Altered Folate Homeostasis in Children with Down Syndrome: A Potential Basis for Enhanced Methotrexate Toxicity.

Methotrexate is used to treat autoimmune and oncologic diseases in children with Down syndrome. However, increased methotrexate-related toxicity is reported in this population. We evaluated differences in the concentrations and distribution of erythrocyte folates in children with Down syndrome as a potential basis for this enhanced toxicity.

Nicotinamide Phosphoribosyltransferase Deficiency Potentiates the Antiproliferative Activity of Methotrexate through Enhanced Depletion of Intracellular ATP.

Lower plasma nicotinamide phosphoribosyltransferase (NAMPT) levels are associated with improved response to methotrexate (MTX) in patients with juvenile idiopathic arthritis. Cell-based studies confirmed that reduced cellular NAMPT activity potentiates the pharmacologic activity of MTX; however, the mechanism of this interaction has yet to be defined. Therefore, in this study, we investigate the mechanism of enhanced pharmacologic activity of MTX in NAMPT-deficient A549 cells. Small interfering RNA-based silencing of NAMPT expression resulted in a greater than 3-fold increase in sensitivity to MTX (P < 0.005) that was completely reversed by supplementation with folinic acid. Despite a 68% reduction in cellular NAD levels in NAMPT-deficient cells, no change in expression or activity of dihydrofolate reductase was observed and uptake of MTX was not significantly altered. MTX did not potentiate the depletion of cellular NAD levels, but NAMPT-deficient cells had significant elevations in levels of intermediates of de novo purine biosynthesis and were 4-fold more sensitive to depletion of ATP by MTX (P < 0.005). Supplementation with hypoxanthine and thymidine completely reversed the antiproliferative activity of MTX in NAMPT-deficient cells and corresponded to repletion of the cellular ATP pool without any effect on NAD levels. Together, these findings demonstrate that increased MTX activity with decreased NAMPT expression is dependent on the antifolate activity of MTX and is driven by enhanced sensitivity to the ATP-depleting effects of MTX. For the first time, these findings provide mechanistic details to explain the increase in pharmacological activity of MTX under conditions of reduced NAMPT activity.

Identification of Plasma Metabolomic Biomarkers of Juvenile Idiopathic Arthritis.

Identification of disease and therapeutic biomarkers remains a significant challenge in the early diagnosis and effective treatment of juvenile idiopathic arthritis (JIA). In this study, plasma metabolomic profiling was conducted to identify disease-related metabolic biomarkers associated with JIA. Plasma samples from treatment-naïve JIA patients and non-JIA reference patients underwent global metabolomic profiling across discovery (60 JIA, 60 non-JIA) and replication (49 JIA, 38 non-JIA) cohorts. Univariate analysis identified significant metabolites (q-value ≤ 0.05), followed by enrichment analysis using ChemRICH and metabolic network mapping with MetaMapp and Cytoscape. Receiver operating characteristic (ROC) analysis determined the top discriminating biomarkers based on area under the curve (AUC) values. A total of over 800 metabolites were measured, consisting of 714 known and 155 unknown compounds. In the discovery cohort, 587 metabolites were significantly altered in JIA patients compared with the reference population (q < 0.05). In the replication cohort, 288 metabolites were significantly altered, with 78 overlapping metabolites demonstrating the same directional change in both cohorts. JIA was associated with a notable increase in plasma levels of sphingosine metabolites and fatty acid ethanolamides and decreased plasma levels of sarcosine, iminodiacetate, and the unknown metabolite X-12462. Chemical enrichment analysis identified cycloparaffins in the form of naproxen and its metabolites, unsaturated lysophospholipids, saturated phosphatidylcholines, sphingomyelins, ethanolamines, and saturated ceramides as the top discriminating biochemical clusters. ROC curve analysis identified 11 metabolites classified as highly discriminatory based on an AUC > 0.90, with the top discriminating metabolite being sphinganine-1-phosphate (AUC = 0.98). This study identifies specific metabolic changes in JIA, particularly within sphingosine metabolism, through both discovery and replication cohorts. Plasma metabolomic profiling shows promise in pinpointing JIA-specific biomarkers, differentiating them from those in healthy controls and Crohn's disease, which may improve diagnosis and treatment.

