An LC/ESI-MS/MS method to quantify the PI3K inhibitor GDC-0084 in human plasma and cerebrospinal fluid: Validation and clinical application.

A liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated to measure GDC-0084 in human plasma and cerebrospinal fluid (CSF). Reverse-phase chromatography with gradient elution was performed using a C18 column (50 × 2.0 mm, 3 µm). Solid-phase extraction of plasma and CSF was employed to give excellent recovery. MS detection was performed with positive ion screening in multiple reaction monitoring mode. The precursor to the product ions (Q1 → Q3) selected for GDC-0084 and GDC-0084-d6 were 383.2 → 353.2 and 389.2 → 353.2, respectively. A separate calibration curve was established for human plasma and CSF. Both calibration curves, ranging from 0.2 to 200 ng/mL, were linear and had acceptable intra- and inter-day precision and accuracy. The lower limit of quantitation and limit of detection for GDC-0084 in human plasma were 0.2 ng/mL (signal/noise ≥47) and 0.005 ng/mL (signal/noise ≥3.5), respectively, and for GDC-0084 in human CSF were 0.2 ng/mL (signal/noise ≥19.7) and 0.04 ng/mL (signal/noise ≥7.2). This method was successfully applied to analyze serial plasma samples obtained from children with diffuse intrinsic pontine gliomas and other midline gliomas who participated in pharmacokinetic studies as part of a phase I clinical trial of GDC-0084.

Comprehensive quantification of purine and pyrimidine metabolism in Alzheimer's disease postmortem cerebrospinal fluid by LC-MS/MS with metal-free column.

The metabolome presence of nucleobases, nucleosides, nucleotides and related phosphorylated metabolites has been examined for Alzheimer's disease (AD). Although reversed-phase liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used for the determination of these analytes, they were limited in chromatographic signal intensity and reproducibility owing to significant peak tailing caused by complexing with metallic cations and phosphate groups. In this work, we applied LC-MS/MS analysis with a metal-free column for comprehensive quantification of 40 analytes regarding to purine and pyrimidine metabolism in postmortem cerebrospinal fluid (pCSF) from AD patients. For the analytical column, an InertSustain AQ-C18 metal-free PEEK column was used. MS detection was by electrospray positive ionization. The metal-free column allowed for sharp peak detection of highly polar metabolites within a running time of 17 min. In validation, the limits of detection (LOD), the limit of quantitation (LOQ) and recovery value using a pooled pCSF sample are 1-500 nM, 0.5-250 nM and a range of 53.1-144.0% (RSD ranged from 0.4 to 19.6%). The developed LC-MS/MS method utilizing a metal-free column provides an accurate quantification of some metabolites regarding purine and pyrimidine metabolism in pCSF samples obtained from AD patients.

Pharmacokinetics and pharmacodynamics of a single dose Nilotinib in individuals with Parkinson's disease.

Nilotinib is a broad-based tyrosine kinase inhibitor with the highest affinity to inhibit Abelson (c-Abl) and discoidin domain receptors (DDR1/2). Preclinical evidence indicates that Nilotinib reduces the level of brain alpha-synuclein and attenuates inflammation in models of Parkinson's disease (PD). We previously showed that Nilotinib penetrates the blood-brain barrier (BBB) and potentially improves clinical outcomes in individuals with PD and dementia with Lewy bodies (DLB). We performed a physiologically based population pharmacokinetic/pharmacodynamic (popPK/PD) study to determine the effects of Nilotinib in a cohort of 75 PD participants. Participants were randomized (1:1:1:1:1) into five groups (n = 15) and received open-label random single dose (RSD) 150:200:300:400 mg Nilotinib vs placebo. Plasma and cerebrospinal fluid (CSF) were collected at 1, 2, 3, and 4 hours after Nilotinib administration. The results show that Nilotinib enters the brain in a dose-independent manner and 200 mg Nilotinib increases the level of 3,4-Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), suggesting alteration to dopamine metabolism. Nilotinib significantly reduces plasma total alpha-synuclein and appears to reduce CSF oligomeric: total alpha-synuclein ratio. Furthermore, Nilotinib significantly increases the CSF level of triggering receptors on myeloid cells (TREM)-2, suggesting an anti-inflammatory effect. Taken together, 200 mg Nilotinib appears to be an optimal single dose that concurrently reduces inflammation and engages surrogate disease biomarkers, including dopamine metabolism and alpha-synuclein.

Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs.

