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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.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.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).Implementing a flipped classroom approach based on cognitive science of learning strategies positively impacted student performance and experience in a pharmacotherapy course.

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 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.

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.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.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).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.

Aims: In order to better predict the pharmacokinetics (PK) of antibodies in children, and to facilitate dose optimization of antibodies in pediatric patients, there is a need to develop systems PK models that integrate ontogeny related changes in human physiological parameters. Methods: A population-based physiological-based PK (PBPK) model to characterize antibody PK in pediatrics has been developed, by incorporating age-related changes in body weight, organ weight, organ blood flow rate, and interstitial volumes in a previously published platform model. The model was further used to perform Monte Carlo simulations to investigate clearance vs. age and dose-exposure relationship for infliximab. Results: By estimating only one parameter and associated interindividual variability, the model was able to characterize clinical PK of infliximab from two pediatric cohorts (n=141, 4-19 years) reasonably well. Model simulations demonstrated that only 50% of children reached desired trough concentrations when receiving FDA-labelled dosing regimen for infliximab, suggesting that higher doses and/or more frequent dosing are needed to achieve target trough concentrations of this antibody. Conclusion: The pediatric PBPK model presented here can serve as a framework to characterize the PK of antibodies in pediatric patients. The model can also be applied to other protein therapeutics to advance precision medicine paradigm and optimize antibody dosing regimens in children.

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.

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.

Inside cells, the immunomodulator methotrexate (MTX) undergoes the addition of glutamates to form methotrexate polyglutamates (MTX-Glu)—promising biomarkers of systemic exposure and treatment response to MTX in rheumatology. MTX-Glu are underexplored in Inflammatory Bowel Disease (IBD), with no data in pediatrics. In this cross-sectional secondary analysis, we assessed the relationships between MTX-Glu and MTX dose and treatment response in pediatric IBD. Twenty-one children with IBD, receiving maintenance therapy with infliximab (IFX) and MTX, had MTX-Glu1–6 concentrations and IFX troughs/antibodies measured and disease activity assessed for comparison in remission vs. active IBD using non-parametric tests, with associations explored using Spearman’s correlation (ρ) and regression analyses, SASv9.4 (α = 0.05). Total and long-chain MTX-Glu correlated with MTX dose (ρ = 0.51 and 0.56, respectively, p ≤ 0.02). In children with Crohn’s disease (n = 19), short-chain MTX-Glu1–2 were 2.5-fold higher in remission vs. active disease, approaching statistical significance (p = 0.066), with no statistical differences in IFX trough (p = 0.549) between groups. Our study highlights a potential role for long-chain MTX-Glu in the therapeutic drug monitoring of MTX in IBD. It is the first study in pediatric IBD and, although statistical significance was not reached, our findings also suggest that higher short-chain MTX-Glu levels may be associated with IBD treatment response to MTX in children.

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.

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.

Background: Kawasaki’s disease is a pediatric medium vessel vasculitis causing coronary arteritis and potentially coronary artery aneurysms. Higher systemic levels of Interleukin-6 (IL-6) in the first week of disease has been shown to be associated with a higher risk of coronary artery aneurysms (1, 2). Mice injected with Candida albicans water-soluble fraction (CAWS) develop coronary and aortic arteritis like that seen in Kawasaki’s disease. Our prior work has demonstrated that mast cell degranulation can inhibit lipopolysaccharide (LPS)-induced IL-6 gene expression in the aorta and systemic IL-6 production (3). Objectives: The objective of this study was to determine if mast cells play a role in the IL-6 homeostasis in an established mouse model of Kawasaki’s disease (CAWS model). Methods: 8-10-week-old male wild type controls (WT) and kitW-sh/W-sh mice (mast cell deficient, MCD) of C57Bl/6 background were randomly distributed into four groups (WT-PBS, WT-CAWS, MCD-PBS, MCD-CAWS). They were either injected intraperitoneally with PBS or CAWS (2 mg/mouse) daily for a period of 5 days. Eight animals from each group were sacrificed at either 7, 14 or 30 days after the last injection and blood, aorta and heart specimens were harvested. A pathologist (OT), blinded to treatment groups, examined aortic root and adjacent myocardium specimens and assigned a score for the degree of inflammation (0-6). Results: Seven MCD-CAWS mice died unexpectedly within 24 hours of the first CAWS injection. Autopsies reviewed marked liver sinusoidal dilation in all and patchy necrosis of the liver in 4. Similar, liver findings were seen in 75% of MCD-CAWS after 7 days, but not in other groups. MCD-CAWS mice had higher systemic IL-6 compared to WT-CAWS at both 7 days (208± 21 vs 152 ± 12 pg/ml; p Conclusion: Mast cell deficiency resulted in higher systemic levels of IL-6, TNFα and INFγ in the CAWS mouse model of Kawasaki’s disease. Similarly, mast cell deficiency resulted in more intense inflammation at the root of the aorta in this model. These results support the novel concept that mast cells play a protective role in reducing the initial systemic inflammatory response in Kawasaki’s disease. References: [1] Ueno Y, Takano N, Kanegane H, Yokoi T, Yachie A, Miyawaki T, et al. The acute phase nature of interleukin 6: studies in Kawasaki disease and other febrile illnesses. Clinical and experimental immunology. 1989;76(3):337-42. [2] Chow YM, Lin CY, Hwang B. Serum and urinary interleukin-6 (IL-6) levels as predicting factors of Kawasaki disease activity. Zhonghua Minguo xiao er ke yi xue hui za zhi [Journal] Zhonghua Minguo xiao er ke yi xue hui. 1993;34(2):77-83. [3] Springer J, Raveendran V, Smith D, Maz M, Dileepan K. OP0138 Novel Role of Mast Cells in the Regulation of Large Vessel Vasculitis. EULAR Paris, France: Ann Rheum Dis; 2014. p. 112-3. Acknowledgement: Acknowledgements: Joseph & Elizabeth Carey Endowment Fund and Lied Basic Science Research Development Award from University of Kansas Medical Center Disclosure of Interests: None declared