Low-dose methotrexate results in the selective accumulation of aminoimidazole carboxamide ribotide in an erythroblastoid cell line.

Therapeutic and toxic response to low-dose methotrexate (MTX) in the treatment of autoimmune disease continues to be highly variable, resulting in a critical need to identify predictive biomarkers of response. Biomarker development has been hampered by an incomplete understanding of the molecular pharmacology of low-dose MTX. To address this issue, accumulation of the substrates for aminoimidazole carboxamide ribonucleotide transformylase (AICART) and thymidylate synthase (TS) was measured as markers of pharmacological activity of MTX in an erythroblastoid cell line. A 115-fold increase in the AICART substrate and anti-inflammatory mediator, 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (ZMP), was observed following exposure to 10 nM MTX but subsequently decreased with increasing MTX concentrations, declining to baseline levels with 1000 nM MTX. In contrast, the TS substrate, 2'-deoxyuridine 5'-monophosphate disodium salt (dUMP), displayed concentration-dependent accumulation, increasing 29-, 342-, and 471-fold over baseline with 10, 100, and 1000 nM MTX, respectively. Cellular levels of dUMP correlated with levels of the parent drug (MTX-PG1; r = 0.66, P < 0.001) and its polyglutamates (MTX-PG2-6) (r = 0.81, P < 0.001), whereas cellular levels of ZMP were only moderately correlated with MTX-PG1 (r = 0.34, P < 0.01). In contrast, accumulation of ZMP at 10 nM MTX was associated with a 2.9-fold increase in the AICART inhibitor dihydrofolate (DHF), represented primarily by long-chain DHF polyglutamates. Selectivity, defined as the ratio of ZMP to dUMP, was maximal following exposure to 6 nM MTX. Characterizing the range of MTX concentrations that selectively promote ZMP accumulation while preserving pyrimidine biosynthesis may lead to optimization of low-dose MTX therapy.

Lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (Fa2N-4 cells).

Lipophilic (logP > 1) and amphiphilic drugs (also known as cationic amphiphilic drugs) with ionizable amines (pKa > 6) can accumulate in lysosomes, a process known as lysosomal trapping. This process contributes to presystemic extraction by lysosome-rich organs (such as liver and lung), which, together with the binding of lipophilic amines to phospholipids, contributes to the large volume of distribution characteristic of numerous cardiovascular and central nervous system drugs. Accumulation of lipophilic amines in lysosomes has been implicated as a cause of phospholipidosis. Furthermore, elevated levels of lipophilic amines in lysosomes can lead to high organ-to-blood ratios of drugs that can be mistaken for active drug transport. In the present study, we describe an in vitro fluorescence-based method (using the lysosome-specific probe LysoTracker Red) to identify lysosomotropic agents in immortalized hepatocytes (Fa2N-4 cells). A diverse set of compounds with various physicochemical properties were tested, such as acids, bases, and zwitterions. In addition, the partitioning of the nonlysosomotropic atorvastatin (an anion) and the lysosomotropics propranolol and imipramine (cations) were quantified in Fa2N-4 cells in the presence or absence of various lysosomotropic or nonlysosomotropic agents and inhibitors of lysosomal sequestration (NH4Cl, nigericin, and monensin). Cellular partitioning of propranolol and imipramine was markedly reduced (by at least 40%) by NH4Cl, nigericin, or monensin. Lysosomotropic drugs also inhibited the partitioning of propranolol by at least 50%, with imipramine partitioning affected to a lesser degree. This study demonstrates the usefulness of immortalized hepatocytes (Fa2N-4 cells) for determining the lysosomal sequestration of lipophilic amines.

Cationic amphiphilic drugs cause a marked expansion of apparent lysosomal volume: implications for an intracellular distribution-based drug interaction.