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

Development and validation of an UHPLC-MS/MS method for simultaneous quantification of ibrutinib and its dihydrodiol-metabolite in human cerebrospinal fluid.

Ibrutinib is an orally administered first-in-class irreversible Bruton's tyrosine kinase (BTK) covalent inhibitor for the treatment of patients with B-cell malignancies. Several isolated clinical observations reported its efficacy in central nervous system dissemination. Herein, we described the development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure for the quantification of ibrutinib and its active metabolite PCI-45227 in cerebrospinal fluid (CSF). This is the first complete validated method for quantification of ibrutinib and PCI-45227 in CSF. The compounds were eluted on a Waters BEH C18 column (50.0 × 2.1 mm; 1.7 µm) using a gradient elution with a mobile phase composed of ammonium formate buffer 5 mM pH 3.2 and acetonitrile +0.1% formic acid with a flow rate of 400 µL·min-1. Two deuterated internal standards were used to obtain the most accurate quantification. The CSF samples were prepared by a simple and rapid dilution. The method was validated by testing the selectivity, response function, intra-day and inter-day precisions, trueness, limits of detection (LOD) and lower limits of quantification (LLOQ). The validation results proved that the methods were suitable to quantify ibrutinib and PCI-45227 in real biological CSF samples from 0.50 (ibrutinib) or 1.00 (PCI-45227) to 30.00 ng·mL-1. Furthermore, the developed method was adapted to allow the quantification of both compounds in plasma and the results were compared to those reported in literature. The plasmatic samples were treated by protein precipitation and the method was validated to quantify ibrutinib and PCI-45227 in real biological plasmatic samples from 5.00 to 491 ng·mL-1. Lastly, for both matrices, accuracy profiles were plotted from the trueness and precision results using a 20% α-risk (ß = 80%) and the tolerance intervals were comprised within the acceptance limits fixed at ±25% for the LLOQ and ±15% for the other concentrations. Finally, these methods were successfully applied to quantify ibrutinib and PCI-45227 in real human CSF and plasma samples.

Selective and interactive effects of D2 receptor antagonism and positive allosteric mGluR4 modulation on waiting impulsivity.

BACKGROUND: Metabotropic glutamate receptor 4 (mGluR4) and dopamine D2 receptors are specifically expressed within the indirect pathway neurons of the striato-pallidal-subthalamic pathway. This unique expression profile suggests that mGluR4 and D2 receptors may play a cooperative role in the regulation and inhibitory control of behaviour. We investigated this possibility by testing the effects of a functionally-characterised positive allosteric mGluR4 modulator, 4-((E)-styryl)-pyrimidin-2-ylamine (Cpd11), both alone and in combination with the D2 receptor antagonist eticlopride, on two distinct forms of impulsivity. METHODS: Rats were trained on the five-choice serial reaction time task (5-CSRTT) of sustained visual attention and segregated according to low, mid, and high levels of motor impulsivity (LI, MI and HI, respectively), with unscreened rats used as an additional control group. A separate group of rats was trained on a delay discounting task (DDT) to assess choice impulsivity. RESULTS: Systemic administration of Cpd11 dose-dependently increased motor impulsivity and impaired attentional accuracy on the 5-CSRTT in all groups tested. Eticlopride selectively attenuated the increase in impulsivity induced by Cpd11, but not the accompanying attentional impairment, at doses that had no significant effect on behavioural performance when administered alone. Cpd11 also decreased choice impulsivity on the DDT (i.e. increased preference for the large, delayed reward) and decreased locomotor activity. CONCLUSIONS: These findings demonstrate that mGluR4s, in conjunction with D2 receptors, affect motor- and choice-based measures of impulsivity, and therefore may be novel targets to modulate impulsive behaviour associated with a number of neuropsychiatric syndromes.

A reference laboratory experience of clinically achievable voriconazole, posaconazole, and itraconazole concentrations within the bloodstream and cerebral spinal fluid.

