A phase I dose-finding clinical pharmacokinetic study of an oral formulation of irinotecan (CPT-11) administered for 5 days every 3 weeks in patients with advanced solid tumours.

BACKGROUND: Oral administration of irinotecan (CPT-11) should allow sustained exposure to the drug without the inconvenience of intravenous delivery and with fewer side-effects. PATIENTS AND METHODS: The present phase I trial of CPT-11, administered orally as a powder-filled capsule for 5 consecutive days every 3 weeks at doses ranging from 30 to 90 mg/m(2)/day, was conducted in 47 patients for whom a satisfactory standard treatment option was no longer available (24 males/23 females; median age 51 years, range 26-85). Tumour types included melanoma (11), colorectal (4), urinary tract (3), lung/pleura (4), thyroid (3), liver (3), gallbladder (2), cervix/uterus (3), breast (2), pancreas (2), carcinoma and other cancer types (10). RESULTS: A total of 171 cycles were administered (median 3, range 1-11). Dose limiting toxicities (DLTs) occurred during the first cycle in five of 31 patients in the dose-escalation part of the study: one patient at the 50 mg/m(2)/day dose level (diarrhoea grade 4); one patient at the 80 mg/m(2)/day dose level (prolonged neutropenia grade 4 and diarrhoea grade 3); and three patients at the 90 mg/m(2)/day dose level (diarrhoea, vomiting and neutropenia). The 80 mg/m(2)/day dose level was expanded, as a feasibility study, to include 16 additional patients, five of whom had received extensive prior pelvic irradiation. A further three patients in this cohort experienced DLTs, two of whom had received extensive prior pelvic irradiation. One patient died on study day 15 during the first cycle of oral CPT-11 following grade 3 diarrhoea, febrile neutropenia and a necrotic enterocolitis. Overall the grade 3/4 toxicities in 47 patients were asthenia (19%), anorexia (17%), neutropenia (14.9 %), diarrhoea (13%), nausea (12.7%), vomiting (8.5%) and thrombocytopenia (8.5%). Partial responses were observed in two melanoma patients and disease stabilisation was noted in 17 (36.1%) patients. Pharmacokinetic parameters were recorded for 46 patients. CONCLUSIONS: At the maximum tolerated dose, defined as 80 mg/m(2)/day for 5 days every 3 weeks, oral CPT-11 was shown to be well tolerated and safe with few of the haematological toxicities associated with the intravenous formulation.

Development of a rapid and sensitive method for the quantitation of benzodiazepines in Calliphora vicina larvae and puparia by LC-MS-MS.

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is emerging as the tool of choice for rapid analysis and the detection of biologically active compounds in complex mixtures. We describe the development of a sensitive method for the simultaneous quantitation of 10 benzodiazepines in Calliphora vicina (Diptera: Calliphoridae) larvae and puparia. The use of larvae for toxicological analyses offers some technical advantages over putrefied tissue. Four sample pretreatment methods for isolating the benzodiazepines out of larvae were evaluated. A simple homogenization, followed by acetonitrile precipitation yielded the highest recoveries. Puparia were pulverized and extracted by ultrasonification in methanol. All extracts were subsequently analyzed using reversed-phase LC-MS-MS. Larvae and puparia calibrators containing benzodiazepines at concentrations ranging from 25 to 750 pg/mg and 50 to 500 pg/mg, respectively, were prepared and analyzed. The method was demonstrated to be linear over the ranges investigated. Limits of detection were from 1.88 to 5.13 pg/mg larva and from 6.28 to 19.03 pg/mg puparium. The developed method was applied to the determination of nordiazepam and its metabolite oxazepam in larvae and puparia of the Calliphora vicina fly that had been reared on artificial foodstuff (beef heart) spiked with 1 microg/g nordiazepam. The larvae were harvested at day 5 for analysis of drug content. The method was sufficiently sensitive to allow the detection of nordiazepam and oxazepam in a single larva or puparium.