How a drug distributes within highly compartmentalized mammalian cells can affect both the activity and pharmacokinetic behavior. Many commercially available drugs are considered to be lysosomotropic, meaning they are extensively sequestered in lysosomes by an ion trapping-type mechanism. Lysosomotropic drugs typically have a very large apparent volume of distribution and a prolonged half-life in vivo, despite minimal association with adipose tissue. In this report we tested the prediction that the accumulation of one drug (perpetrator) in lysosomes could influence the accumulation of a secondarily administered one (victim), resulting in an intracellular distribution-based drug interaction. To test this hypothesis cells were exposed to nine different hydrophobic amine-containing drugs, which included imipramine, chlorpromazine and amiodarone, at a 10 µM concentration for 24 to 48 h. After exposure to the perpetrators the cellular accumulation of LysoTracker Red (LTR), a model lysosomotropic probe, was evaluated both quantitatively and microscopically. We found that all of the tested perpetrators caused a significant increase in the cellular accumulation of LTR. Exposure of cells to imipramine caused an increase in the cellular accumulation of other lysosomotropic probes and drugs including LyosTracker Green, daunorubicin, propranolol and methylamine; however, imipramine did not alter the cellular accumulation of non-lysosomotropic amine-containing molecules including MitoTracker Red and sulforhodamine 101. In studies using ionophores to abolish intracellular pH gradients we were able to resolve ion trapping-based cellular accumulation from residual pH-gradient independent accumulation. Results from these evaluations in conjunction with lysosomal pH measurements enabled us to estimate the relative aqueous volume of lysosomes of cells before and after imipramine treatment. Our results suggest that imipramine exposure caused a 4-fold expansion in the lysosomal volume, which provides the basis for the observed drug interaction. The imipramine-induced lysosomal volume expansion was shown to be both time- and temperature-dependent and reversed by exposing cells to hydroxypropyl-ß-cyclodextrin, which reduced lysosomal cholesterol burden. This suggests that the expansion of lysosomal volume occurs secondary to perpetrator-induced elevations in lysosomal cholesterol content. In support of this claim, the cellular accumulation of LTR was shown to be higher in cells isolated from patients with Niemann-Pick type C disease, which are known to hyperaccumulate cholesterol in lysosomes.

Evaluating the impact of a flipped classroom model based on cognitive science of learning strategies in a pharmacotherapy course.

INTRODUCTION: The purpose of this study was to determine the impact of a flipped classroom method based on cognitive science of learning strategies on student performance and experience in a third-year pharmacotherapy course. METHODS: The cognitive science of learning flipped classroom (CSL-FC) strategies in this study included pre-class learning (Preview), in-class application to cases (Retrieval), after-class learning (Spaced Retrieval), and post-module reflection (Deliberate Reflection) in a required pharmacotherapy course. During fall 2017, one instructor piloted the CSL-FC method. During fall 2018, this method expanded to four instructors. All other instructors used traditional lecture-based methods. The same multiple-choice exam questions were used both years. The average exam question scores between teaching methods were compared by independent t-test. Student focus groups were conducted after the 2017 semester. In 2018, students were surveyed using a 5-point Likert rating (1 = strongly agree, 5 = strongly disagree) to evaluate their experience. RESULTS: The 2017 and 2018 classes included 132 and 137 students, respectively. During the two years, exam question scores were significantly better with CSL-FC (n = 136 questions) compared to traditional (n = 110 questions) (88.8% vs 84.9%, respectively; P = .02). The focus group analysis revealed three main themes including a "love-hate relationship," "time," and "it works." Student agreement to the survey question "the cognitive science of learning flipped classroom helped me learn" was 2.18 (SD 1.12). CONCLUSIONS: Implementing a flipped classroom approach based on cognitive science of learning strategies positively impacted student performance and experience in a pharmacotherapy course.

Plasma metabolomic profiling as a tool to identify predictive biomarkers of methotrexate efficacy in rheumatoid arthritis.