Interest in antifungal therapeutic-drug monitoring has increased due to studies demonstrating associations between concentrations and outcomes. We reviewed the antifungal drug concentration database at our institution to gain a better understanding of achievable triazole drug levels. Antifungal concentrations were measured by high-performance liquid chromatography (HPLC), ultraperformance liquid chromatography and single-quadrupole mass spectrometry (UPLC/MS), or a bioassay. For this study, only confirmed human bloodstream (serum or plasma) and cerebral spinal fluid (CSF) concentrations of voriconazole, posaconazole, and itraconazole were analyzed. The largest numbers of bloodstream and CSF samples were found for voriconazole (14,370 and 173, respectively). Voriconazole bloodstream concentrations within the range of 1 to 5.5 µg/ml represented 50.6% of samples. Levels below the lower limit of quantification (0.2 µg/ml) were observed in 14.6% of samples, and 10.4% of samples had levels of ≥5.5 µg/ml. CSF voriconazole levels ranged from undetectable to 15.3 µg/ml and were <0.2 µg/ml in 11% of samples. Posaconazole bloodstream concentrations were ≥0.7 and ≥1.25 µg/ml in 41.6% and 18.9% of samples, respectively. Posaconazole was detected in only 4 of 22 CSF samples (undetectable to 0.56 µg/ml). Itraconazole levels, as measured by UPLC/MS, were ≥0.5 µg/ml in 43.3% and were undetectable in 33.9% of bloodstream samples. In contrast, when measured by a bioassay, itraconazole/hydroxyitraconazole bloodstream concentrations were ≥1.0 µg/ml in 72.9% of samples and were undetectable in 18% of samples. These results indicate that there is marked variability in bloodstream concentrations achieved with these three azoles. In addition, many levels within the bloodstream for each azole and for voriconazole and posaconazole in the CSF were undetectable or below thresholds associated with efficacy.

Measurement of antifungal drug levels in cerebrospinal fluid for cryptococcal meningoencephalitis.

We report a rare case of cryptococcal meningoencephalitis in which antifungal therapy was monitored by measuring the cerebrospinal fluid (CSF) levels of the antifungal drugs. A 78-year-old man with diabetes mellitus being treated with oral agents. He had no history of human immunodeficiency virus infection. The patient showed abnormal behavior and fever (>38°C) on November 20, 2009, and was admitted for disturbance of consciousness on November 24. CSF examination showed an increased cell count, and a yeast-like fungus, suggesting cryptococcal meningoencephalitis, was observed by India ink staining. Initial treatment was liposomal amphotericin B (L-AMB) plus flucytosine. Cryptococcus neoformans was isolated by CSF culture on day 2. MIC was 0.25 µg/ml for amphotericin B (AMPH-B), 4 µg/ml for flucytosine, 4 µg/ml for fluconazole (FLCZ), and 0.03 µg/ml for voriconazole (VRCZ). Despite treatment, his disturbance of consciousness persisted. The CSF level of AMPH-B was ≤0.05 µg/ml on day 8. Therefore, L-AMB was switched to fosfluconazole. The CSF level of FLCZ was sufficient (22.6 µg/ml) on day 25, but there was a decrease in glucose and the fungus could still be detected in CSF smears. Consequently, FLCZ was switched to VRCZ. On day 47, CSF level of VRCZ was 1.97 µg/ml, exceeding its MIC, so treatment was continued. On day 77, the patient was generally lucid, and CSF smears did not detect any fungi. The patient was then transferred for rehabilitation. On day 84, voriconazole was discontinued, with no evidence of fungal recurrence.

Plasma and cerebrospinal fluid pharmacokinetics of MP470 in non-human primates.

PURPOSE: MP470 is a multi-targeted tyrosine kinase inhibitor with potent activity against mutant c-Kit, PDGFRα, Flt3, c-Met and c-Ret that is being evaluated as an anticancer agent. The plasma and cerebrospinal fluid (CSF) pharmacokinetics of MP470 were studied in a non-human primate model that is highly predictive of CSF penetration in humans. METHODS: Oral MP470, 300 mg, was administered to four non-human primates. Serial samples of blood were collected from four animals and CSF samples from three animals for pharmacokinetic studies. Plasma and CSF concentrations were measured using an LC-MS/MS assay. Both model-independent and model-dependent methods were used to analyze the pharmacokinetic data. RESULTS: Following a one-time oral dose of 300 mg, the MP470 plasma area under the curve (AUC) was 1,690 ± 821 nM h (mean ± SD). The half-life of MP470 in the plasma was 11.0 ± 3.4 h. There was no measurable MP470 in the CSF. CONCLUSIONS: Although CSF penetration is minimal, MP470 has demonstrated potent activity against cancer cell lines in vitro and in vivo, and further clinical investigation is warranted.

Cerebral aspergillosis: tissue penetration is the key.