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.

Mast cells play an important role in immunomodulation and in the maintenance of vascular integrity. Interleukin-6 (IL-6) is one of the key biomarkers and therapeutic target in systemic vasculitis. The objective of the current study is to describe the role of mast cells in arterial IL-6 homeostasis. Eight- to ten-week-old male C57BL/6 (wild-type) mice were injected with either (a) saline, (b) compound 48/80 (a systemic mast cell degranulating agent), (c) lipopolysaccharide (LPS), or (d) a combination of C48/80 and LPS. Twenty-four hours after the injections, mice were sacrificed and serum samples and aortic tissues were analyzed for determining inflammatory response and cytokine expression profile. The results revealed that induction of mast cell degranulation significantly lowers serum IL-6 levels and aortic expression of IL-6 in LPS-treated mice. Significantly higher aortic expression of toll-like receptor-2 (TLR-2) and TNF-α was seen in the LPS and LPS+C48/80 groups of mice compared to controls. Aortic expression of TLR-4 was significantly decreased in LPS+C48/80 compared to C48/80 alone. LPS+C48/80-treated mice presented with a 3-fold higher aortic expression of suppressor of cytokine signaling (SOCS-1) compared to saline-injected groups. The inhibition of LPS-induced increase in serum IL-6 levels by mast cell degranulation was not seen in H1R knockout mice which suggests that mast cell-derived histamine acting through H1R may participate in the regulatory process. To examine whether the mast cell-mediated downregulation of LPS-induced IL-6 production is transient or cumulative in nature, wild-type mice were injected serially over a period of 10 days (5 injections) and serum cytokine levels were quantified. We found no significant differences in serum IL-6 levels between any of the groups. While mice injected with C48/80 or LPS had higher IL-10 compared to vehicle-injected mice, there was no difference between C48/80- and LPS+C48/80-injected mice. In conclusion, in an in vivo setting, mast cells appear to partially and transiently regulate systemic IL-6 homeostasis. This effect may be regulated through increased systemic IL-10 and/or aortic overexpression of SOCS-1.

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 ± S.E.M. of 8.9 ± 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.

Disease-Modifying Anti-Rheumatic Drug (DMARD) use in the treatment of juvenile idiopathic arthritis (JIA) has experienced a dramatic evolution since the early introduction of methotrexate in the 1970’s. This renaissance has been primarily driven by innovation in drug discovery and development that has resulted in the approval of a number of protein-based therapeutics targeting disease-specific pathways. Despite the expansion in the number of drugs available in the treatment of JIA, interindividual variation in therapeutic response and drug-associated toxicities continue to be a major concern and has driven efforts towards individualized therapy. Recent advances in pediatric drug development and innovative approaches to identifying a priori predictors of drug response hold the promise for an individualized approach to therapy that will yield the highest efficacy and safety potential for each JIA patient.