OBJECTIVE: Methotrexate (MTX) remains the first-choice disease-modifying therapy in rheumatoid arthritis (RA). However, clinical response is variable, and no reliable predictive biomarkers of efficacy currently exist. In this study, plasma metabolomic profiling is evaluated as a tool to identify pretreatment biomarkers of MTX response in RA. METHODS: Plasma collected from RA patients initiating MTX therapy (n = 20) were analyzed by metabolomic profiling totaling 648 identified metabolites. Pretreatment metabolomic profiles were compared based on clinical response after 16-weeks of MTX therapy. Clinical response to MTX was defined by a clinically meaningful reduction in disease activity score in 28 joints (DAS28-ESR) of greater than 1.2. RESULTS: Pretreatment plasma levels of 19 metabolites were found to differ (p < 0.05) between RA patients based on response to MTX at 16-weeks. Spearman's correlation of pretreatment plasma metabolite levels with change in DAS28-ESR over the treatment period further supported three of the identified metabolites as associated with MTX response (p < 0.05). The identified metabolite levels were all found to be lower in RA patients responsive to MTX but were not found to be intercorrelated. Receiver operating characteristic analysis of each of the identified metabolites, alone or in combination, demonstrated an excellent discrimination between responders and non-responders based on pretreatment plasma levels of nornicotine (AUC = 0.84), N-methylisoleucine (AUC = 0.82), 2,3-dihydroxybutanoic acid (AUC = 0.82), and a combination biomarker panel score (AUC = 0.98). CONCLUSION: Pretreatment plasma metabolomic profiling identified multiple metabolites associated with early response to MTX therapy in RA and represents a promising approach for the identification of clinical biomarkers of MTX response in RA.

Dose-dependent Pharmacological Response to Rituximab in the Treatment of Antineutrophil Cytoplasmic Antibody-associated Vasculitis.

OBJECTIVE: Rituximab (RTX) is effective in the induction and maintenance of remission in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). However, uncertainty remains regarding the optimal maintenance dosing regimen. This work evaluates the relationship between variability in RTX dosing and pharmacological response in AAV. METHODS: A prospective cohort of patients with AAV (n = 28) with either granulomatosis with polyangiitis (n = 23) or microscopic polyangiitis (n = 5) receiving maintenance RTX therapy were followed in a single tertiary care academic medical center over a 2-year period. Patient demographics, RTX dosing information, and trough plasma RTX levels were collected along with laboratory measures of pharmacologic response, including B cell counts and ANCA titers. RESULTS: RTX dosing information from 94 infusions with 59 trough samples were collected with a mean ± SD dose of 640 ± 221 mg, dosing interval of 210 ± 88 days, and trough plasma RTX concentration of 622 ± 548 ng/mL. RTX trough concentrations were associated with RTX dose (ρ = 0.60, P < 0.0001) and dosing interval (ρ = -0.55, P < 0.0001). RTX dosing intensity (mg/d) was associated with RTX trough concentrations (ρ = 0.57, P < 0.0001). Higher dosing intensities were associated with undetectable B cell repopulation (P < 0.0001), but not negative ANCA titers (P = 0.60). Stratification of dosing intensities based on the standard dosing regimen of 500 mg every 6 months (2.4-3.3 mg/d) demonstrated that this regimen was associated with B cell repopulation in 8 of 17 doses (47%) compared to 0 of 23 doses (0%) with the high-dose regimen (> 3.3 mg/d; P < 0.0001). CONCLUSION: RTX maintenance dosing of 500 mg every 6 months may be inadequate to maintain B cell depletion in the treatment of AAV.

Low Etanercept Concentrations in Children With Obesity and Juvenile Idiopathic Arthritis.

OBJECTIVE: To evaluate the impact of obesity on etanercept (ETN) drug exposure in children with juvenile idiopathic arthritis (JIA). METHODS: We conducted a pilot, cross-sectional, observational study in a real-world cohort of children with JIA receiving ETN as standard of care from a single center. We analyzed the relationship between body size and ETN plasma concentrations, adjusting for dosage. Body size was analyzed as a continuous measure using weight and body mass index (BMI) percentiles and categorically using BMI percentile classifications according to the CDC guidelines. RESULTS: We enrolled a total of 29 children. Each child provided one plasma sample for ETN concentration measurement, and all participants were receiving subcutaneous ETN dosed weekly. We observed that the ETN concentration normalized for dose decreased significantly as a function of weight (p = 0.004) and BMI percentile (p = 0.04). Similarly, we observed a progressive decline in mean and median dose-normalized concentrations across higher body size categories. Because of reaching maximum ETN dosage (50 mg), 7 of 8 children (87.5%) with obesity received a weight-based dosage < 0.8 mg/kg/dose. CONCLUSIONS: We found that higher body weight and BMI percentile are significantly and negatively associated with ETN drug serum concentration, accounting for differences in dosing. Our data suggest that children who are obese may be routinely under-dosed using current dosing strategies. Inadequate dosing may increase the risk for therapeutic failure and long-term morbidity in a developing child. As a result, characterizing adequate drug exposure in children of all sizes is an important step toward precision dosing.

Methotrexate Disposition, Anti-Folate Activity, and Metabolomic Profiling to Identify Molecular Markers of Disease Activity and Drug Response in the Collagen-Induced Arthritis Mouse Model.

Methotrexate (MTX) is widely used in the treatment of autoimmune arthritis but is limited by its unpredictable and variable response profile. Currently, no biomarkers exist to predict or monitor early therapeutic responses to MTX. Using a collagen-induced arthritis (CIA) mouse model, this study aimed to identify biochemical pathways and biomarkers associated with MTX efficacy in autoimmune arthritis. Following arthritis disease induction, DBA/1J mice were treated with subcutaneous MTX (20 mg/kg/week) and disease activity was assessed based on disease activity scores (DAS) and paw volume (PV) measurements. Red blood cell (RBC) and plasma samples were collected at the end of the study and were assessed for folate and MTX content. Plasma samples were analyzed by semitargeted global metabolomic profiling and analyzed by univariate and multivariate analysis. Treatment with MTX was associated with significant reductions in disease activity based on both DAS (p = 0.0006) and PV (p = 0.0006). MTX therapy resulted in significant reductions in 5-methyltetrahydrofolate (5mTHF) levels in plasma (p = 0.02) and RBCs (p = 0.001). Reductions in both RBC and plasma 5mTHF were associated with lower DAS (p = 0.0007, p = 0.01, respectively) and PV (p = 0.001, p = 0.005, respectively). Increases in RBC MTX were associated with lower DAS (p = 0.003) but not PV (p = 0.23). Metabolomic analysis identified N-methylisoleucine (NMI) and quinolone as metabolites significantly altered in disease mice, which were corrected towards healthy control levels in mice treated with MTX. Reductions in plasma NMI were associated with lower DAS (p = 0.0002) and PV (p = 9.5 × 10-6). Increases in plasma quinolone were associated with lower DAS (p = 0.02) and PV (p = 0.01). Receiver-operating characteristic curve analysis identified plasma NMI (AUC = 1.00, p = 2.4 × 10-8), RBC 5mTHF (AUC = 0.99, p = 2.4 × 10-5), and plasma quinolone (AUC = 0.89, p = 0.01) as top discriminating metabolites of MTX treatment. Our data support a relationship between MTX efficacy and its effect on circulating folates and identified 5mTHF, NMI, and quinolone as potential therapeutic biomarkers of disease activity and MTX response in the CIA mouse model of autoimmune arthritis.

Plasma Metabolome Normalization in Rheumatoid Arthritis Following Initiation of Methotrexate and the Identification of Metabolic Biomarkers of Efficacy.

Methotrexate (MTX) efficacy in the treatment of rheumatoid arthritis (RA) is variable and unpredictable, resulting in a need to identify biomarkers to guide drug therapy. This study evaluates changes in the plasma metabolome associated with response to MTX in RA with the goal of understanding the metabolic basis for MTX efficacy towards the identification of potential metabolic biomarkers of MTX response. Plasma samples were collected from healthy control subjects (n = 20), and RA patients initiating MTX therapy (n = 20, 15 mg/week) before and after 16 weeks of treatment. The samples were analyzed by a semi-targeted metabolomic analysis, and then analyzed by univariate and multivariate methods, as well as an enrichment analysis. An MTX response was defined as a clinically significant reduction in the disease activity score in 28 joints (DAS-28) of greater than 1.2; achievement of clinical remission, defined as a DAS-28 < 2.6, was also utilized as an additional measure of response. In this study, RA is associated with an altered plasma metabolome that is normalized following initiation of MTX therapy. Metabolite classes found to be altered in RA and corrected by MTX therapy were diverse and included triglycerides (p = 1.1 × 10-16), fatty acids (p = 8.0 × 10-12), and ceramides (p = 9.8 × 10-13). Stratification based on responses to MTX identified various metabolites differentially impacted in responders and non-responders including glucosylceramides (GlcCer), phosphatidylcholines (PC), sphingomyelins (SM), phosphatidylethanolamines (PE), choline, inosine, hypoxanthine, guanosine, nicotinamide, and itaconic acid (p < 0.05). In conclusion, RA is associated with significant alterations to the plasma metabolome displaying at least partial normalization following 16 weeks of MTX therapy. Changes in multiple metabolites were found to be associated with MTX efficacy, including metabolites involved in fatty acid/lipid, nucleotide, and energy metabolism.