Cerebral aspergillosis is increasingly recognized in severely immunocompromised patients and, until recently, this type of fungal infection was associated with a mortality approaching 100%. The central nervous system is a protected environment and penetration of drugs across the blood-brain barrier is mainly limited by their molecular size and physicochemical properties, as well as drug interaction with transporter systems (e.g., P-glycoprotein) at the blood-brain barrier. Most antifungal agents are large molecules (>700 Da), which makes sufficient penetration into the central nervous system unlikely. In fact, the available data indicate low levels of most antifungal agents in cerebrospinal fluid and brain tissue, except for fluconazole and voriconazole. Concentrations of voriconazole exceeding inhibitory concentrations for Aspergillus species were found repeatedly in cerebrospinal fluid and brain tissue, including brain abscess material. A recent retrospective study confirmed that voriconazole treatment resulted in improved response and survival rates in patients with cerebral aspergillosis. Data from animal models, which explored escalated doses or combinations of antifungal agents in experimental neuroaspergillosis, suggest that selected combination or dose-escalated therapies might further improve the still unsatisfactory prognosis in this particular type of Aspergillus infection.

Inhibition of voriconazole metabolism by chloramphenicol in an adolescent with central nervous system aspergillosis.

For an adolescent with bacterial meningitis and subsequent cerebral aspergillosis, intravenous voriconazole dose requirements substantially decreased during coadministration with intravenous chloramphenicol and considerably rose after discontinuation of the antibiotic. In agreement with in vitro evidence, these data suggest that chloramphenicol is a rather significant inhibitor of hepatic CYP3A4 and/or CYP2C19.

Successful treatment with voriconazole of Aspergillus brain abscess in a boy with medulloblastoma.

Invasive aspergillosis is an increasing problem in immuno-incompetent patients after prolonged steroid therapy, cancer radio-chemotherapy, and bone marrow or solid organ transplantation. Cerebral aspergillosis is a well-described complication of the invasive aspergillosis but only in rare cases, the brain is the sole site of infection. Despite increasing availability of antifungal drugs, the prognosis of cerebral aspergillosis is poor. We report on an 11-year-old boy with medulloblastoma in the area of the fourth ventricle. Following tumor surgery and radio-chemotherapy, several abscess-like structures occurred in the operating field. After incomplete abscess, resection histology and culture confirmed a localized Aspergillus fumigatus infection. The initial treatment of the Aspergillus fumigatus infection with conventional amphotericin B failed, and treatment with the triazole voriconazole was started. Intravenous treatment with voriconazole resulted in a reduction of the Aspergillus fumigatus abscess. After switching to oral ambulatory therapy, the Aspergillus fumigatus abscess increased in size. To improve treatment, voriconazole dosage was adapted to reach drug concentrations in cerebrospinal fluid (CSF) above the minimal fungicidal concentration and plasma specimens. During the concentration-controlled voriconazole therapy for a period of 18 months, a complete response was achieved.

Purine and pyrimidine metabolism and electrocortical brain activity during hypotension in near-term lambs.

BACKGROUND: Insufficient cerebral O2 supply leads to cellular energy failure and loss of brain cell function. The relationship between the severity of cellular energy failure due to hemorrhagic hypotension and the loss of electrocortical brain activity (ECBA), as a measure of brain cell function, is not yet fully elucidated in near-term born lambs. OBJECTIVES: To study the relationship between cerebral purine and pyrimidine metabolism, as a measure of brain cell energy failure, and brain cell function after hemorrhagic hypotension in near-term born lambs. METHODS: Eight near-term lambs (term 147 days) were delivered at 131 days of gestation. After a stabilization period, mean arterial blood pressure was reduced till 30% of baseline by withdrawal of blood. Cerebrospinal fluid (CSF) was obtained at the end of the hypotensive period (2.5 h). CSF from 8 siblings was used for comparison. HPLC was used to determine purine and pyrimidine metabolites in CSF, as a measure of cellular energy failure. ECBA was calculated as the root mean square value of a band-filtered (2-16 Hz) one-channel EEG. RESULTS: Values of guanosine, inosine, hypoxanthine, xanthine and uridine were significantly higher, while ECBA was significantly lower after hemorrhagic hypotension than control values. The concentrations of inosine, hypoxanthine, xanthine and uridine were significantly negatively linearly related to ECBA. CONCLUSIONS: Brain cell function is negatively related to concentrations of inosine, hypoxanthine, xanthine and uridine in the CSF after hemorrhagic hypotension in near-term born lambs.

