Preemptive pharmacogenetic testing to guide chemotherapy dosing in patients with gastrointestinal malignancies: a qualitative study of barriers to implementation.

BACKGROUND: Pharmacogenetic (PGx) testing for germline variants in the DPYD and UGT1A1 genes can be used to guide fluoropyrimidine and irinotecan dosing, respectively. Despite the known association between PGx variants and chemotherapy toxicity, preemptive testing prior to chemotherapy initiation is rarely performed in routine practice. METHODS: We conducted a qualitative study of oncology clinicians to identify barriers to using preemptive PGx testing to guide chemotherapy dosing in patients with gastrointestinal malignancies. Each participant completed a semi-structured interview informed by the Consolidated Framework for Implementation Research (CFIR). Interviews were analyzed using an inductive content analysis approach. RESULTS: Participants included sixteen medical oncologists and nine oncology pharmacists from one academic medical center and two community hospitals in Pennsylvania. Barriers to the use of preemptive PGx testing to guide chemotherapy dosing mapped to four CFIR domains: intervention characteristics, outer setting, inner setting, and characteristics of individuals. The most prominent themes included 1) a limited evidence base, 2) a cumbersome and lengthy testing process, and 3) a lack of insurance coverage for preemptive PGx testing. Additional barriers included clinician lack of knowledge, difficulty remembering to order PGx testing for eligible patients, challenges with PGx test interpretation, a questionable impact of preemptive PGx testing on clinical care, and a lack of alternative therapeutic options for some patients found to have actionable PGx variants. CONCLUSIONS: Successful adoption of preemptive PGx-guided chemotherapy dosing in patients with gastrointestinal malignancies will require a multifaceted effort to demonstrate clinical effectiveness while addressing the contextual factors identified in this study.

Temporal evolution of electron cloud in a cylindrical Penning trap at room temperature.

The temporal evolution of the electron cloud at room temperature has been recorded through a resonance circuit by observing the axial oscillation frequency of its center of mass. The electron cloud undergoes radial expansion by interacting with the residual gas molecules, and it is finally lost upon hitting the Penning trap electrodes. It has been confirmed through detailed experimental investigations that the unique temporal pattern of frequency variation is a consequence of the cloud's radial expansion. Consequently, this approach offers a non-destructive means for single-shot detection, enabling continuous monitoring of the electron cloud's radial expansion during the confinement time. This technique offers a significant advantage over its destructive alternatives.

Design of a helical resonator with improved figure of merit.

A helical resonator serves as a key element for the detection of the trapped charged particles in a Penning trap. In order to compare the performance of the helical resonators, the concept of figure of merit (FOM) was introduced by Ulmer et al. [Nucl. Instrum. Methods Phys. Res., Sect. A 705, 55-60 (2013)]. In this work, we optimized the geometrical parameters of a resonator by numerical simulations keeping its outer dimensions and the diameter of the copper wire fixed and obtained the best possible value of FOM under these constraints. The corresponding 95 MHz helical resonator has been designed and fabricated, and its measured value of FOM is in good agreement with the simulated values. An empirical relationship between the total length of the wire to make the helical coil and the resonance frequency has been obtained. The simulations show that the FOM increases considerably with the increase in the conductivity of the building material, and this would be useful in detecting the feeble trap signal in cryogenic environment.

Implementing Pharmacogenetic Testing in Gastrointestinal Cancers (IMPACT-GI): Study Protocol for a Pragmatic Implementation Trial for Establishing DPYD and UGT1A1 Screening to Guide Chemotherapy Dosing.

