P-glycoprotein (MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) restrict brain accumulation of the JAK1/2 inhibitor, CYT387.

CYT387 is an orally bioavailable, small molecule inhibitor of Janus family of tyrosine kinases (JAK) 1 and 2. It is currently undergoing Phase I/II clinical trials for the treatment of myelofibrosis and myeloproliferative neoplasms. We aimed to establish whether the multidrug efflux transporters P-glycoprotein (P-gp; MDR1; ABCB1) and breast cancer resistance protein (BCRP;ABCG2) restrict oral availability and brain penetration of CYT387. In vitro, CYT387 was efficiently transported by both human MDR1 and BCRP, and very efficiently by mouse Bcrp1 and its transport could be inhibited by specific MDR1 inhibitor, zosuquidar and/or specific BCRP inhibitor, Ko143. CYT387 (10 mg/kg) was orally administered to wild-type (WT), Bcrp1(-/-), Mdr1a/1b(-/-) and Bcrp1;Mdr1a/1b(-/-) mice and plasma and brain concentrations were analyzed. Over 8h, systemic exposure of CYT387 was similar between all the strains, indicating that these transporters do not substantially limit oral availability of CYT387. Despite the similar systemic exposure, brain accumulation of CYT387 was increased 10.5- and 56-fold in the Bcrp1;Mdr1a/1b(-/-) mice compared to the WT strain at 2 and 8h after CYT387 administration, respectively. In single Bcrp1(-/-) mice, brain accumulation of CYT387 was more substantially increased than in Mdr1a/1b(-/-) mice, suggesting that CYT387 is a slightly better substrate of Bcrp1 than of Mdr1a at the blood-brain barrier. These results indicate a marked and additive role of Bcrp1 and Mdr1a/1b in restricting brain penetration of CYT387, potentially limiting efficacy of this compound against brain (micro) metastases positioned behind a functional blood-brain barrier.

PI3K/mTOR inhibitor omipalisib prolongs cardiac repolarization along with a mild proarrhythmic outcome in the AV block dog model.

Background: The phosphoinositide 3-kinase (PI3K) signaling pathway is an interesting target in cancer treatment. The awareness of the proarrhythmic risk of PI3K inhibitors was raised because PI3K is also involved in regulating signaling toward cardiac ion channels. Canine cardiomyocytes treated with PI3K inhibitors show an increased action potential duration and reduced cardiac repolarizing currents. Now, the potential proarrhythmic effect of chronic treatment of PI3K/mTOR inhibitor GSK2126458 (omipalisib) was investigated in the atrioventricular (AV) block dog model. Methods: Purpose-bred Mongrel dogs received complete AV block by ablation of the bundle of His and their hearts were paced in the right ventricular apex at VDD-mode (RVA-VDD). In this way, sinus rhythm was maintained for 15 ± 1 days and thereby bradycardia-induced cardiac remodeling was prevented. Dogs received 1 mg/kg omipalisib once (n = 3) or twice (n = 10) a day via oral administration for 7 days. Under standardized conditions (anesthesia, bradycardia at 60 beats/min, and a dofetilide challenge), potential proarrhythmic effects of omipalisib were investigated. Results: Twice daily dosing of omipalisib increased accumulative plasma levels compared to once daily dosing accompanied with adverse events. Omipalisib prolonged the QT interval at baseline and more strongly after the dofetilide challenge (490 ± 37 to 607 ± 48 ms). The arrhythmic outcome after omipalisib resulted in single ectopic beats in 30% of dogs perpetuating in multiple ectopic beats and TdP arrhythmia in 20% of dogs. Isolated ventricular cardiomyocytes from omipalisib-treated dogs showed a diminished IKs current density. Conclusion: Chronic treatment of PI3K/mTOR inhibitor omipalisib prolonged the QT interval in a preclinical model under standardized proarrhythmic conditions. Furthermore, this study showed that electrical remodeling induced by omipalisib had a mild proarrhythmic outcome.

Bioanalysis of EGFRm inhibitor osimertinib, and its glutathione cycle- and desmethyl metabolites by liquid chromatography-tandem mass spectrometry.

Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000 nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6 nM due to low accuracy at 2 nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80 mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.

Bioanalysis of ibrutinib, and its dihydrodiol- and glutathione cycle metabolites by liquid chromatography-tandem mass spectrometry.

Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma. The method emphasizes on simplicity, the thiol-conjugates were synthesized by a simple one step synthesis, followed by LC-purification. Sample preparation was done by a simple protein crash with acetonitrile containing labeled internal standards, evaporation of solvents, and reconstitution in eluent. Finally, the compounds were quantified using UHPLC-MS/MS. The assay was successfully validated in a 0.5-500nM calibration range for all compounds, and also a lower range of 0.05-50 nM was demonstrated for ibrutinib to accommodate for even the lowest trough levels. This assay has a considerably higher sensitivity than previous published assays, with the previous lowest LLOQ being 1.14 nM. Both, ibrutinib, dihydrodiol-ibrutinib and the cysteine conjugate were deemed stable under refrigerated or frozen storage conditions. At room temperature, the glutathione conjugate showed rapid degradation into the cysteinylglycine conjugate in plasma. Finally, the applicability of the assay was demonstrated in patient samples.

Hepatic artery infusion of high-dose melphalan at reduced flow during isolated hepatic perfusion for the treatment of colorectal metastases confined to the liver: a clinical and pharmacologic evaluation.

Isolated hepatic perfusion (IHP) offers the advantage of high local drug exposure with limited systemic toxicity. To increase local drug exposure, we administered melphalan at a reduced flow in the hepatic artery during IHP (hepatic artery infusion, hepatic artery-portal vein perfusion, HI-HPP). Between December 2001 and December 2004, 30 patients with colorectal cancer liver metastases underwent HI-HPP with 200mg melphalan. Samples of the perfusate were taken for pharmacokinetic analysis. Patients were monitored for response, toxicity and survival. Perfusion was aborted prematurely in 2 patients due to leakage. During melphalan administration in the hepatic inflow cannula a mean flow rate of 121.3 mL/min and mean pressure of 62.5mm Hg were achieved. One patient died within 30 days after HI-HPP. Four patients developed veno-occlusive disease (VOD), while 2 patients showed signs of VOD. Twelve patients showed hepatic response, with a median duration of response of 11.5 months, according to WHO criteria. Although HI-HPP results in high perfusate melphalan concentration levels, it is associated with a relatively high level of hepatotoxicity and a limited response rate. We believe that the low flow and pressure rates found in this study can result in reduced drug penetration of the tumour and thus limited tumour response.

Development and validation of a quantitative assay for the measurement of two HIV-fusion inhibitors, enfuvirtide and tifuvirtide, and one metabolite of enfuvirtide (M-20) in human plasma by liquid chromatography-tandem mass spectrometry.

A method for the quantification of two peptide HIV-1 fusion inhibitors (enfuvirtide, T-20 and tifuvirtide, T-1249) and one metabolite of enfuvirtide (M-20) in human plasma has been developed and validated, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). The analytes were extracted from plasma by solid-phase extraction (SPE) on vinyl-copolymer cartridges. Chromatographic separation of the peptides was performed on a Symmetry 300 C(18) column (50mmx2.1mm I.D., particle size 3.5 microm), using a water-acetonitrile gradient containing 0.25% (v/v) formic acid. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for peak detection. Deuterated (d60) enfuvirtide and (d50) tifuvirtide were used as internal standards. The assay was linear over a concentration range of 20-10,000 ng/ml for enfuvirtide and tifuvirtide and of 20-2000 ng/ml for M-20. Intra- and inter-assay precisions and deviations from the nominal concentrations were

High-performance ion-exchange chromatography with in-line complexation of bisphosphonates and their quality control in pharmaceutical preparations.

