iMPAQT reveals that adequate mitohormesis from TFAM overexpression leads to life extension in mice.

Mitochondrial transcription factor A, TFAM, is essential for mitochondrial function. We examined the effects of overexpressing the TFAM gene in mice. Two types of transgenic mice were created: TFAM heterozygous (TFAM Tg) and homozygous (TFAM Tg/Tg) mice. TFAM Tg/Tg mice were smaller and leaner notably with longer lifespans. In skeletal muscle, TFAM overexpression changed gene and protein expression in mitochondrial respiratory chain complexes, with down-regulation in complexes 1, 3, and 4 and up-regulation in complexes 2 and 5. The iMPAQT analysis combined with metabolomics was able to clearly separate the metabolomic features of the three types of mice, with increased degradation of fatty acids and branched-chain amino acids and decreased glycolysis in homozygotes. Consistent with these observations, comprehensive gene expression analysis revealed signs of mitochondrial stress, with elevation of genes associated with the integrated and mitochondrial stress responses, including Atf4, Fgf21, and Gdf15. These found that mitohormesis develops and metabolic shifts in skeletal muscle occur as an adaptive strategy.

Interlaboratory evaluation of LC-MS-based biomarker assays.

Validation of biomarker assays is crucial for effective drug development and clinical applications. Interlaboratory reproducibility is vital for reliable comparison and combination of data from different centers. This review summarizes interlaboratory studies of quantitative LC-MS-based biomarker assays using reference standards for calibration curves. The following points are discussed: trends in reports, reference and internal standards, evaluation of analytical validation parameters, study sample analysis and normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studies is limited, necessitating further research. Some reports suggest methods to address variations in biomarker assay data among laboratories, facilitating organized studies and data combination. Method validation across laboratories is crucial for reducing interlaboratory differences and reflecting target biomarker responses.

The association of circulating endocannabinoids with cancer cachexia: A cross-sectional study.

BACKGROUND & AIMS: Endocannabinoids (eCBs) are involved in various physiological functions such as appetite, metabolism, and inflammation. Although deterioration of these functions is often observed in patients with refractory cancer cachexia (RCC), the relationship between circulating eCBs and cancer cachexia remains unknown. This study aimed to evaluate the relationship between circulating levels of eCBs and clinical findings in patients with RCC. METHODS: Circulating N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG) levels were measured in 39 patients with RCC (36% females, median age and IQR: 79 and 69-85), and 18 age- and sex-matched controls who received medical therapy for non-communicable diseases, using liquid chromatography with tandem mass spectrometry. In the RCC group, relationships between eCB levels and clinical findings-such as anorexia, awareness of pain, performance status, and survival period-were also examined. As anti-inflammatory drugs can influence the action and metabolism of eCBs, the following two analyses were conducted. In analysis 1, all participants were included, and in analysis 2, participants receiving any anti-inflammatory drugs were excluded. RESULTS: Serum AEA and 2-AG levels were more than twice as high in the RCC group than in those in the control group in both analyses. In analysis 1, only 8% of patients reported normal appetite assessed using the numerical rating scale (NRS), and serum AEA levels were negatively correlated with the NRS scores (R = -0.498, p = 0.001). Serum 2-AG levels were positively correlated with serum triglyceride levels (R = 0.419, p = 0.008). Both AEA and 2-AG levels were positively correlated with serum C-reactive protein (CRP) levels (AEA: R = 0.516, p < 0.001; 2-AG: R = 0.483, p = 0.002). Multiple linear regression analysis in the form of a stepwise procedure was performed; NRS scores and CRP levels showed a significant association with AEA levels (NRS: p = 0.001; CRP: p < 0.001), with an adjusted R2 value of 0.426. Similarly, triglyceride and CRP levels showed a significant association with the log of 2-AG levels (triglycerides: p < 0.001; CRP: p < 0.001), with an adjusted R2 value of 0.442. In analysis 2, serum AEA levels were negatively correlated with the NRS scores (R = -0.757, p < 0.001), whereas serum triglyceride levels were positively correlated with 2-AG levels (R = 0.623, p = 0.010). CONCLUSIONS: Circulating eCB levels were significantly higher in patients with RCC than those in controls. In patients with RCC, circulating AEA may play a role in anorexia, whereas 2-AG may play a role in serum triglyceride levels.

Multi-laboratory evaluation of immunoaffinity LC-MS-based glucagon-like peptide-1 assay.

Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.

Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

No obvious toxicological influences of 50 µL microsampling from rats administered phenacetin as a drug with hematological toxicity.