Background: Fluoropyrimidines (fluorouracil [5-FU], capecitabine) and irinotecan are commonly prescribed chemotherapy agents for gastrointestinal (GI) malignancies. Pharmacogenetic (PGx) testing for germline DPYD and UGT1A1 variants associated with reduced enzyme activity holds the potential to identify patients at high risk for severe chemotherapy-induced toxicity. Slow adoption of PGx testing in routine clinical care is due to implementation barriers, including long test turnaround times, lack of integration in the electronic health record (EHR), and ambiguity in test cost coverage. We sought to establish PGx testing in our health system following the Exploration, Preparation, Implementation, Sustainment (EPIS) framework as a guide. Our implementation study aims to address barriers to PGx testing. Methods: The Implementing Pharmacogenetic Testing in Gastrointestinal Cancers (IMPACT-GI) study is a non-randomized, pragmatic, open-label implementation study at three sites within a major academic health system. Eligible patients with a GI malignancy indicated for treatment with 5-FU, capecitabine, or irinotecan will undergo PGx testing prior to chemotherapy initiation. Specimens will be sent to an academic clinical laboratory followed by return of results in the EHR with appropriate clinical decision support for the care team. We hypothesize that the availability of a rapid turnaround PGx test with specific dosing recommendations will increase PGx test utilization to guide pharmacotherapy decisions and improve patient safety outcomes. Primary implementation endpoints are feasibility, fidelity, and penetrance. Exploratory analyses for clinical effectiveness of genotyping will include assessing grade ≥3 treatment-related toxicity using available clinical data, patient-reported outcomes, and quality of life measures. Conclusion: We describe the formative work conducted to prepare our health system for DPYD and UGT1A1 testing. Our prospective implementation study will evaluate the clinical implementation of this testing program and create the infrastructure necessary to ensure sustainability of PGx testing in our health system. The results of this study may help other institutions interested in implementing PGx testing in oncology care. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT04736472, identifier [NCT04736472].

Fabrication of non-magnetic multi-pin coaxial vacuum feedthrough system for cryogenic applications.

We have developed and tested a compact non-magnetic feedthrough made of epoxy resin and capable of maintaining vacuum leak tightness over a wide temperature range (300 K-4 K). It is equipped with 15 electrical pins and three 50 Ω coaxial lines. The feedthrough has been designed to apply a high voltage (up to 5 kV) and transmit radio-frequency signals for operating a Penning trap over a wide temperature range (300 K-4 K). The characteristic impedances of the coaxial lines have been measured at 300 K and 77 K and found to remain ∼50 Ω over the frequency range of our interest (10 MHz-80 MHz). The details of its fabrication and performance over a wide temperature range have been discussed.

Aspirin potentiates prestimulated acid secretion and mobilizes intracellular calcium in rabbit parietal cells.

The effects of aspirin on gastric acid secretion were studied in isolated rabbit parietal cells (PC). Aspirin (10(-5) M) potentiated histamine-, dibutyryl cyclic AMP (dbcAMP)-, forskolin- and 3-isobutyl-1-methylxanthine-stimulated acid secretion without affecting basal acid secretion. Augmentation of secretagogue-stimulated acid secretion by aspirin was dependent on calcium (Ca2+) since potentiation was blocked by removal of extracellular Ca2+ ([Ca2+]o) or addition of the calcium antagonist lanthanum chloride. Using the Ca2+ probe fura-2, aspirin (10(-6) - 2 X 10(-5) M) rapidly increased intracellular free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The source of released Ca2+ was intracellular as demonstrated by depletion of intracellular Ca2+ and [Ca2+]o with EGTA washing. Aspirin did not affect several other signal transduction sites involved in stimulus-secretion coupling, including the H2 receptor, intracellular cyclic AMP (cAMP), inositol 1,4,5, triphosphate (IP3) and H+,K(+)-ATPase. Aspirin decreased PC prostaglandin E2 (PGE2) content by 98%. Exogenous dimethyl PGE2 (dmPGE2) inhibited both histamine-stimulated acid secretion and its enhancement by aspirin. In contrast, dmPGE2 abolished aspirin-induced potentiation of dbcAMP-stimulated acid secretion by augmenting the dbcAMP-stimulated response. These results indicate that aspirin acts at a site beyond the adenylate cyclase/cAMP system and before the proton pump, presumably by releasing Ca2+ from an IP3-independent intracellular storage pool and by inhibiting PGE2 generation.

The effects of lapatinib on CYP3A metabolism of midazolam in patients with advanced cancer.

PURPOSE: The potential inhibition of CYP3A4 by lapatinib was studied using midazolam as a probe substrate in patients with cancer. METHODS: This was a partially randomized, 4-period, 4-sequence, 4-treatment, cross-over study in 24 patients with advanced cancer. Single 1-mg IV and 3-mg oral doses of midazolam were given 2 days apart, in a partially random order, on study days 1, 3, 9, and 11. Lapatinib 1500-mg was administered orally once daily on study days 4 through 11. Midazolam plasma concentrations were measured up to 24-h post dosing, and lapatinib plasma concentrations measured prior to each midazolam dose. RESULTS: Lapatinib increased the geometric mean (95% CIs) midazolam AUC(o-∞) by 45% (31-60%) after the oral dose and by 14% (0-29%) after the IV dose, and prolonged the midazolam elimination half-life by 48% (22-81%) after the oral dose and by 20% (2-40%) after the IV dose. Lapatinib decreased midazolam total clearance by 13% (1-23%), while total bioavailability was increased 23% (4-46%) without changes in apparent volume of distribution or hepatic bioavailability. CONCLUSION: These data show that lapatinib caused weak inhibition of gastrointestinal CYP3A4 in vivo. This suggests that oral CYP3A4 drug substrates with a narrow therapeutic index may need dose reduction if lapatinib is to be co-prescribed.

Endogenous protease mediated manifestations of a Ca2+-stimulated ATPase in purified dog gastric microsomes.

An endogenous soluble protease has been demonstrated to unmask a Ca2+-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca2+-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca2+-stimulated ATPase occurs without affecting the microsomal (H+ + K+)-ATPase activity and associated H+ uptake ability. The unmasked Ca2+-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H+ transport have been discussed.

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties.

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.

Modulation of gastric H+,K+-transporting ATPase function by sodium.

Gastric H+,K+-ATPase activity is not affected by Na+ at pH 7.0 but is significantly stimulated by Na+ at pH 8.5. For the stimulation at the latter pH, the presence of both Na+ and K+ were essential. Contrary the H+,K+-ATPase, the associated K+-pNPPase was inhibited by Na+ at both pH values. Sodium competes with K+ for the K+-pNPPase reaction. Also, unlike the H+, K+-ATPase activity the ATPase-mediated transport of H+ within the gastric microsomal vesicles was inhibited by Na+. For the latter event only the extravesicular and not the intravesicular Na+ was effective. The data suggest that the K+-pNPPase activity does not represent the phosphatase step of the H+,K+-ATPase reaction. In addition, the observed inhibition of vesicular H+ uptake by Na+ appears to be due to the displacement by Na+ of a cytosolic (extravesicular) H+ site responsible for the vectorial translocation of H+.

Induction of an ATPase inhibitor protein by propylthiouracil and protection against paracetamol (acetaminophen) hepatotoxicity in the rat.

1. The purpose of the present study was to test the following hypothesis: propylthiouracil (PTU) treatments of rats induces an increase in the concentration and activity of the mitochondrial ATPase (m-ATPase) inhibitor protein (IF1). The PTU-induced elevated baseline levels of this inhibitor protein inactivated m-ATPase, and prevented hepatotoxicity by a toxic dose of acetaminophen (AAP) (paracetamol), by maintaining hepatic adenosine 5'-triphosphate (ATP) levels. 2. Male Wistar rats were either gavaged with a toxic dose of AAP alone, or after pretreatment with PTU for periods of 3 and 12 days. 3. Twenty four hours after acetaminophen treatment alone, toxicity was manifested by: an approximately 10 fold increase in serum transaminase levels (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase); depletion of hepatic reduced glutathione (GSH) and ATP levels; loss of inhibitor protein activity, and extensive pericentral necrosis of the hepatocytes. Propylthiouracil pretreatment for 12 days enhanced the concentration of the following metabolites in the liver: ATP (1.5 fold), ATPase inhibitor protein (IF1) (4.5 fold), and reduced glutathione (1.3 fold), while the activity of the inhibitor protein increased 2 fold. When the PTU treated rats were challenged with AAP, transaminases were not elevated, and only sporadic areas of necrosis were detected by histological examination of the liver tissue. In contrast to the 12 day treatment with PTU the 3 day treatment had no protection against AAP. No histological evidence of protection was manifested and the transaminases were not different from AAP treated controls. Most of the protective metabolites were depleted. 4. Our findings suggest that PTU-induced increased concentration of inhibitor protein and GSH, are contributing factors in the prevention of hepatotoxicity by maintaining hepatic m-ATP levels and reducing the harmful effect of the toxic metabolite of AAP.

Mode of food intake reduction in Lewis rats with indomethacin-induced ulcerative ileitis.

The mechanism of anorexia in inflammatory bowel disease is poorly understood. To gain insight into possible pathophysiologic mechanisms, the feeding indices and food intake were studied in an animal model of Crohn's disease. The anorexia of indomethacin-induced ulcerative ileitis was compared with that of the well-known anorexia of total parenteral nutrition (TPN). Forty-five female Lewis rats were randomized to four groups: Control, Indomethacin, Indomethacin + TPN, and TPN. Feeding indices and food intake were continuously measured using the Automated Computerized Rat Eater Meter. Interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) were assayed in plasma, mononuclear cell culture, or ileum to determine their role in mediating anorexia. In the TPN group, spontaneous food intake (SFI) decreased (52%; p < 0.05), primarily via reduction in meal number (MN, 54%; p < 0.05) and, to a lesser extent, meal size (MZ, 35%; p < 0.05). In comparison, in the Indomethacin group SFI decreased (74%; p < 0.05) primarily via reduction in MZ (67%, p < 0.05); MN also decreased but to a lesser extent (27%; p < 0.05). In the Indomethacin + TPN group, SFI decreased (55%; p > 0.05) primarily via reduction in MN (79%; p < 0.05), whereas MZ decreased slightly (19%; p < 0.05). Only in the Indomethacin group were IL-1 alpha and TNF-alpha detected in the mononuclear cell culture and plasma, respectively. In the Indomethacin group, an inverse correlation existed between MZ and TNF-alpha (p < 0.05). In the Indomethacin group, IL-1 alpha, PGE2, and LTB4 concentrations did not correlate with feeding indices. SFI reduction in this model was mediated primarily via a decrease in MZ. TNF-alpha is proposed to mediate this effect and TPN was shown to overcome the effect on MZ.

Expression and characterization of protein kinase C in isolated rabbit parietal cells.

We investigated the expression, characterization and distribution of protein kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibodies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane and cytoskeletal fractions of basal and histamine-stimulated PC, whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractions. Cytosolic and membrane fractions were partially purified by DEAE cellulose column chromatography with elution of increasing NaCl concentration. Eluates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha and -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) treatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-alpha and increased membrane-associated PKC-alpha. In contrast to the distribution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-epsilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-associated PKC-alpha suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurosporine, produced a concentration-dependent enhancement of histamine-stimulated H+ secretion. These findings suggest that PKC-alpha plays a negative modulatory role, rather than an obligatory role, in H+ secretion. The localization and distribution of PKC-epsilon into the cytoskeletal fraction of PC also suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.

In vitro and in vivo evaluation of versicolin, an antifungal antibiotic.

Versicolin is mainly active against Trichophyton rubrum. It is inactivated by serum. However, attainable blood level can be fifteen to twenty times higher than the fungicidal concentration for as long as 4 hours after a single intravenous administration of the maximum tolerable dose of 25 mg/kg body weight of mouse. It has no haemolytic activity like other polypeptide antibiotics. Skin does not show the presence of any measurable amount of the antibiotic. It is excreted through urine to the extent of 65% of the maximum tolerable dose of the antibiotic administered intravenously. Versicolin is effective against experimental infection by T. rubrum in guinea pigs at dose as low as 2.5 mg/kg body weight. The antibiotic shows no sign of subacute toxicity at these curative doses.

Evaluation of mycobacillin and versicolin as agricultural fungicides. II. Stability in soil.

The effect of paddy soils on mycobacillin and versicolin was investigated. Soil inactivated mycobacillin as determined by spectral analysis and microbiological assay. Soil can inactive mycobacillin only at or above the threshold concentration (125 approximately 130 mug per 10 mg of soil), the excess being unreacted. No new peak appears in the ultraviolet spectrum (240 approximately 300 nm) while mycobacillin is inactivated. Soil is without any effect on versicolin.

Evaluation of mycobacillin and versicolin as agricultural fungicides. I. Antifungal spectrum and phytotoxicity.

Two antifungal antibiotics, mycobacillin and versicolin, were studied as agricultural fungicides in the control of fungal infection of rice and jute. Mycobacillin is especially active against Piricularia oryzae at a concentration of 10 mug/ml, and versicolin against Colletotrichum gloeosporioides at a concentration of 2.5 mug/ml. Mycobacillin has no adverse effect on germination of seeds and growth of seedlings of rice and jute plants as a concentration of 500ppm, even for prolonged exposure. (24 or 48 hours); in fact, it is stimulatory. On the other hand, versicolin has showed detectable phytotoxicity at 500ppm for prolonged exposure

Evaluation of mycobacillin and versicolin as agricultural fungicides. III. Growth pattern and antibiotic production in soil by Aspergillus versicolor.

Soil supports the growth of a jute pathogen Colletotrichum gloeosporioides but only to a limited extent that of its antagonist Aspergillus versicolor. The growth of the sensitive pathogen is considerably checked by the antagonist in mixed soil culture although versicolin production could not be demonstrated within the limits of assay. Both the sensitive and the antagonistic organisms grow well in soil-compost medium and versicolin production by the latter is also enhanced. The antagonistic effect of Aspergillus versicolor on Colletotrichum gloeosporioides is expectedly more marked in soil-compost medium than in soil medium.

Chloroquine versus amodiaquine in the treatment of Plasmodium falciparum malaria in northeast India.

Amodiaquine is being used in India for presumptive treatment as an alternative to chloroquine in areas with chloroquine resistant P. falciparum. Keeping in view the toxicity of amodiaquine, studies have been undertaken to evaluate the advantage of the drug over chloroquine in the treatment of P. falciparum malaria. In vivo drug resistance studies were carried out in the states of Assam and Meghalaya in India. A total of 388 subjects have been studied to compare the efficacy of chloroquine and amodiaquine. The overall cure rate, degree of resistance, mean parasite clearance time and mean parasite recrudescence time were comparable for both the drugs, the differences being not statistically significant. The results indicate no advantage of amodiaquine in the treatment of patients with P. falciparum infection in chloroquine resistant areas of northeast India and development of cross resistance in P. falciparum to these 4-aminoquinolines is complete and parallel.

Alternative mode of replication of human immunodeficiency virus: a hypothesis.

For diagnosis of Human Immunodeficiency Virus (HIV) infection by the recently developed Polymerase Chain Reaction (PCR), the two commonly used clinical samples are either the peripheral blood monocytes (PBMC) or the plasma of the infected individuals. In the former instance, DNA is extracted from PBMC. The integrated proviral DNA is then amplified using HIV specific oligonucleotide primers. In the latter instance, RNA is extracted from plasma. This is reverse transcribed in vitro into cDNA by using extraneous reverse transcriptase. This cDNA is then used as a target in PCR experiments with HIV specific primers. In contrast we have recently used DNA directly extracted from plasma of infected individuals. This DNA was used for amplification of HIV genome with primer pairs specific for HIV. An interesting outcome of this study was a model to explain the presence of DNA of HIV in the plasma. We suggest that possibly there is an alternative mode of replication of HIV. Apart from the obligatory integration of the DNA of HIV into the DNA of lymphocytes as provirus, several additional copies of the DNA are also made which remain unintegrated. These probably exist as a housekeeping repertoire of the viral genome. These DNA molecules may be released into the circulation along with the newly formed mature virion particles during the usual course of replication and release of the virus. In our experiments with direct extraction of DNA from plasma, these unintegrated DNA of HIV may act as the target for PCR to give positive signals with HIV specific primers.