P-glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Main Active Heroin Metabolites 6-monoacetylmorphine (6-MAM) and Morphine in Mice.

Background & Purpose: Heroin (diacetylmorphine; diamorphine) is a highly addictive opioid prodrug. Heroin prescription is possible in some countries for chronic, treatment-refractory opioid-dependent patients and as a potent analgesic for specific indications. We aimed to study the pharmacokinetic interactions of heroin and its main pharmacodynamically active metabolites, 6-monoacetylmorphine (6-MAM) and morphine, with the multidrug efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2 using wild-type, Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice.

Natural deletion of mouse carboxylesterases Ces1c/d/e impacts drug metabolism and metabolic syndrome development.

Mammalian carboxylesterase 1 enzymes can hydrolyze many xenobiotic chemicals and endogenous lipids. We here identified and characterized a mouse strain (FVB/NKI) in which three of the eight Ces1 genes were spontaneously deleted, removing Ces1c and Ces1e partly, and Ces1d entirely. We studied the impact of this Ces1c/d/e deficiency on drug and lipid metabolism and homeostasis.

Carboxylesterase 1 family knockout alters drug disposition and lipid metabolism.

The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated cluster knockout ( ) mice, and a hepatic human CES1 transgenic model in the background (TgCES1). mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues.

Bottom-up sample preparation for the LC-MS/MS quantification of anti-cancer monoclonal antibodies in bio matrices.

Therapeutic monoclonal antibodies (mAbs) are rapidly taking over the treatment of many malignancies, and an astonishing number of mAbs is in development. This causes a high demand for quantification of mAbs in biomatrices both for measuring therapeutic mAb concentrations and to support pharmacokinetics and pharmacodynamics studies. Conventionally, ligand-binding assays are used for these purposes, but LC-MS is gaining popularity.

Development and validation of a bioanalytical method for the quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib in human and mouse matrices using liquid chromatography-tandem mass spectrometry.

A novel method was developed and validated for the quantification of the three approved CDK4/6 inhibitors (abemaciclib, palbociclib, and ribociclib) in both human and mouse plasma and mouse tissue homogenates (liver, kidney, spleen, brain, and small intestine) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For all matrices, pretreatment was performed using 50 μL of sample by protein precipitation with acetonitrile, followed by dilution of the supernatant. Chromatographic separation of the analytes was done on a C18 column using gradient elution.

Validation of a liquid chromatographic method for the pharmaceutical quality control of products containing elacridar.

Many anticancer drugs have an impaired bioavailability and poor brain penetration because they are substrates to drug efflux pumps such as P-glycoprotein and Breast Cancer Resistance Protein. Elacridar is a strong inhibitor of these two drug efflux pumps and therefore has great potential to improve oral absorption and brain penetration of many anticancer drugs. Currently, a clinical formulation of elacridar is unavailable and therefore the pharmaceutical development of a drug product is highly warranted.

Development of an LC-MS/MS assay for the quantitative determination of the intracellular 5-fluorouracil nucleotides responsible for the anticancer effect of 5-fluorouracil.

5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP).

Quantitative determination of azacitidine triphosphate in peripheral blood mononuclear cells using liquid chromatography coupled with high-resolution mass spectrometry.

Azacitidine is a cytidine analog used in the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia and acute myeloid leukemia. The pharmacological effect of azacitidine arises after incorporation into the DNA and RNA. To this end, the drug first has to be converted into its triphosphate forms.

Quantitative determination of capecitabine and its six metabolites in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry.

Capecitabine is the oral prodrug of the anticancer drug 5-fluorouracil (5-FU). The purpose of this study was to quantify capecitabine and its metabolites including 5'-deoxy-5-fluorocytidine (5'-dFCR), 5'-deoxy-5-fluorouridine (5'-dFUR), 5-FU, dihydro-5-fluorouracil (FUH(2)), α-fluoro-ureidopropionic acid (FUPA) and fluoro-β-alanine (FBAL) in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. To this end two individual assays were developed: one for the simultaneous quantification of capecitabine, 5'-dFCR and 5'-dFUR using reversed phase chromatography and gradient elution, and one assay for 5-FU, FUH(2), FUPA and FBAL using hydrophilic interaction chromatography and isocratic elution.