An ion-exchange chromatographic method using an anion-exchange column was developed for the analysis of a number of bisphosphonates. The bisphosphonates were in-line complexed by copper(II) ions and added to the acidic eluent, to yield an UV-absorbing complex. Chromatographic parameters were studied for eight different bisphosphonates, particularly amino-1-hydroxyalkyl-1,1-bisphosphonates; special attention was paid to the relationship between retention and complex formation. The method was applied to the quality control of pamidronate injection concentrate and olpadronate tablets. The lower detection limit was 8 ng of disodium pamidronate, and the inter-assay precision was 1.0% for both pamidronate and olpadronate standard solutions and 1.8% for a 3 mg ml-1 disodium pamidronate injection concentrate. The method was compared with a previously described ion-exchange chromatographic method with conductivity detection, without copper(II) ions in the eluent.

The determination of pamidronate in pharmaceutical preparations by ion-pair liquid chromatography after derivatization with phenylisothiocyanate.

An analytical method was developed for the determination of pamidronate [(3-amino-1-hydroxypropylidene)bisphosphonate] by ion-pair liquid chromatography. The analyte was derivatized with phenylisothiocyanate into an UV-absorbing derivative. The reaction product was cleaned-up by a double ion-pair extraction and treated with hydrogen peroxide prior to injection. Both, the detection limit and the lower limit of quantification of pamidronate in water were 0.1 microgram ml-1 disodium pamidronate. The intra-day precision was 3% for a 5-microgram ml-1 pamidronate standard solution and the inter-day precision 6% for a 3-microgram ml-1 solution. The method was applied in the quality control of pamidronate injection concentrates and tablets.

Isolated lung perfusion with melphalan: pharmacokinetics and toxicity in a pig model.

BACKGROUND: In patients with unresectable lung cancer or pulmonary metastases, isolated lung perfusion (ILP) has been described as an alternative method to deliver high-dose chemotherapy to the lungs, thereby minimizing systemic toxicity. Pharmacokinetics of ILP have not been extensively investigated. Therefore, we studied the feasibility of ILP with melphalan in a pig model with emphasis on pharmacokinetics and acute lung damage. METHODS: Five pigs underwent ILP with melphalan. Blood and tissue samples were obtained for determination of melphalan levels. Tissue biopsies were taken for microscopic evaluation of lung damage. RESULTS: During ILP, no hemodynamic effects of importance were noted. No systemic leakage of melphalan was observed in any of the animals. Compared with normal lung tissue, microscopic examination of lung tissue after perfusion without melphalan showed pulmonary edema. Directly after melphalan perfusion additional hemorrhagic areas were seen; however, electron microscopy displayed no irreversible endothelial damage. CONCLUSION: This study on pigs proved to be a well reproducible model for ILP with melphalan. Pharmacokinetics show a safety profile with no systemic toxicity, which could justify further patient studies, necessary to determine its effect on pulmonary metastases in humans, especially in case of adjuvant therapy after surgical resection or in unresectable disease.

Phase I and pharmacological study of the farnesyltransferase inhibitor tipifarnib (Zarnestra, R115777) in combination with gemcitabine and cisplatin in patients with advanced solid tumours.

This phase I trial was designed to determine the safety and maximum tolerated dose (MTD) of tipifarnib in combination with gemcitabine and cisplatin in patients with advanced solid tumours. Furthermore, the pharmacokinetics of each of these agents was evaluated. Patients were treated with tipifarnib b.i.d. on days 1-7 of each 21-day cycle. In addition, gemcitabine was given as a 30-min i.v. infusion on days 1 and 8 and cisplatin as a 3-h i.v. infusion on day 1. An interpatient dose-escalation scheme was used. Pharmacokinetics was determined in plasma and white blood cells. In total, 31 patients were included at five dose levels. Dose-limiting toxicities (DLTs) consisted of thrombocytopenia grade 4, neutropenia grade 4, febrile neutropenia grade 4, electrolyte imbalance grade 3, fatigue grade 3 and decreased hearing grade 2. The MTD was tipifarnib 200 mg b.i.d., gemcitabine 1000 mg m(-2) and cisplatin 75 mg m(-2). Eight patients had a confirmed partial response and 12 patients stable disease. No clinically relevant pharmacokinetic interactions were observed. Tipifarnib can be administered safely at 200 mg b.i.d. in combination with gemcitabine 1000 mg m(-2) and cisplatin 75 mg m(-2). This combination showed evidence of antitumour activity and warrants further evaluation in a phase II setting.