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.

tudy Objective To evaluate the relationship between plasma cytokine levels with disease activity and therapeutic response in patients with juvenile idiopathic arthritis 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 Sixty-one patients diagnosed with juvenile idiopathic arthritis who started therapy with standard-dose methotrexate (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, 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-1β, 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 etanercept 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-1β (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 3 months (p=0.005). Reductions in JADAS by 6 months were observed following either the addition of etanercept (p=0.009) or an increase in MTX dose (p=0.007), but the addition of etanercept 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 juvenile idiopathic arthritis, 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 etanercept resulted in increased TNF-α levels that corresponded with therapeutic response, which suggests a potential clinical benefit of monitoring TNF-α levels as a pharmacodynamic marker of etanercept activity. This article is protected by copyright. All rights reserved.

The purpose of this study was to develop extemporaneously compounded oral liquid formulations of aripiprazole for use in pediatric patients and those patients unable to swallow the solid oral dosage forms. Aripiprazole tablets(30 mg) were ground to a fine powder and suspended at a concentration of 1.0 mg/mL in either a 1:1 blend of Ora-Plus and Ora-Sweet, or 1% methylcellulose and Simple Syrup NF. Five amber, plastic liquid prescription bottles of each formulation were stored at 4°C, and aripiprazole content was measured by ultra-performance liquid chromatography time-of-flight mass spectrometry at 0, 14, 32, 67, and 91 days. Formulations were visually inspected at each time point for color change and precipitation. Forced degradation studies were conducted under oxidizing, acidic, basic, and thermal conditions. Concentrations of aripiprazole in the formulation containing 1:1 Ora-Plus and Ora-Sweet were unchanged over the study period with no signs of degradation over 91 days. In the 1:1 1% methylcellulose and Simple Syrup NF formulation, aripiprazole concentrations were 95% of labeled levels at 67 days, but failed to maintain greater than 90% of labeled levels at 91 days, with an average of only 84% of the labeled content. No apparent physical changes in the formulations were noted over the study period. In the forced degradation studies, loss of aripiprazole was notable under extreme oxidizing and alkaline conditions. Extemporaneously compounded oral suspensions of 1.0 mg/mL aripiprazole in 1:1 Ora-Plus and Ora-Sweet are stable for at least 91 days when stored in amber, plastic prescription bottles at 4°C, whereas suspensions in 1:1 1% methylcellulose and Simple Syrup NF are stable for up to 67 days.

Objective Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply. Methods Blood, plasma, and red blood cell (RBC) measurements of MTX and folates were obtained from previously published data sets and an additional analysis of JIA patients receiving MTX (n = 98) and those not receiving MTX (n = 78). Erythroblastoid cells maintained in culture were exposed to MTX and grown under varying levels of folic acid supplementation. Samples were analyzed for cellular folate and MTX content. Results Circulating folate levels were lower in JIA patients receiving MTX, with reduced levels of blood, plasma, and RBC 5-methyl-tetrahydrofolate (5mTHF) (P < 0.0001). Average polyglutamate chain length (Gluavg) of RBC 5mTHF was elevated in JIA patients receiving MTX (median ± interquartile range 5.63 ± 0.15 versus 5.54 ± 0.11 in those not receiving MTX; P < 0.001) and correlated with both RBC MTX accumulation (P = 0.02) and reduced plasma 5mTHF levels (P = 0.008). MTX exposure and folate deprivation in erythroblastoid cells resulted in a depletion of bioactive folate species that was associated with a shift to higher Gluavg values for several species, most notably tetrahydrofolate (THF) and 5,10-methylene-tetrahydrofolate (CH2THF). Increased Gluavg resulted from the depletion of short-chain and the accumulation of long-chain glutamate species. Conclusion Our findings indicate that folate content and polyglutamate distribution are responsive markers of MTX activity and folate supply in vivo and in vitro, and may provide novel clinical markers of pharmacologic activity of MTX.

Variability in response to methotrexate (MTX) in the treatment of juvenile idiopathic arthritis (JIA) remains unpredictable and poorly understood. Based on previous studies implicating an interaction between nicotinamide phosphoribosyltransferase (NAMPT) expression and MTX therapy in inflammatory arthritis, we hypothesized that increased NAMPT expression would be associated with reduced therapeutic response to MTX in patients with JIA. A significant association was found between increased plasma concentrations of NAMPT and reduced therapeutic response in patients with JIA treated with MTX. Inhibition of NAMPT in cell culture by either siRNA-based gene silencing or pharmacological inhibition with FK-866 was found to result in a fourfold increase in the pharmacological activity of MTX. Collectively, these findings provide evidence that NAMPT inhibits the pharmacological activity of MTX and may represent a predictive biomarker of response, as well as a therapeutic target, in the treatment of JIA with MTX.