A sensitive combined assay for the quantification of paclitaxel, docetaxel and ritonavir in human plasma using liquid chromatography coupled with tandem mass spectrometry.

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human plasma is described. The drugs were extracted from 200 μL human plasma using liquid-liquid extraction with tertiar-butylmethylether, followed by high performance liquid chromatography analysis using 10 mM ammonium hydroxide pH 10:methanol (3:7, v/v) as mobile phase. Chromatographic separation was obtained using a Zorbax Extend C(18) column.

Brain accumulation of sunitinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by oral elacridar and sunitinib coadministration.

Sunitinib is an orally active, multitargeted tyrosine kinase inhibitor which has been used for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. We aimed to investigate the in vivo roles of the ATP-binding cassette drug efflux transporters ABCB1 and ABCG2 in plasma pharmacokinetics and brain accumulation of oral sunitinib, and the feasibility of improving sunitinib kinetics using oral coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. We used in vitro transport assays and Abcb1a/1b(-/-) , Abcg2(-/-) and Abcb1a/1b/Abcg2(-/-) mice to study the roles of ABCB1 and ABCG2 in sunitinib disposition.

Lethal morphine intoxication in a patient with a sickle cell crisis and renal impairment: case report and a review of the literature.

Morphine-6-glucuronide, the active metabolite of morphine, and to a lesser extent morphine itself are known to accumulate in patients with renal failure. A number of cases on non-lethal morphine toxicity in patients with renal impairment report high plasma concentrations of morphine-6-glucuronide, suggesting that this metabolite achieves sufficiently high brain concentrations to cause long-lasting respiratory depression, despite its poor central nervous system penetration. We report a lethal morphine intoxication in a 61-year-old man with sickle cell disease and renal impairment, and we measured concentrations of morphine and morphine-6-glucuronide in blood, brain and cerebrospinal fluid.

Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment.

Purpose: Imatinib, a BCR-ABL tyrosine kinase inhibitor, is a substrate of the efflux transporters P-glycoprotein (P-gp; ABCB1) and ABCG2 (breast cancer resistance protein), and its brain accumulation is restricted by both transporters. For dasatinib, an inhibitor of SCR/BCR-ABL kinases, in vivo interactions with P-gp and ABCG2 are not fully established yet.

Experimental Design: We used Abcb1a/1b(-/-), Abcg2(-/-), and Abcb1a/1b;Abcg2(-/-) mice to establish the roles of P-gp and ABCG2 in the pharmacokinetics and brain accumulation of dasatinib.

Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.

Fast and simultaneous determination of darunavir and eleven other antiretroviral drugs for therapeutic drug monitoring: method development and validation for the determination of all currently approved HIV protease inhibitors and non-nucleoside reverse transcriptase inhibitors in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

For the quantification of all currently approved non-nucleoside reverse transcriptase inhibitors and protease inhibitors, including the new protease inhibitor darunavir and the active nelfinavir metabolite M8, an assay was developed, using liquid chromatography coupled with tandem mass spectrometry. The sample pretreatment consisted of a protein precipitation with a mixture of methanol and acetonitrile using only 100 microL plasma. Chromatographic separation was performed on a reversed-phase C18 column (150 x 2.

Multidrug resistance proteins 2 and 3 provide alternative routes for hepatic excretion of morphine-glucuronides.

Glucuronidation is a major hepatic detoxification pathway for endogenous and exogenous compounds, resulting in the intracellular formation of polar metabolites that require specialized transporters for elimination. Multidrug resistance proteins (MRPs) are expressed in the liver and can transport glucuronosyl-conjugates. Using morphine as a model aglycone, we demonstrate that morphine-3-glucuronide (M3G), the predominant metabolite, is transported in vitro by human MRP2 (ABCC2), a protein present in the apical membrane of hepatocytes.