Chromatographic analysis of bisphosphonates.

Chromatographic analysis of bisphosphonates in the past has been based primarily on reversed-phase liquid chromatography (RPLC) and ion-exchange chromatography. Gas chromatography (GC) and recently even capillary electrophoresis have also been employed. For bioanalysis, pre-treatment of the sample is a major part of the analysis; protein precipitation, calcium precipitation, solid-phase extraction (SPE) and derivatization have demonstrated to play an important role in bisphosphonate assays. For some of these treatments, for example SPE and derivatization, automation may be possible. Derivatization is a prerequisite for GC analysis of bisphosphonates; a volatile derivative has to be formed. For liquid chromatography, two types of derivatization are known for bisphosphonates. First, the bisphosphonate side chain can be modified by a chemical reaction to yield a derivative with advantageous chromatographic and spectroscopic properties. Secondly, by complexation of both phosphonate groups or of phosphate after decomposition of the analyte, a coloured complex can be formed. The most sensitive bioanalytical methods are based on RPLC and fluorescence detection, if necessary after derivatization. If low detection limits are not required, for example for analysis of pharmaceutical preparations, non-specific detection methods can be applied.

Bisphosphonates in bone diseases.

Bisphosphonates are a class of drugs which are strongly attracted to the bone where they influence the calcium metabolism, mainly by inhibition of the osteoclast-mediated bone resorption. This property makes these compounds suited for the treatment of several diseases of the bone. In Paget's disease, several bisphosphonates can reduce bone pain and decrease the bone turnover 60-70%. Cyclical oral etidronate and daily oral alendronate both proved to reduce the vertebral fracture rate for postmenopausal osteoporotic woman, while most investigated bisphosphonates can increase spinal bone mass in osteoporosis. Bisphosphonates can help lowering serum calcium and reverse skeletal complications in malignancy mediated bone diseases. Oral and intravenous administration of therapeutic doses is relatively safe. In general, gastrointestinal disturbances are described most often and the oldest, least potent, bisphosphonate etidronate can induce osteomalacia. The various characteristics of bisphosphonates: physicochemical, biological, therapeutic and toxicological, vary greatly depending on the structure of the individual bisphosphonate. Even small changes in the structure can lead to enormous differences in potency. Overall, this class of drugs offers several prospects for the future.

Bioanalysis of m-iodobenzylguanidine in plasma by high-performance liquid chromatography after derivatization with benzoin.

A sensitive chromatographic assay has been developed for m-iodobenzylguanidine (MIBG) in human plasma based on the derivatization with benzoin. MIBG is first isolated from plasma using solid-phase extraction on a cyanopropyl-modified silica phase. After evaporation of the eluate, a fluorescent derivative is formed using benzoin. The derivative is analysed by reversed-phase liquid chromatography using a mixture 60% (v/v) acetonitrile, 30% (v/v) water and 10% (v/v) of the 0.5 M Tris buffer (pH 8.0) as the eluent and fluorescence detection at 320 nm for excitation and 435 nm for emission, respectively. In the evaluated concentration range (2-200 ng/ml) precisions < or = 10% and accuracies in between 90 and 100% have been found, with 2 ng/ml being the lower limit of quantification using a 0.5-ml plasma sample volume. The assay can also be used without the internal standard benzylguanidine. The assay was successfully used to obtain a pharmacokinetic curve of MIBG.

Sensitive liquid chromatographic assay for amprenavir, a human immunodeficiency virus protease inhibitor, in human plasma, cerebrospinal fluid and semen.