Factors associated with reduced infliximab exposure in the treatment of pediatric autoimmune disorders: a cross-sectional prospective convenience sampling study.

BACKGROUND: Inadequate systemic exposure to infliximab (IFX) is associated with treatment failure. This work evaluated factors associated with reduced IFX exposure in children with autoimmune disorders requiring IFX therapy. METHODS: In this single-center cross-sectional prospective study IFX trough concentrations and anti-drug antibodies (ADAs) were measured in serum from children diagnosed with inflammatory bowel disease (IBD) (n = 73), juvenile idiopathic arthritis (JIA) (n = 16), or uveitis (n = 8) receiving maintenance IFX infusions at an outpatient infusion clinic in a tertiary academic pediatric hospital. IFX concentrations in combination with population pharmacokinetic modeling were used to estimate IFX clearance. Patient demographic and clinical data were collected by chart review and evaluated for their relationship with IFX clearance. RESULTS: IFX trough concentrations ranged from 0 to > 40 µg/mL and were 3-fold lower in children with IBD compared to children with JIA (p = 0.0002) or uveitis (p = 0.001). Children with IBD were found to receive lower IFX doses with longer dosing intervals, resulting in dose intensities (mg/kg/day) that were 2-fold lower compared to children with JIA (p = 0.0002) or uveitis (p = 0.02). Use of population pharmacokinetic analysis to normalize for variation in dosing practices demonstrated that increased IFX clearance was associated with ADA positivity (p = 0.004), male gender (p = 0.02), elevated erythrocyte sedimentation rate (ESR) (p = 0.02), elevated c-reactive protein (CRP) (p = 0.001), reduced serum albumin concentrations (p = 0.0005), and increased disease activity in JIA (p = 0.009) and IBD (p ≤ 0.08). No significant relationship between diagnosis and underlying differences in IFX clearance was observed. Multivariable analysis by covariate population pharmacokinetic modeling confirmed increased IFX clearance to be associated with anti-IFX antibody positivity, increased ESR, and reduced serum albumin concentrations. CONCLUSIONS: Enhanced IFX clearance is associated with immunogenicity and inflammatory burden across autoimmune disorders. Higher systemic IFX exposures observed in children with rheumatologic disorders are driven primarily by provider drug dose and interval selection, rather than differences in IFX pharmacokinetics across diagnoses. Despite maintenance IFX dosing at or above the standard recommended range for IBD (i.e., 5 mg/kg every 8 weeks), the dosing intensity used in the treatment of IBD is notably lower than dosing intensities used to treat JIA and uveitis, and may place some children with IBD at risk for suboptimal maintenance IFX exposures necessary for treatment response.

Leveraging innovative technology to generate drug response phenotypes for the advancement of biomarker-driven precision dosing.

Although traditional approaches to biomarker discovery have elucidated key molecular markers that have improved drug selection (precision medicine), the discovery of biomarkers that inform optimal dose selection (precision dosing) continues to be a challenge in many therapeutic areas. Larger and more diverse study populations are necessary to discover additional biomarkers that provide the resolution needed for a more tailored dose. To generate and accommodate large datasets of drug response phenotypes, time- and cost-efficient strategies are necessary. In particular, a multitude of technological advances that originated for purposes outside of biomedical research (electronic health records, direct-to-consumer genetic testing, social media, mobile devices, and machine learning) have made it easier to communicate, connect, and gather information from consumers. Although these technologies have been used with success in the health sciences for an array of purposes, these resources have not been fully capitalized on for precision dosing. This perspective will touch on how these innovations can be used as data sources, data collection tools, and data processing tools for drug-response phenotypes with a unique focus on advancing biomarker-driven precision dosing.

Metabolomic Profiling to Identify Molecular Biomarkers of Cellular Response to Methotrexate In Vitro.

Variation in methotrexate (MTX) efficacy represents a significant barrier to early and effective disease control in the treatment of autoimmune arthritis. We hypothesize that the utilization of metabolomic techniques will allow for an improved understanding of the biochemical basis for the pharmacological activity of MTX, and can promote the identification and evaluation of novel molecular biomarkers of MTX response. In this work, erythroblastoid cells were exposed to MTX at the physiologic concentration of 1,000 nM and analyzed using three metabolomic platforms to give a broad spectrum of cellular metabolites. MTX pharmacological activity, defined as cellular growth inhibition, was associated with an altered cellular metabolomic profile based on the analysis of 724 identified metabolites. By discriminant analysis, MTX treatment was associated with increases in ketoisovaleric acid, fructose, galactose, and 2-deoxycytidine, and corresponding reductions in 2-deoxyuridine, phosphatidylinositol 32:0, orotic acid, and inosine monophosphate. Inclusion of data from analysis of folate metabolism in combination with chemometric and metabolic network analysis demonstrated that MTX treatment is associated with dysregulated folate metabolism and nucleotide biosynthesis, which is in line with its known mechanism of action. However, MTX treatment was also associated with alterations in a diversity of metabolites, including intermediates of amino acid, carbohydrate, and lipid metabolism. Collectively, these findings support a robust metabolic response following exposure to physiologic concentrations of MTX. They also identify various metabolic intermediates that are associated with the pharmacological activity of MTX, and are, therefore, potential molecular biomarker candidates in future preclinical and clinical studies of MTX efficacy in autoimmune arthritis.

Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model.

Methotrexate (MTX) efficacy in autoimmune arthritis is variable and unpredictable resulting in the need for the identification of biomarkers to guide drug therapy. This study utilizes the collagen-induced arthritis mouse model to investigate erythrocyte MTX disposition and anti-folate activity as biochemical markers of efficacy in autoimmune arthritis. Following induction of arthritis, DBA/1J mice were treated with once-weekly subcutaneous MTX at varying doses over a period of 40 days. At the completion of the study tissue samples were analyzed for MTX and folate content and assessed for their relationship with MTX efficacy. MTX treatment resulted in a reduction in disease activity that was variable and dose-dependent. Erythrocyte accumulation of MTX and its polyglutamate metabolites were dose proportionate, however, polyglutamate metabolites represented a mean ±â€¯S.E.M. of 8.9 ±â€¯0.4% of total erythrocyte MTX, which is markedly lower than previously observed in humans and failed to display any significant association with MTX efficacy. MTX treatment resulted in reductions in erythrocyte 5-methyl-tetrahydrofolate (5mTHF) levels that were similar to those previously observed in human studies. Disease induction was associated with a decrease in liver 5mTHF and increased formyl-tetrahydrofolate (fTHF) that was normalized in MTX treated mice. MTX efficacy was associated with reductions in erythrocyte 5mTHF (P = 0.04) and increases in liver 5mTHF (P = 0.0001). Together, these findings demonstrate a relationship between alterations in tissue folate levels and MTX efficacy, and supports erythrocyte levels of 5mTHF as a marker of MTX efficacy in autoimmune arthritis.

Precision Pharmacotherapy: Integrating Pharmacogenomics into Clinical Pharmacy Practice.

Precision pharmacotherapy encompasses the use of therapeutic drug monitoring; evaluation of liver and renal function, genomics, and environmental and lifestyle exposures; and analysis of other unique patient or disease characteristics to guide drug selection and dosing. This paper articulates real-world clinical applications of precision pharmacotherapy, focusing exclusively on the emerging field of clinical pharmacogenomics. This field is evolving rapidly, and clinical pharmacists now play an invaluable role in the clinical implementation, education, and research applications of pharmacogenomics. This paper provides an overview of the evolution of pharmacogenomics in clinical pharmacy practice, together with recommendations on how the American College of Clinical Pharmacy (ACCP) can support the advancement of clinical pharmacogenomics implementation, education, and research. Commonalities among successful clinical pharmacogenomics implementation and education programs are identified, with recommendations for how ACCP can leverage and advance these common themes. Opportunities are also provided to support the research needed to move the practice and application of pharmacogenomics forward.