Breast cancer resistance protein (Bcrp1/Abcg2) is expressed in the harderian gland and mediates transport of conjugated protoporphyrin IX.

Proper regulation of intracellular levels of protoporphyrin IX (PPIX), the direct precursor of heme, is important for cell survival. A deficiency in ferrochelatase, which mediates the final step in heme biosynthesis, leads to erythropoietic protoporphyria (EPP), a photosensitivity syndrome caused by the accumulation of PPIX in the skin. We have previously shown that mice with a deficiency in the ABC transporter Bcrp1/Abcg2 display a novel type of protoporphyria.

Metabolism of trabectedin (ET-743, Yondelis) in patients with advanced cancer.

Purpose: Trabectedin (ET-743, Yondelis) is a novel anti-cancer drug currently undergoing phase II-III evaluation, that has shown remarkable activity in pre-treated patients with soft tissue sarcoma. Despite extensive pharmacokinetic studies, the human disposition and metabolism of trabectedin remain largely unknown. We aimed to determine the metabolic profile of trabectedin and to identify its metabolites in humans.

Quantitative analysis of EO9 (apaziquone) and its metabolite EO5a in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray tandem mass spectrometry.

A sensitive and specific LC-MS/MS assay for the quantitative determination of EO9 and its metabolite EO5a is presented. A 200-microl human plasma aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 ml ethyl acetate.

Phase I and pharmacokinetic study of combined treatment with perifosine and radiation in patients with advanced solid tumours.

Purpose: Perifosine is an orally applicable, membrane-targeted alkylphosphocholine analogue with antitumour activity and radiosensitising properties in preclinical models. The purpose of this phase I study was to determine the feasibility and tolerability of concurrent daily perifosine and radiation in patients with advanced cancer.

Patients And Methods: Starting dose of perifosine was 50 mg/day; dose escalation was in steps of 50mg.

Screening for illicit heroin use in patients in a heroin-assisted treatment program.

The aim of this study was to investigate the use of illicit heroin among patients in a heroin-assisted treatment program. In this program, pharmaceutical-grade heroin was administered to heroin-addicted patients. Monitoring of illicit heroin use was considered important for the evaluation of this treatment program.

Analysis of diacetylmorphine, caffeine, and degradation products after volatilization of pharmaceutical heroin for inhalation.

Pharmaceutical smokable heroin was developed for a clinical trial on medical co-prescription of heroin and methadone. This product, consisting of 75% w/w diacetylmorphine base and 25% w/w caffeine anhydrate, was intended for use via "chasing the dragon", that is, inhalation after volatilization. This procedure involves heating the powder mixture, which may lead to formation of degradation products that could subsequently be inhaled.

Pharmacokinetics and pharmacodynamics of high doses of pharmaceutically prepared heroin, by intravenous or by inhalation route in opioid-dependent patients.

A pharmacokinetic-pharmacodynamic study was performed in opioid-dependent patients in the Netherlands, who were currently treated with high doses of pharmaceutically prepared heroin on medical prescription. Besides intravenous heroin, heroin was prescribed for inhalation by "chasing the dragon" method. In this technique, heroin base is heated on aluminium foil, and heroin vapours are inhaled into the lungs.

Deuterodiacetylmorphine as a marker for use of illicit heroin by addicts in a heroin-assisted treatment program.

In preparation for a treatment program concerning the medical coprescription of heroin and methadone to treatment-resistant addicts in the Netherlands, we studied a novel strategy for monitoring co-use of illicit (nonprescribed) heroin. A deuterated analogue of heroin was added (1:20) to pharmaceutical, smokable heroin (a powder mixture of 75% w/w diacetylmorphine base and 25% w/w caffeine anhydrate), to be used by inhalation after volatilization ("chasing the dragon"). Plasma and urine samples were collected from nine male patients who had used pharmaceutical, smokable heroin during a four-day stay in a closed clinical research unit, and these samples were analyzed by liquid chromatography coupled with tandem mass spectrometry.

The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry.

For a pharmacokinetic-pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC-MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards.