Differential distribution of ivacaftor and its metabolites in plasma and human airway epithelia.

Ivacaftor is the first clinically approved monotherapy potentiator to treat CFTR channel dysfunction in people with cystic fibrosis. Ivacaftor (Iva) is a critical component for all current modulator therapies, including highly effective modulator therapies. Clinical studies show that CF patients on ivacaftor-containing therapies present various clinical responses, off-target effects, and adverse reactions, which could be related to metabolites of the compound. In this study, we reported the concentrations of Iva and two of its major metabolites (M1-Iva and M6-Iva) in capillary plasma and estimated M1-Iva and M6-Iva metabolic activity via the metabolite parent ratio in capillary plasma over 12 h. We also used the ratio of capillary plasma versus human nasal epithelial cell concentrations to evaluate entry into epithelial cells in vivo. M6-Iva was rarely detected by LC-MS/MS in epithelial cells from participants taking ivacaftor, although it was detected in plasma. To further explore this discrepancy, we performed in vitro studies, which showed that M1-Iva, but not M6-Iva, readily crossed 16HBE cell membranes. Our studies also suggest that metabolism of these compounds is unlikely to occur in airway epithelia despite evidence of expression of metabolism enzymes. Overall, our data provide evidence that there are differences between capillary and cellular concentrations of these compounds that may inform future studies of clinical response and off-target effects.

Pharmacokinetic variability of CFTR modulators from standard and alternative regimens.

Elexacaftor, tezacaftor, ivacaftor (ETI) is a CFTR modulator combination approved for use in ∼90 % of people with cystic fibrosis (pwCF) over 2 years old. While most pwCF tolerate this therapy well, some are intolerant to standard dosing, and others show little response. Clinical providers may adjust ETI dosing to combat these issues, but these adjustments are not well guided by pharmacokinetic evidence. Our post-approval study aimed to describe pharmacokinetic variability of ETI plasma concentrations in 15 participants who were administered a standard or reduced dose. ETI were quantified by LC-MS/MS in plasma samples taken prior to the morning dose. Results showed non-significant differences for each compound regardless of dosing regimen and after dose equivalence normalization. The majority of participants in both dosing groups had concentrations expected to elicit clinical response to ETI therapy. These findings indicate that dose reduction may be a viable strategy to maintain clinical benefit while managing intolerance.

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate.

The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of -5.7 to 3.5% (ELX), -3.2 to 6.0% (TEZ), and -3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Response of germ-free mice to colonization with O. formigenes and altered Schaedler flora.

Colonization with Oxalobacter formigenes may reduce the risk of calcium oxalate kidney stone disease. To improve our limited understanding of host/O.formigenes and microbe/O.formigenes interactions, germ-free or altered Schaedler flora (ASF) mice were colonized with O.formigenes Germ-free mice were stably colonized with O.formigenes suggesting O.formigenes does not require other organisms to sustain its survival. Examination of intestinal material indicated no viable O.formigenes in the small intestine, ∼4 × 106 O.formigenes per 100mg contents in the cecum and proximal colon, and ∼0.02% of total cecal O. formigenes cells were tightly associated to the mucosa. O.formigenes did not alter the overall microbial composition of ASF, and ASF did not impact O.formigenes capacity to degrade dietary oxalate in the cecum. 24-hour urine and fecal collections within metabolic cages in semi-rigid isolators demonstrated that introduction of ASF into germ-free mice significantly reduced urinary oxalate excretion. These experiments also showed that mono-colonized O.formigenes mice excrete significantly more urinary calcium compared to germ-free mice, which may be due to degradation of calcium oxalate crystals by O.formigenes and the subsequent intestinal absorption of free calcium. In conclusion, the successful establishment of defined-flora O.formigenes mouse models should improve our understanding of O.formigenes host and microbe interactions. These data support the use of O.formigenes as a probiotic that has limited impact on the composition of the resident microbiota but providing efficient oxalate degrading function. IMPORTANCE: Despite evidence suggesting a lack of O. formigenes colonization is a risk factor for calcium oxalate stone formation, little is known about O. formigenes biology. This study is the first to utilize germ-free mice to examine the response to mono-colonization with O. formigenes and the impact of a defined bacterial cocktail, altered Schaedler flora, on O. formigenes colonization. This study demonstrates that germ-free mice on their regular diet remain mono-colonized with O. formigenes, and suggests that further studies with O. formigenes gnotobiotic mouse models could improve our understanding of O. formigenes biology and host/O. formigenes and microbe/O. formigenes interactions.

