characterization and noninvasive, temporal monitoring within the same subject. This rapidly evolving technology could powerfully augment TB research by advancing fundamental knowledge and accelerating the development of novel diagnostics, biomarkers, and therapeutics.

An optimized method for the detection and spatial distribution of aminoglycoside and vancomycin antibiotics in tissue sections by mass spectrometry imaging.

Suboptimal antibiotic dosing has been identified as one of the key drivers in the development of multidrug-resistant (MDR) bacteria that have become a global health concern. Aminoglycosides and vancomycin are broad-spectrum antibiotics used to treat critically ill patients infected by a variety of MDR bacterial species. Resistance to these antibiotics is becoming more prevalent. In order to design proper antibiotic regimens that maximize efficacy and minimize the development of resistance, it is pivotal to obtain the in situ pharmacokinetic-pharmacodynamic profiles at the sites of infection. Mass spectrometry imaging (MSI) is the ideal technique to achieve this. Aminoglycosides, due to their structure, suffer from poor ionization efficiency. Additionally, ion suppression effects by endogenous molecules greatly inhibit the detection of aminoglycosides and vancomycin at therapeutic levels. In the current study, an optimized method was developed that enabled the detection of these antibiotics by MSI. Tissue spotting experiments demonstrated a 5-, 15-, 35-, and 54-fold increase in detection sensitivity in the washed samples for kanamycin, amikacin, streptomycin, and vancomycin, respectively. Tissue mimetic models were utilized to optimize the washing time and matrix additive concentration. These studies determined the improved limit of detection was 40 to 5 µg/g of tissue for vancomycin and streptomycin, and 40 to 10 µg/g of tissue for kanamycin and amikacin. The optimized protocol was applied to lung sections from mice dosed with therapeutic levels of kanamycin and vancomycin. The washing protocol enabled the first drug distribution investigations of aminoglycosides and vancomycin by MSI, paving the way for site-of-disease antibiotic penetration studies.

Therapeutic Potential of Fosmanogepix (APX001) for Intra-abdominal Candidiasis: from Lesion Penetration to Efficacy in a Mouse Model.

Intra-abdominal candidiasis (IAC) is one of the most common yet underappreciated forms of invasive candidiasis. IAC is difficult to treat, and therapeutic failure and drug-resistant breakthrough infections are common in some institutions despite the use of echinocandins as first-line agents. Fosmanogepix (FMGX, formerly APX001) is a first-in-class antifungal prodrug that can be administered both intravenously and orally. FMGX is currently in phase 2 clinical development for the treatment of life-threatening invasive fungal infections. To explore the pharmacological properties and therapeutic potential of FMGX for IAC, we evaluated both drug penetration and efficacy of the active moiety manogepix (MGX, formerly APX001A) in liver tissues in a clinically relevant IAC mouse model infected with Candida albicans Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM)-directed absolute drug quantitation were employed to evaluate drug penetration into liver abscess lesions both spatially and quantitatively. The partitioning of MGX into lesions occurred slowly after a single dose; however, robust accumulation in the lesion was achieved after 3 days of repeated dosing. Associated with this drug penetration pattern, reduction in fungal burden and clearance in the liver were observed in mice receiving the multiday FMGX regimen. In comparison, administration of micafungin resulted in marginal reduction in fungal burden at the end of 4 days of treatment. These results suggest that FMGX is a promising candidate for the treatment of IAC.

MALDI-MSI spatially maps N-glycan alterations to histologically distinct pulmonary pathologies following irradiation.

Radiation-induced lung injury is a highly complex combination of pathological alterations that develop over time and severity of disease development is dose-dependent. Following exposures to lethal doses of irradiation, morbidity and mortality can occur due to a combination of edema, pneumonitis and fibrosis. Protein glycosylation has essential roles in a plethora of biological and immunological processes. Alterations in glycosylation profiles have been detected in diseases ranging from infection, inflammation and cancer. We utilized mass spectrometry imaging to spatially map N-glycans to distinct pathological alterations during the clinically latent period and at 180 days post-exposure to irradiation. Results identified alterations in a number of high mannose, hybrid and complex N-glycans that were localized to regions of mucus and alveolar-bronchiolar hyperplasia, proliferations of type 2 epithelial cells, accumulations of macrophages, edema and fibrosis. The glycosylation profiles indicate most alterations occur prior to the onset of clinical symptoms as a result of pathological manifestations. Alterations in five N-glycans were identified as a function of time post-exposure. Understanding the functional roles N-glycans play in the development of these pathologies, particularly in the accumulation of macrophages and their phenotype, may lead to new therapeutic avenues for the treatment of radiation-induced lung injury.

Tissue Distribution of Doxycycline in Animal Models of Tuberculosis.

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

Penetration of Ibrexafungerp (Formerly SCY-078) at the Site of Infection in an Intra-abdominal Candidiasis Mouse Model.

Ibrexafungerp (IBX), formerly SCY-078, is a novel, oral and intravenous, semisynthetic triterpenoid glucan synthase inhibitor in clinical development for treating multiple fungal infections, including invasive candidiasis. Intra-abdominal candidiasis (IAC) is one of the most common types of invasive candidiasis associated with high mortality largely due to poor drug exposure in infected lesions. To better understand the potential of IBX to treat such infections, we investigated its penetration at the site of infection. Using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) and laser capture microdissection (LCM)-directed high-pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we investigated tissue distribution and lesion-specific drug exposure of IBX in a clinically relevant IAC mouse model. After a single-dose treatment, IBX quickly distributed into tissues and efficiently accumulated within lesions. Drug concentrations of IBX within the liver abscesses were almost 100-fold higher than the serum concentration. In addition, drug penetration after repeated treatment of IBX was compared with micafungin. IBX exhibited robust and long-lasting lesion penetration after repeated treatment. These data indicate that IBX penetrates into intra-abdominal abscesses highly efficiently and holds promise as a potential therapeutic option for IAC patients.