Application of solid phase extraction and high-performance liquid chromatography to qualitative and quantitative analysis of nucleotides and nucleosides in human cerebrospinal fluid.

New method of qualitative and quantitative analysis of nucleotides in human cerebrospinal fluid (CSF), based on the combination of extraction of purines and pyrimidines to the solid phase (SPE) and high-performance liquid chromatography (HPLC), was proposed. Use of SPE and lyophilization of samples allowed for the first time to detect the presence of di- and triphosphonucleotides in human CSF. Concentration of those compounds varied from 0.003 to 5.0 microM. Differences in the nucleotide mixture composition in human CSF detected with the new method are coupled with the neurological disorders and might be a basis for an efficient diagnostic tool.

Behavioral, adrenal, and sympathetic responses to long-term administration of an oral corticotropin-releasing hormone receptor antagonist in a primate stress paradigm.

CRH is a main regulator of the stress response. This neuropeptide and its specific receptors, CRHR-1 and CRHR-2, are disseminated throughout the central nervous system. There is a significant interspecies difference in the distribution of CRHR within the central nervous system. CRH-R1 antagonists may attenuate stress-related behavior in rats without compromising adrenal function, but few studies have addressed the same question in higher mammals. Antalarmin (AA) is a specific CRHR-1 antagonist suitable for oral administration. Social separation is a potent stressor for rhesus monkeys. Therefore, we sought to investigate the hormonal responses to chronic administration of AA using a primate stress model. Eight preadolescent (4-6 kg) male rhesus monkeys received AA (20 mg/kg.d) or placebo (PBO) orally. All animals were on a regular day/light cycle and were fed with standard monkey chow daily. The study (114 d) was comprised of the following consecutive phases: adaptation, baseline, separation (stress), recovery, and cross-over. During social separation, solid panels separated the individuals. Cerebrospinal fluid (CSF) and femoral venous blood samples were obtained once a week on the fourth day of separation under ketamine anesthesia. Serum samples were also obtained 1 and 2 h after separation. CSF samples were assayed for CRH, AA, norepinephrine (NE) and epinephrine (EPI). Plasma was assayed for ACTH, cortisol, NE, and EPI. AA was detected in the plasma of each monkey while they were taking the active drug and in none of the animals on PBO. Among the behaviors assessed, environmental exploration, a behavior inhibited by stress, was increased during AA administration. However, AA at this dose did not affect other anxiety-related behavioral end points, including self-directed behavior, vocalization, or locomotion. We also observed that: 1) ACTH decreased between adaptation and baseline, indicating that the animals had adjusted to the novel environment; 2) ACTH and cortisol increased significantly after social separation, indicating that social separation was an adequate model for acute stress; 3) NE and EPI increased significantly during acute stress in the AA and PBO groups (P < 0.005, NE; P < 0.001, EPI); 4) after chronic stress, by d 4 of separation, ACTH levels were no longer significantly different from baseline, and NE and EPI remained slightly elevated when compared with baseline (P < 0.05, NE; P < 0.01, EPI); and 5) all the animals remained healthy and gained the expected weight during the study. In summary, oral chronic administration of a specific CRH-R1 antagonist to rhesus monkeys does not blunt the sympathoadrenal response to stress while increasing environmental exploration, a behavior that is normally suppressed during stressful events. Taken together, these findings suggest that CRHR-1 antagonists may be a valid treatment for stress-related disorders.

Central nervous system failure in patients with chronic myelogenous leukemia lymphoid blast crisis and Philadelphia chromosome positive acute lymphoblastic leukemia treated with imatinib (STI-571).

Isolated central nervous system (CNS) relapse occurred in 5 out of 24 patients (20.8%) with chronic myeloid leukemia (CML) lymphoid blast crisis (2), Philadelphia (Ph) chromosome positive acute lymphoblastic leukemia (ALL) (2) or CML with biphenotypic markers (1) treated on imatinib mesylate (IM) protocols at our institution. CNS relapse occurred despite peripheral blood (5) and bone marrow (3) complete responses. Median time to CNS relapse was day 32 (range 23 to 100). This observation raised the possibility that IM may not penetrate into the CNS. Simultaneous plasma and cerebral spinal fluid (CSF) IM levels were determined in four subsequent patients by liquid chromatography and mass spectrophotometric assay. Levels of IM were found to be approximately two logs lower in CSF than in plasma (0.044 microg/ml (0.088 +/- 0.029 micrro) vs 3.27 microg/ml (6.54 +/- 0.93 microM)). CSF levels were substantially below the concentration required for inhibition of BCR-ABL and killing of cell lines in vitro. These results suggest that IM may not penetrate the intact blood/brain barrier and its implications are discussed.