Variability in Potency Among Commercial Preparations of Berberine.

Berberine is an isoquinoline alkaloid plant extract that is widely available as a dietary supplement in the United States and has demonstrated efficacy in the treatment of type 2 diabetes mellitus and dyslipidemia. Because of its increased use and purported pharmacological properties, potential variations in product quality could pose a barrier to berberine's safety and effectiveness in clinical practice. Thus, this study evaluated the potency of dietary supplements containing berberine available in the U.S. commercial market. Fifteen unique dietary supplements containing berberine were purchased through U.S. dietary supplement vendors. For each product, berberine was extracted from 3 unique capsules and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry. Percentage content based on the product label claim was determined for each product. The average berberine content across the products was found to be 75% ± 25% of the product label claim, with product potency ranging from 33% to 100%. Nine of the 15 tested products (60%) failed to meet the potency standards of 90% to 110% of labeled content claim, as commonly required of pharmaceutical preparations by the U.S. Pharmacopeial Convention. Evaluation of the relationship between product cost and the measured potency failed to demonstrate an association between quality and cost. Variability in product quality may significantly contribute to inconsistencies in the safety and effectiveness of berberine. In addition, the quality of the berberine product cannot be inferred from its cost.

Cytokine Biomarkers of Disease Activity and Therapeutic Response after Initiating Methotrexate Therapy in Patients with Juvenile Idiopathic Arthritis.

STUDY OBJECTIVE: To evaluate the relationship between plasma cytokine levels with disease activity and therapeutic response in patients with juvenile idiopathic arthritis (JIA) after initiating methotrexate (MTX) therapy. DESIGN: Single-center observational prospective cohort study. SETTING: Outpatient pediatric rheumatology clinic at a tertiary care academic pediatric hospital. PATIENTS: The study included 61 patients diagnosed with JIA who started therapy with standard-dose MTX 15 mg/m2 /week. At 3 months, treating physicians were given the option of maintaining the MTX dose, increasing the MTX dose, or adding etanercept (ETN), based on their clinical judgment. MEASUREMENTS AND MAIN RESULTS: Patients were evaluated at baseline, 3 months (51 patients), and 6 months (35 patients). Plasma samples from each visit were analyzed for interleukin (IL)-1α, IL-1ß, IL-1Ra, IL-6, and tumor necrosis factor-α (TNF-α). Cytokine concentrations were evaluated for relationships with disease activity using the 71-joint count Juvenile Arthritis Disease Activity Score (JADAS). Therapeutic response was assessed by changes in JADAS. Failure to respond to standard-dose MTX was defined as the need for the addition of ETN or a MTX dose increase at or before the 3-month visit. Increased disease severity at baseline was associated with increased IL-6 (p=0.01) and TNF-α (p=0.008) levels. Initiation of MTX was associated with reductions in IL-1α (p=0.009), IL-1ß (p=0.01), IL-1Ra (p=0.007), and IL-6 (p=0.03) levels; however, reductions in JADAS were only associated with reductions in IL-6 (p=0.009) and TNF-α levels (p=0.02). Compared with responders, patients failing to respond to standard-dose MTX had increased TNF-α levels at baseline (p=0.02) and at 3 months (p=0.005). Reductions in JADAS by 6 months were observed following either the addition of ETN (p=0.009) or an increase in MTX dose (p=0.007), but the addition of ETN was associated with a median 7-fold increase in TNF-α levels (p=0.003) that corresponded with clinical response. CONCLUSION: Plasma cytokine levels were responsive to MTX therapy in patients with JIA, but only TNF-α and IL-6 levels were consistently associated with disease activity and therapeutic response. Increased TNF-α levels at baseline were associated with failure to respond to standard-dose MTX and the need for more aggressive drug therapy. Initiation of ETN resulted in increased TNF-α levels that corresponded with therapeutic response, suggesting a potential clinical benefit of monitoring TNF-α levels as a pharmacodynamic marker of etanercept activity.