Effects of methylphenidate, desipramine, and L-dopa on attention and inhibition in children with Attention Deficit Hyperactivity Disorder.

The objective of this study was to investigate the effects of methylphenidate (MPH) on attention and inhibition in children with Attention Deficit Hyperactivity Disorder (ADHD) and to establish what the relative contributions of the noradrenergic and dopaminergic systems to this effect were. In addition to MPH, two other drugs were administered in order to affect both transmitter systems more selectively, L-dopa (dopamine (DA) agonist) and desipramine (DMI) (noradrenaline (NA) re-uptake inhibitor). Sixteen children with ADHD performed a stop-task, a laboratory task that measures the ability to inhibit an ongoing action, in a double-blind randomized within-subjects design. Each child received an acute clinical dose of MPH, DMI, L-dopa, and placebo; measures of performance and plasma were determined. The results indicated that inhibition performance was improved under DMI but not under MPH or L-dopa. The response-time to the stop-signal was marginally shortened after intake of DMI. MPH decreased omission and choice-errors and caused faster reaction times to the trials without the stop-tone. No effects of L-dopa whatsoever were noted. Prolactin levels were increased and 5-HIAA levels were lowered under DMI relative to placebo. It is suggested that the effects of MPH on attention are due to a combination of noradrenergic and dopaminergic mechanisms. The improved inhibition under DMI could be serotonergically mediated.

Development of a rapid and sensitive method for the quantitation of amphetamines in human plasma and oral fluid by LC-MS-MS.

Target analysis of amphetamines in biological samples is of great importance for clinical and forensic toxicologists alike. At present, most laboratories analyze such samples by gas chromatography-mass spectrometry. However, this procedure is labor-intensive and time-consuming, particularly as a preliminary extraction and derivatization are usually unavoidable. Here we describe the development of an alternative method. Amphetamines were isolated from human plasma and oral fluid using a simple methanol precipitation step and subsequently analyzed using reversed-phase liquid chromatography-tandem mass spectrometry. Quantitation of the drugs was performed using multiple reaction monitoring. The developed method, which requires only 50 microL of biological sample, has a total analysis time of less than 20 min (including sample preparation) and enables the simultaneous quantitation of 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, amphetamine, methamphetamine, and ephedrine in a single chromatographic run. Limits of detection of 2 microg/L or better were obtained. The method has been validated and subsequently applied to the analysis of plasma and oral fluid samples collected from current drug users.

Seizure of illicitly produced para-fluorofentanyl: quantitative analysis of the content of capsules and tablets.

A gas chromatography/mass spectrometry (GC/MS) method for the quantification of para-fluorofentanyl (pFF) in powder and powdered samples was developed and validated. The method was applied on a seizure of capsules and tablets, that had been confiscated at an illicit production site in the Netherlands. The investigated capsules and tablets contained pFF in the range of 33.8-408.7 microg. As caffeine was detected as being an adulterant, a HPLC/UV method for the quantification of caffeine in capsules and tablets was also validated and applied. Caffeine was detected in the range of 25.6-108 mg per capsule or tablet. Based on an extrapolation of pharmacological and toxicological data of fentanyl, it can be argued that the highest detected single dose of pFF could be lethal, when administered orally. However, the large variability of the doses observed for pFF could mislead abusers, potentially leading to multiple doses and thus overdosing.

Hyphenated techniques in anticancer drug monitoring. I. Capillary gas chromatography-mass spectrometry.