A sensitive bio-analytical assay for amprenavir, a human immunodeficiency virus protease inhibitor, based on reversed-phase liquid chromatography and fluorescence detection, is reported. The analyte is extracted from the matrix, plasma, cerebrospinal fluid (CSF) or semen, with chloroform using propyl-p-hydroxybenzoate as an internal standard. After centrifugation, evaporation of the organic phase and reconstitution in the eluent, the sample is injected into the chromatograph. The analyte is detected spectrofluorometrically at 270 and 340 nm for excitation and emission, respectively. The method has been validated in the 1-1000 ng/ml range for a 50-microl volume of plasma and in the 0.5-50 ng/ml range for a 100-microl volume of CSF and semen. The lower limit of quantification was 0.5 ng/ml in CSF and 1 ng/ml in both plasma and semen. Precision and accuracy both meet the current requirements for a bio-analytical assay and are <15% in the validated ranges. The assay was successfully used to obtain a concentration-time curve of amprenavir in plasma.

Liquid chromatographic assay for simultaneous determination of abacavir and mycophenolic acid in human plasma using dual spectrophotometric detection.

A combined bio-analytical assay for abacavir, a reversed transcriptase inhibitor, and mycophenolic acid (MPA), based on reversed-phase liquid chromatography and both ultraviolet (UV) absorption and fluorescence detection, is reported. Both analytes are extracted from plasma with acetonitrile. After centrifugation, evaporation of the supernatant and reconstitution in water, the sample is injected into the chromatograph. Abacavir is detected using UV detection at 285 nm and MPA spectrofluorometrically at 345 and 430 nm for excitation and emission, respectively. The method has been validated in the 80-2000 ng/ml range for abacavir and in the 10-10,000 ng/ml range for MPA for 200-microl plasma samples. The lower limits of quantification are 80 and 10 ng/ml for abacavir and MPA, respectively. Precisions and accuracies are < or = 8% in the valid concentration ranges of both analytes.

Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate.

The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid-liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5-8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.

Determination of pamidronate in urine by ion-pair liquid chromatography after derivatization with 1-naphthylisothiocyanate.

A sensitive method for the determination of pamidronate disodium [(3-amino-1-hydroxypropylidene)bisphosphonate, APD] in urine has been developed and validated. The procedure involves a triple co-precipitation with calcium phosphate, solid-phase extraction on a quaternary ammonium column, derivatization with 1-naphthylisothiocyanate and ion-pair liquid-liquid extraction. From the two reaction products, naphthylthiocarbamyl-APD is converted into the other, naphthylcarbamyl-APD, by an oxidative desulphuration with hydrogen-peroxide prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The method has a coefficient of variation of 7% for the intra-assay precision of 99 ng ml-1 APD and 11% for the inter-assay precision. The lower limit of quantification is 3 ng ml-1 APD in 2.5 ml of human urine.

Semi-automatic liquid chromatographic analysis of pamidronate in urine after derivatization with 1-naphthylisothiocyanate.

An existing sensitive chromatographic assay for pamidronate in urine has considerably been automated. Using the same sample processor, the solid-phase extraction (SPE) was automated separately from the derivatization with 1-naphthylisothiocyanate, the two-fold ion-pair liquid-liquid-extraction and the treatment with hydrogen peroxide for the 2-20 ng/ml concentration range. The automatic procedure was preceded by a triple calcium precipitation and interrupted by evaporation of the SPE eluate under nitrogen. For the 0.5-5 microg/ml concentration range one automatic sequence was used by avoiding evaporation during the sample treatment. In addition to the labour-saving of the semi-automatic procedure, the daily sample-throughput was improved compared to the existing manual assay. Further, the validation showed marginal improvements in the precision, accuracy and lower limit of quantification.

Semi-automatic liquid chromatographic analysis of pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate.

The semi-automatic method for the determination of the bisphosphonate pamidronate in serum and citrate plasma involves a manual protein precipitation with trichloroacetic acid and a manual coprecipitation of the bisphosphonate with calcium phosphate, followed by an automated solid-phase extraction on anion-exchange columns. After off-line evaporation of the extract under nitrogen and reconstitution in water, the automatic procedure is continued by automatic derivatization with 1-naphthylisothiocyanate, ion-pair liquid-liquid extraction and a treatment with hydrogen peroxide, prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The intra- and inter-day precisions are 1.3 and 7%, respectively, for a standard of 100 ng ml(-1) pamidronate in serum; the average accuracy for this standard is 107%. The lower limit of quantification is 20 ng ml(-1) pamidronate in 1 ml of human serum.