Plasma and cerebrospinal fluid pharmacokinetics of imatinib after administration to nonhuman primates.

PURPOSE: Imatinib mesylate (Gleevec, Glivec, STI571, imatinib) is a potent tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. The role of imatinib in the treatment of malignant gliomas and other solid tumors is being evaluated. We used a nonhuman primate model that is highly predictive of the cerebrospinal fluid penetration of drugs in humans to study the pharmacokinetics of imatinib in plasma and cerebrospinal fluid (CSF) after i.v. and p.o. administration. EXPERIMENTAL DESIGN: Imatinib, 15 mg/kg i.v. over 30 min (n = 3) or 30 mg/kg p.o. (n = 3), was administered to nonhuman primates. Imatinib was measured in serial samples of plasma and CSF using high-pressure liquid chromatography with UV absorbance or mass spectroscopic detection. Pharmacokinetic parameters were estimated using model-independent methods. RESULTS: Peak plasma imatinib concentrations ranged from 6.4 to 9.5 microM after i.v. dosing and 0.8 to 2.8 microM after p.o. dosing. The mean +/-SD area under the plasma concentration versus time curve was 2480 +/-1340 microM.min and 1191 +/-146 microM.min after i.v. and p.o. dosing, respectively. The terminal half-life was 529 +/-167 min after i.v. dosing and 266 +/-88 min after p.o. dosing. After i.v. dosing the steady state volume of distribution was 5.9 +/-2.8 liter/kg, and the total body clearance was 12 +/-5 ml/min/kg. The mean peak CSF concentration was 0.25 +/-0.07 microM after i.v. dosing and 0.07 +/-0.04 microM after p.o. dosing. The mean CSF:plasma area under the plasma concentration versus time curve ratio for all of the animals was 5% +/-2%. CONCLUSIONS: There is limited penetration of imatinib into the CSF of nonhuman primates after i.v. and p.o. administration.

CNS blast crisis of chronic myelogenous leukemia in a patient with a major cytogenetic response in bone marrow associated with low levels of imatinib mesylate and its N-desmethylated metabolite in cerebral spinal fluid.

Imatinib mesylate (STI571) is a very effective treatment option for Ph(+) chronic myeloid leukemia (CML) in chronic phase. Secondary treatment failures have mostly been observed in patients with advanced stages of disease. We report the case of a patient who unexpectedly experienced blast crisis of the central nervous system although having achieved complete cytogenetic remission in the bone marrow. The levels of STI571 and its metabolite N-desmethyl STI were 40-fold lower in the cerebral spine fluid than in plasma. The risk of CNS disease has to be kept in mind when patients with CML in chronic phase who are at an increased risk for blastic transformation are treated with imatinib mesylate.

Pharmacokinetics and cellular uptake of imatinib and its main metabolite CGP74588.

Despite the remarkable clinical response rates to imatinib in the treatment of bcr-abl leukemic patients, pharmacokinetic data on this relatively novel substance are needed to improve our understanding of the emergence of resistance, the interindividual variations of clinical response and the clinical and biologic relevance of its main metabolite N-desmethyl-imatinib. We present here pharmacokinetic data obtained with a newly designed HPLC approach in 97 patients with chronic myeloid leukemia or acute lymphatic leukemia (ALL) under treatment with imatinib that allowed us to calculate the AUC (39.5 microg.h/ml for an oral dose of 400 mg daily), the t(1/2) (18.2 h) and the peak concentration (1.92 micro/ml for an oral dose of 400 mg daily) of imatinib in plasma. In a subgroup of patients, the same parameters were analyzed for N-desmethyl-imatinib. We also provide data on the imatinib concentration in the cerebrospinal fluid (CSF) of ALL patients and demonstrate that oral administration of imatinib resulted only in a marginal flux across the blood-brain barrier. Finally, in an in vitro setting, we determined cellular concentrations of imatinib in HL-60 cells and showed an over-proportional uptake both in RPMI medium and in human plasma. Using an arithmetical approach combining all parameters obtained in imatinib-treated patients, we finally provide a conclusive approximation of basic pharmacokinetic data for both imatinib and its main metabolite N-desmethyl-imatinib.

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations.

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.