Most anticancer agents are relatively unstable substances and are subjected to intensive metabolism in vivo and radiation during sample pretreatment. Hyphenated techniques including a separation technique and, most frequently, mass spectrometry are therefore chosen to obtain insight into the in vivo behavior of anticancer agents. Once established, simpler assays can be derived from those based on hyphenation, which are less expensive. Capillary gas chromatography (cGC)-mass spectrometry (MS) is amongst the most frequently applied hyphenated analytical technologies in anticancer drug monitoring. Here a selection has been made of: (i) cGC-MS applied to the analysis of agents frequently used in clinical oncology (e.g. tamoxifen, oxazaphosphorines); (ii) cGC-MS applied to the development of new agents (Swainsonine and Brefeldin); (iii) cGC-MS applied to the analysis of agents for which comparisons with other frequently applied hyphenation technologies are possible (see Part I of this series). cGC-MS played a key role in the elucidation of the in vivo behavior of the oxazaphosphorine cyclophosphamide, historically the most frequently applied anticancer agent. cGC-MS appeared to be of special interest in the analysis of cyclophosphoramide and congeners in human erythrocytes by coupling of the hyphenated technique with a measurement of sediment technique. This resulted in the quantitative and qualitative analysis of oxaphosphorine-related mustard gas moieties in human erthrocytes for the first time.

Hyphenated techniques in anticancer drug monitoring. II. Liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry.

High-performance liquid chromatography has become the separation technique of choice for the monitoring of generally thermolabile anticancer agents. With the introduction of electrospray mass spectrometry, the coupling of liquid chromatogaphy and mass spectrometry has opened the way to widely and routinely applied anticancer drug monitoring. Real-time metabolism versus degradation can now be distinguished, since derivatization is no longer obligatory. This is important for the monitoring of the anabolic and catabolic pathways of the same agent, such as 5-fluorouracil. Detection limits almost equal to those obtained with capillary gas chromatography-mass spectrometry are realistic with the latest generation of mass spectrometers, enabling quantitative analysis of various anticancer agents and their metabolites down to the low ng/ml level. Furthermore, sample clean-up and chromatography can be downscaled markedly using the latest column technologies, such as the generally applied 10 cm x 2.8 mm I.D. RP 18 columns. The coupling of capillary electrophoresis to mass spectrometry is today far from a routine application in anticancer drug monitoring. Nevertheless, interesting applications have been reported and are selected for the present review.

Artifact formation due to ethyl thio-incorporation into silylated steroid structures as determined in doping analysis.

Trimethylsilylation of target substances in a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and ethanethiol is frequently applied for the application of gas chromatography-mass spectrometry (GC-MS) in steroid analysis. However, artifacts were formed when using this mixture to silylate the steroids androsterone and etiocholanolone obtained from a urine matrix. The artifacts were identified as ethyl thio-containing products of the respective trimethylsilyl derivatives. The conversion of the studied products increased slowly as a function of time, was dependent on the presence of the urine matrix and was significantly accelerated by adding diethyl disulfide to the reagent before incubation. Also ethyl thio-incorporation into testosterone and epitestosterone was established. A mechanism for ethyl thio-incorporation is proposed. The conversion achieved after 120-h sample storage at room temperature was insufficient to significantly influence the analysis of androsterone and etiocholanolone under the studied conditions. However, the results provide fundamental insight into the mechanism of silylation and the occurring side-reactions. Moreover, when investigating the formation of new metabolites, the ethyl thio-incorporation can lead to misinterpretation.

Hair as a biological specimen for therapeutic drug monitoring.

To study the potential of hair as a biological specimen in therapeutic drug monitoring (TDM), a review of the correlation between hair concentration, blood levels, dose and therapeutic effects of various drugs is presented. The results indicate there is a fair correlation between plasma and hair for several medicines. Moreover, hair samples have the advantage of providing information of more prolonged drug intake, thus allowing a better evaluation of patient compliance. Using segmental hair analysis, only a single sample is necessary. We conclude that hair has potential as a biological specimen in TDM--at least as far as compliance is concerned and possibly as a longer term record of drug concentrations--and that correlation between hair concentration, blood levels and clinical efficacy should be investigated for all drugs where TDM is indicated.

Piperazine-like compounds: a new group of designer drugs-of-abuse on the European market.

1-Aryl-piperazine compounds are, depending on their substituents, selective for certain serotonin receptors and together with their easy availability and their so-called legal status, this group of psychoactive compounds are potential designer drugs-of-abuse. Internet in that respect is an important source of information and distribution facilities. Because this development may have consequences for the interpretation of future clinical and forensic toxicological case studies, some analytical aspects of 1-benzyl-piperazine (BZP), 1-[4-methoxyphenyl]-piperazine (pMeOPP) and 1-[3-trifluoromethylphenyl]-piperazine (TFMPP) were studied. BZP was not detected by the AxSYM FPIA technology designed to determine amphetamine-like compounds, but had showed some cross reactivity with EMIT d.a.u.. The cross reactivities at 300 and 12,000ng/ml (RS)-amphetamine equivalents were 0.4 and 1.3%, respectively. Although BZP was not identified directly by the REMEDi HS Drug Profiling System, it can be detected by this HPLC/UV scanning system. Using GC/NPD without derivatisation, BZP, pMeOPP and TFMPP can be analysed for and applying GC/MS without or with acetylation or trifluoroacetylation, these compounds can be identified unambiguously. The usefulness of GC/NPD and GC/MS in this respect was demonstrated by the quantitative and qualitative analysis of the content of a capsule with the synthetic stimulant A2, which proved to contain 86.4mg of BZP.

Erythrocytes and the transfer of anticancer drugs and metabolites: a possible relationship with therapeutic outcome.

Blood functions as a mobile tissue in an exchange system, with the remaining body tissue as a stationary phase. The equilibrium among plasma water, plasma proteins, and blood cells is described by models, but little consideration has been given to the substance-binding capacity of erythrocytes. There are numerous reasons for this, including bioanalytical limitations (ie, it has been difficult to study erythrocytes in the laboratory in their natural state). Erythrocyte monitoring requires accurate blood sampling and quantitative isolation of erythrocytes without disturbing the equilibrium of substances of interest between erythrocytes and plasma or other blood constituents. This became possible with the advent of the measurement of sediment device. The mass of a given substance available in blood can be described by M(Blood) = M(Plasma) + M(ERY) (+ M(REM)). M(ERY) is the mass of a substance present in erythrocytes and it is shown that for several oxazaphosphorines, such as iphosphoramide mustard, that M(ERY) determines M(Blood) with great superiority over M(Plasma). The impact of erythrocyte monitoring on therapeutic outcome has to be defined, but is an important area of research.

Absence of 5-HT(1B) receptors is associated with impaired impulse control in male 5-HT(1B) knockout mice.

BACKGROUND: Serotonin (5-HT) plays a complex regulatory role in processes like anxiety, depression, aggression, and impulse control. Due to the large amount of serotonergic receptors, knockout mice offer an important opportunity to investigate the role of specific receptors. The 5-HT(1B) receptor is thought to mediate aggression and impulse control. This was studied here in mice lacking 5-HT(1B) receptors (5-HT(1B) KO). METHODS: Wild type and 5-HT(1B) KO mice were exposed to several types of entrained and nonentrained stimuli. With telemetry, body temperature, heart rate, and locomotor activity were measured continuously during the different experiments. RESULTS: To nonentrained stimuli like disturbance stress and confrontation with an intruder, 5-HT(1B) KO mice showed exaggerated physiologic and behavioral responses. These mice displayed behavioral disinhibition, measured as increased social interest and aggression to an intruder mouse. However, in response to well-entrained stimuli like daily light transitions, responses were smaller in 5-HT(1B) KO than in wild type mice, suggesting that hyperreactivity is stimulus specific. CONCLUSIONS: Serotonin 1B receptors are essential in impulse control by inhibiting responses to nonentrained stimuli. Therefore, the 5-HT(1B) KO mouse might be an important additional model for studying aspects of disinhibition in aggression and impulse control.

Stress-induced hyperthermia in the 5-HT(1A) receptor knockout mouse is normal.

BACKGROUND: Several studies on serotonin 1A (5-HT(1A)) receptor knockout mice in different genetic backgrounds indicate that such mice display a more anxious phenotype than their corresponding wild types. We hypothesized that the 5-HT(1A) receptor knockout mice would show a different phenotype than the wild type mice in the stress-induced hyperthermia (SIH) paradigm, which tests putative anxiolytic effects of drugs. Moreover, on pharmacologic challenges with the 5-HT(1A) receptor agonist flesinoxan we expected an absence of the functional response in knockout mice relative to wild type mice. METHODS: Effects of the 5-HT(1A) receptor agonist flesinoxan, alone or in combination with the 5-HT(1A) receptor antagonist WAY-100635, and the gamma-aminobutyric acid A (GABA(A))-benzodiazepine receptor agonist diazepam were studied in the SIH paradigm in male 129/Sv 5-HT(1A) receptor knockout and wild type mice. In addition, the effects of flesinoxan on plasma corticosterone concentrations were determined. RESULTS: Plasma corticosterone concentrations were dose dependently elevated by flesinoxan in wild type mice but not in knockout mice. Flesinoxan dose dependently decreased SIH in wild type mice but not in knockout mice. The flesinoxan effect in wild type mice was blocked by WAY-100635. Furthermore, diazepam decreased SIH in both genotypes. There were no differences in basic SIH responses between wild type and knockout mice. CONCLUSIONS: 5 -HT(1A) receptor knockout mice display a normal SIH response, and results indicate, based on the SIH, that the GABA(A)-benzodiazepine receptor complex functions normally.

Corticosterone responses in 5-HT1B receptor knockout mice to stress or 5-HT1A receptor activation are normal.

RATIONALE: Previous research found no adaptations in presynaptic 5-HT1A receptors in mice lacking 5-HT1B receptors (5-HT1B KO). Stress and 5-HT1A receptor agonists induce corticosterone release in mice via hypothalamus-pituitary-adrenal (HPA) axis activation. 5-HT1B KO mice are hyperreactive to mild stressors and this might be reflected in altered postsynaptic 5-HT1A receptor sensitivity. OBJECTIVES: Our aim was to determine whether the activity of the HPA axis was increased in 5-HT1B KO mice in response to mild stress and pharmacological activation of 5-HT1A receptors as an indication of putative adaptive changes in postsynaptic 5-HT1A receptor function. METHODS: The effect of mild stress [i.e., the stress-induced hyperthermia (SIH) paradigm], induced by rectal temperature measurement, was determined on temperature and corticosterone over time (0, 5, 10, 20, 30, 60, and 90 min) in 5-HT1B KO and wildtype mice. In addition, corticosterone was measured 60 min after 5-HT1A receptor activation by flesinoxan (0, 0.03, 0.1, 0.3, 1, and 3 mg/kg s.c.). Blood was collected and plasma corticosterone levels were determined by radioimmunoassay. RESULTS: Both genotypes showed comparable time-dependent SIH responses, whereas basal temperature was higher in 5-HT1B KO mice. The effect of SIH on temperature was mirrored by mild increases in plasma corticosterone. Activation of 5-HT1A receptors caused a strong dose-dependent release of corticosterone in both genotypes. Neither response observed showed differences between both genotypes. CONCLUSIONS: Although 5-HT1B KO mice are hyperreactive to mild stress, this reactivity is not reflected by stronger corticosterone responses in the SIH paradigm. The lack of shift in dose-response curves for flesinoxan suggests that postsynaptic 5-HT1A receptor function is unaffected in 5-HT1B KO mice.

Male and female 5-HT(1B) receptor knockout mice have higher body weights than wildtypes.

5-HT(1B) receptors have a regulatory role in serotonergic activity and influence feeding behavior and body weight. Because the absence of 5-HT(1B) receptors may cause changes in this regulation, body weight was measured in male and female 5-HT(1B) receptor knockout (5-HT(1B) KO) and wildtype (WT) mice from weaning until the age of 30 weeks. In both genders, 5-HT(1B) KO mice had a higher body weight than WT mice (17% and 9%, respectively). Body weight was significantly higher for males over the entire period and for females from Week 18 onwards. Absolute food and water consumption were related to body weight. However, relative to body weight, males consumed more than females. 5-HT(1B) KO males drank strikingly more water. Housing mice singly reduced food and water intake in males, but not in females. Plasma leptin levels and most organ weights did not differ between genotypes, indicating that higher body weight in 5-HT(1B) KO mice is not related to obesity. Relative to body weight, brains and adrenals were larger in females, while heart and liver were smaller. Kidneys were smaller in females, but larger in 5-HT(1B) KO mice, while lungs showed opposite effects. Spleen and testes were smaller in 5-HT(1B) KO mice. Although 5-HT(1B) KO males are more aggressive, testosterone levels were not different from WT mice. Basal corticosterone levels were similar in all groups and increased in response to mild stress, particularly in females. Lifelong absence of 5-HT(1B) receptors in mice resulted in clear phenotypic differences in body weights and food and water intake. Lacking this receptor increases body growth, without signs of obesity. A potential genetic background effect influencing this phenotype is discussed.

Liver and gut mucosa acetylation of mesalazine in healthy volunteers.

AIM: The aim of this investigation was to identify which part of a dose mesalazine is acetylated by enzymes in the gut wall during the absorption process, and which part by the liver enzymes after absorption. METHOD: This study was based on data from four bioequivalence studies of different formulations of tablets (gastro-resistant single dose 500 mg (n = 24) and prolonged-release tablets (single dose 1000 mg, n = 18; multiple dose 1000 mg t.i.d. six days n = 28), suppositories (single 500 mg dose, n = 24) and a study with two i.v. administrations of 100 and 250 mg mesalazine (n = 6). In total, 200 administrations were carried out and plasma concentration-time curves obtained and analyzed. There was a large variability in the absorption of mesalazine for all formulations. The plasma concentration-time curves of parent drug and metabolite acetylmesalazine run nearly parallel, independent of the formulation and the dose. Plasma and urine mesalazine and acetylmesalazine concentrations were determined according to validated methods using HPLC analysis with coulometric or mass-spectrometric detection. RESULTS: As a result of the large variations in release and absorption of mesalazine in the pharmaceutical formulations and administrations, it was possible to demonstrate that acetylation occurs in the gut wall and in the liver. By comparing oral and rectal data to intravenous data, it was possible to indicate where (and to what extent) acetylation occurs in the gut wall, in the liver, or both. Rectal administration of a mesalazine suppository and intravenous administration results in hepatic acetylation. Oral administrations of mesalazine results in both gut wall and hepatic acetylation. Acetylation by the gut wall amounts to 30% of the dose for gastroresistant tablets and to 40% of the dose for prolonged-release tablets.

Ethanol intake is not elevated in male 5-HT(1B) receptor knockout mice.

Recently, the phenotype of increased ethanol intake in mice lacking 5-HT(1B) receptors could not be replicated. We assessed ethanol consumption in male wildtype and 5-HT(1B) receptor knockout mice derived from the original population. Intake of water and ethanol (0%, 3%, 6%, 10% and 20% v/v) from two pipettes was determined daily for 40 days. Ethanol intake (g/kg body weight) did not differ between genotypes, while body weights (20-25%) and water intake (50%) were elevated in 5-HT(1B) receptor knockout mice. Hence, the initial finding of elevated ethanol intake in 5-HT(1B) receptor knockout mice may have been due to phenotypic differences in fluid intake.

Stress-induced hyperthermia in mice: effects of flesinoxan on heart rate and body temperature.

Stress-induced hyperthermia in mice has predictive validity for anxiolytic properties of drugs. In this paradigm, 60 min after drug administration rectal temperature is measured, which causes hyperthermia of 1-1.5 degrees C (DeltaT) in about 10 min. Flesinoxan, a selective 5-HT(1A) receptor agonist with anxiolytic-like properties, causes hypothermia, which complicates interpretation of stress-induced hyperthermia. Therefore, we combined flesinoxan treatment and the stress paradigm with radiotelemetric measurement of body temperature and heart rate, which is also related to anxiety. Subjects were either undisturbed or injected with flesinoxan (0-0.1-0.3-1.0 and 3.0 mg/kg), with or without the stress paradigm. Flesinoxan (1.0 and 3.0 mg/kg) caused a relatively long-lasting hypothermia, but did not lower heart rate. The rectal temperature procedure caused hyperthermia and tachycardia. Flesinoxan reduced the stress-induced hyperthermia and the tachycardia evoked by the stress procedure. Continuous radiotelemetric measurement of heart rate, apart from body temperature, revealed that flesinoxan has anxiolytic-like properties in mice.

Mono- and biphasic plasma concentration-time curves of mesalazine from a 500 mg suppository in healthy male volunteers controlled by the time of defecation before dosing.

This study was based on data from a bioequivalence study (n=24) of two different formulations of suppositories containing 500 mg mesalazine (formulation I and II), with a similar dissolution profile in phosphate buffer pH 6.8. There was a large intra- and intersubject variability in the plasma concentration-time curves of mesalazine from both suppositories. The aim of the investigation was to identify the parameters that caused the observed large variations in release and absorption of mesalazine in the rectum. Plasma mesalazine and acetylmesalazine, and urine acetylmesalazine concentrations were determined according to validated methods involving HPLC analysis with coulometric detection. Lower limit of quantitation values were respectively 10.4 and 19.4 ng mL(-1) in plasma and 0.96 microg mL(-1) in urine. The time of defecation before and after insertion was recorded. There was a clear distinction between subjects who showed monophasic mesalazine release/absorption and those who showed biphasic and more extended release/absorption. With formulation I there was a correlation between time of defecation before dosing and the type of absorption, monophasic and biphasic absorbers showed a significant difference in the time of defecation, e.g. 9.7+/-5.6 h vs 18.8+/-11.9 h (P = 0.0218). The impact of time of defecation before dosing was non-significant with formulation II, 16.7+/-7.2 h vs 15.1+/-4.2 h (P = 0.67). The impact of the time elapsed between administration and time of defecation after the insertion of the suppository was not significant for the type of release/absorption. The plasma concentration-time curves of the metabolite ran parallel to that of the parent drug, the more parent drug was released/absorbed, the more was acetylated (P = 0.0013) and excreted into the urine (P = 0.0004). After absorption the compound was metabolized into acetylmesalazine, and renally excreted (12-13% of the dose). Monophasic release/ absorption resulted in 7.1% metabolite with I and 10.3% with II (P = 0.0004), while biphasic release/absorption gave 16.8% metabolite with I and 15.5% with II. The renal clearance of the metabolite acetylmesalazine was independent of the observed defecation patterns (300 mL min(-1), P > 0.8), stool composition, and type of absorption.

Simultaneous determination of the peptide-mitomycin KW-2149 and its metabolites in plasma by high-performance liquid chromatography.

A gradient high-performance liquid chromatographic (HPLC) method is described for the quantification of KW-2149 and its two major metabolites in plasma. The method involves a sample clean-up by solid-phase extraction on C18 columns, separation of the respective compounds by HPLC on a YMC ODS-AQ column (5-microm particle size, 150x6 mm I.D.), using a methanol-water gradient system as an eluent, and measurement by UV absorbance detection at 375 nm. The limits of quantitation were 10 ng/ml for KW-2149 and M-16, and 15 ng/ml for M-18. Recoveries from plasma were higher than 92% on C18 extraction columns. Intra-day precision, expressed as %C.V., was between 1.4 and 6.5%. Intra-day accuracy ranged from 94 to 107%. Precision and accuracy of variability of inter-assays increased somewhat; however, were still within acceptable ranges. The ability of the method to quantify KW-2149 and two major metabolites simultaneously, with precision, accuracy and sensitivity, make it useful in monitoring the fate of this new mitomycin in cancer patients.