Probiotic properties of Oxalobacter formigenes: an in vitro examination.

Oxalobacter formigenes (O. formigenes) is a nonpathogenic, Gram-negative, obligate anaerobic bacterium that commonly inhabits the human gut and degrades oxalate as its major energy and carbon source. Results from a case-controlled study suggested that lack of O. formigenes colonization is a risk factor for recurrent calcium oxalate stone formation. Hence, O. formigenes colonization may prove to be an efficacious method for limiting calcium oxalate stone risk. However, challenges exist in the preparation of O. formigenes as a successful probiotic due to it being an anaerobe with fastidious growth requirements. Here we examine in vitro properties expected of a successful probiotic strain. The data show that the Group 1 O. formigenes strain OxCC13 is sensitive to pH < 5.0, persists in the absence of oxalate, is aerotolerant, and survives for long periods when freeze-dried or mixed with yogurt. These findings highlight the resilience of this O. formigenes strain to some processes and conditions associated with the manufacture, storage and distribution of probiotic strains.

Circulating microRNA profiling identifies a subset of metastatic prostate cancer patients with evidence of cancer-associated hypoxia.

MicroRNAs (miRNAs) are small (∼22 nucleotide) non-coding RNAs that regulate a myriad of biological processes and are frequently dysregulated in cancer. Cancer-associated microRNAs have been detected in serum and plasma and hold promise as minimally invasive cancer biomarkers, potentially for assessing disease characteristics in patients with metastatic disease that is difficult to biopsy. Here we used miRNA profiling to identify cancer-associated miRNAs that are differentially expressed in sera from patients with metastatic castration resistant prostate cancer (mCRPC) as compared to healthy controls. Of 365 miRNAs profiled, we identified five serum miRNAs (miR-141, miR-200a, miR-200c, miR-210 and miR-375) that were elevated in cases compared to controls across two independent cohorts. One of these, miR-210, is a known transcriptional target of the hypoxia-responsive HIF-1α signaling pathway. Exposure of cultured prostate cancer cells to hypoxia led to induction of miR-210 and its release into the extracellular environment. Moreover, we found that serum miR-210 levels varied widely amongst mCRPC patients undergoing therapy, and correlated with treatment response as assessed by change in PSA. Our results suggest that (i) cancer-associated hypoxia is a frequent, previously under-appreciated characteristic of mCRPC, and (ii) serum miR-210 may be further developed as a predictive biomarker in patients with this distinct disease biology.

Ubiquitous Brms1 expression is critical for mammary carcinoma metastasis suppression via promotion of apoptosis.

Morbidity and mortality of breast cancer patients are drastically increased when primary tumor cells are able to spread to distant sites and proliferate to become secondary lesions. Effective treatment of metastatic disease has been limited; therefore, an increased molecular understanding to identify biomarkers and therapeutic targets is needed. Breast cancer metastasis suppressor 1 (BRMS1) suppresses development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Little is known of Brms1 function throughout the initiation and progression of mammary carcinoma. The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using polyoma middle T oncogene-induced models. Brms1 expression did not significantly alter growth of the primary tumors. When expressed ubiquitously using a ß-actin promoter, Brms1 suppressed pulmonary metastasis and promoted apoptosis of tumor cells located in the lungs but not in the mammary glands. Surprisingly, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in the mammary glands or lung, despite MMTV-induced expression within the lungs. These results strongly suggest that cell type-specific over-expression of Brms1 is important for Brms1-mediated metastasis suppression.