Human development is described by the various anatomic and physiologic changes that occur as the single-celled zygote matures into an adult human being. Concomitant with bodily maturation are changes in the complex interactions between pharmacologic agents and the biologic matrix that is the human body. Profound changes in the manner by which drugs traverse the body during development can have significant implications in drug efficacy and toxicity. Although not a replacement for well-conducted, pediatric, pharmacokinetic studies, an understanding of developmental biology and the mechanisms for drug disposition invariably assists the pediatric clinician with the judicious use of medications in children.

The human body is continuously exposed to small organic molecules containing one or more basic nitrogen atoms. Many of these are endogenous (i.e., neurotransmitters, polyamines and biogenic amines), while others are exogenously supplied in the form of drugs, foods and pollutants. It is well-known that many amines have a strong propensity to specifically and substantially accumulate in highly acidic intracellular compartments, such as lysosomes, through a mechanism referred to as ion trapping. It is also known that cells have acquired the unique ability to sense and respond to amine accumulation in lysosomes in an effort to prevent potential negative consequences associated with hyperaccumulation. We describe here methods that are used to evaluate the dynamics of amine accumulation in, and egress from, lysosomes. Moreover, we highlight specific proteins that are thought to play important roles in these pathways. A theoretical model describing lysosomal amine dynamics is described and shown to adequately fit experimental kinetic data. The implications of this research in understanding and treating disease are discussed.

A number of multidrug-resistant (MDR) cancer cells have been shown to have acquired an increased capacity to sequester weakly basic anticancer drugs in their lysosomes relative to drug-sensitive counterparts. In this report we have comparatively evaluated the concentrations of the anticancer agent daunorubicin (DNR) in intracellular compartments of drug-sensitive and MDR HL-60 cell lines, both of which do not express common efflux transporters such as P-glycoprotein at the plasma membrane. Our results suggest that lysosomal sequestration plays a significant role in the emergence of MDR since it effectively limits the drug's ability to interact with target molecules located in the nucleus. Using a series of weakly basic structural isomers with variable basicity, we illustrate that the magnitude of the pK a value correlates with the degree of lysosomal sequestration. Accordingly, a series of structurally modified forms of DNR with reduced basicity were synthesized, and their intracellular distribution was evaluated. Consistent with model compounds, derivatives of DNR with lowered pK a values showed visibly reduced lysosomal sequestration in two separate MDR cell lines. Collectively, this work highlights the importance of understanding the intracellular localization of drugs and proposes a rational strategy to manipulate it.

Edited by Peter Schuler and Brendan M. Buckley, Published by Elsevier, 1600 John F. Kennedy Boulevard, Suite 1800, Philadelphia, PA 19103, 2015. 360 p. Price $99.95 (hard cover) Description: This book evaluates current methods used in the clinical phase of drug development and presents innovative

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-β-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.

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.

Human development is described by the various anatomic and physiologic changes that occur as the single-celled zygote matures into an adult human being. Concomitant with bodily maturation are changes in the complex interactions between pharmacologic agents and the biologic matrix that is the human body. Profound changes in the manner by which drugs traverse the body during development can have significant implications in drug efficacy and toxicity. Although not a replacement for well-conducted, pediatric, pharmacokinetic studies, an understanding of developmental biology and the mechanisms for drug disposition invariably assists the pediatric clinician with the judicious use of medications in children.

Many commercially available, weakly basic drugs have been shown to be lysosomotropic, meaning they are subject to extensive sequestration in lysosomes through an ion trapping-type mechanism. The extent of lysosomal trapping of a drug is an important therapeutic consideration because it can influence both activity and pharmacokinetic disposition. The administration of certain drugs can alter lysosomes such that their accumulation capacity for co-administered and/or secondarily administered drugs is altered.In this review the authors explore what is known regarding the mechanistic basis for drug-drug interactions involving lysosomes. Specifically, the authors address the influence of drugs on lysosomal pH, volume and lipid processing.Many drugs are known to extensively accumulate in lysosomes and significantly alter their structure and function; however, the therapeutic and toxicological implications of this remain controversial. The authors propose that drug-drug interactions involving lysosomes represent an important potential source of variability in drug activity and pharmacokinetics. Most evaluations of drug-drug interactions involving lysosomes have been performed in cultured cells and isolated tissues. More comprehensive in vivo evaluations are needed to fully explore the impact of this drug-drug interaction pathway on therapeutic outcomes.

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 hours. 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-β-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 hyper-accumulate cholesterol in lysosomes.