According to ICH S3A Q&A focusing on microsampling, its application should be avoided in main study animals for test drugs that could exacerbate hematological parameters with frequent blood sampling. However, no study has reported the effects of microsampling on toxicity parameters of drugs known to induce hematological toxicity. Therefore, we assessed the toxicological effects of serial microsampling on rats treated with phenacetin as a model drug. In a common 28-day study, 50 µL of microsampling was performed at 6-time points on days 1 to 2 and 7-time points on days 27 to 28 from the jugular vein of Sprague Dawley rats. The study was performed independently by two organizations. The toxicological influence of microsampling was evaluated on body weight, food consumption, hematology, blood clinical chemistry, urine parameters, organ weights, and tissue pathology. Phenacetin treatments induced significant changes of various hematological parameters (including hemoglobin and reticulocytes), some organ weights (including liver and spleen), and some hematology-related pathological parameters in the liver, spleen and bone marrow. Meanwhile, serial microsampling exhibited minimal influence on the assessed parameters, although 20 parameters showed statistical differences mostly at one organization. The current results support the notion that serial 50 µL microsampling from the jugular vein had minimal impacts on overall toxicological profiles even in rats treated with a drug inducing hematological toxicity, but the potential adverse effect on certain parameters could not be fully excluded. Accordingly, this microsampling technique has possibility to be employed even for non-clinical rat toxicity studies using drugs with potentially hematological toxicity.

A multilaboratory validation study of LC/MS biomarker assays for three lysophosphatidylcholines.

Aim: Although the fit-for-purpose approach has been proposed for validation procedures and acceptance criteria for biomarker assays, practical biomarker assays to facilitate clinical application and regulatory documents on biomarker assays remain limited. Materials & methods: We assigned six independent laboratories and selected three lysophosphatidylcholines (LPCs): LPC(16:0), LPC(18:0) and LPC(18:1) as model biomarkers. Using LC-MS, the following key validation parameters were evaluated: calibration curve, carryover, parallelism, precision and relative accuracy and these values were similar among all laboratories. Further, we determined LPC levels in six lots of rat plasma at unknown concentrations and compared them among the laboratories. Conclusion: Our multilaboratory validation and reproducibility data are useful for the development of future biomarker assay validation procedures, as well as regulatory documents.

In vivo evaluation of intestinal human CYP3A inhibition by macrolide antibiotics in CYP3A-humanised mice.

It is important to predict drug-drug interactions via inhibition of intestinal cytochrome P450 3A (CYP3A) which is a determinant of bioavailability of orally administered CYP3A substrates. However, inhibitory effects of macrolide antibiotics on CYP3A-mediated metabolism are not entirely identical between humans and rodents.We investigated the effects of macrolide antibiotics, clarithromycin and erythromycin, on in vitro and in vivo metabolism of triazolam, a CYP3A substrate, in CYP3A-humanised mice generated by using a mouse artificial chromosome vector carrying a human CYP3A gene.Metabolic activities of triazolam were inhibited by macrolide antibiotics in liver and intestine microsomes of CYP3A-humanised mice.The area under the plasma concentration-time curve ratios of 4-hydroxytriazolam to triazolam after oral dosing of triazolam were significantly decreased by multiple administration of macrolide antibiotics. The plasma concentrations ratios of α-hydroxytriazolam and 4-hydroxytriazolam to triazolam in portal blood were significantly decreased by multiple administration of clarithromycin in CYP3A-humanised mice.These results suggest that intestinal CYP3A activity was inhibited by macrolide antibiotics in CYP3A-humanised mice in vitro and in vivo. The plasma concentrations of triazolam and its metabolites in the portal blood of CYP3A-humanised mice would be useful for direct evaluation of intestinal CYP3A-mediated drug-drug interactions.

Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.

Overall Similarities and a Possible Factor Affecting Plasma Metabolome Profiles Between Venous and Capillary Blood Samples From 20 Healthy Human Males.

Amino acids and lipids are biomarkers used to assess the presence and severity of disease, as well as the toxicological response to drugs. Although upper-extremity venipuncture is a well-used standard technique, fingertip capillary sampling is a more convenient procedure. Delineating the global differences in amino acid and lipid levels in capillary and venous blood samples is paramount for expanding the application of capillary blood tests in biomarker assays. We recruited 20 healthy male subjects and collected plasma obtained from both fingertip capillary and antecubital venous blood. The samples were analyzed to determine the overall profiles of amino acids and lipids and to test for differences in their levels between both vessel types. The results demonstrated that the differences between capillary and venous blood had a lower impact than interindividual variations; however, trends of separation between them were observed for amino acids. The levels of 5 out of 28 amino acids scored fold changes over 30%, while 9 out of 498 lipids had a fold change over 30%. The time required for fingertip blood collection could be a factor for the differences in 3 metabolites. These findings provide useful information for the application of fingertip capillary blood sampling in biomarker assays.

Evaluation of 4ß-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice.

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4ß-hydroxycholesterol (4ß-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4ß-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4ß-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4ß-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4ß-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4ß-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4ß-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4ß-HC is a more reliable biomarker of hepatic CYP3A activity.

Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver.

Here, we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a-knockout (Cyp3a(-/-)) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a(-/-) mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element-binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a(-/-) mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a(-/-) mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a(-/-) mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7α-hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a(-/-) mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a(-/-) mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo.