Determination of trace lithium in biological fluids using graphite furnace atomic absorption spectrophotometry: Variability of urine matrices circumvented by cation exchange solid phase extraction

A graphite furnace atomic absorption spectrometry method has been developed for the quantitative determination of submicromolar endogenous concentration of lithium in human plasma and urine using pyrolitically-coated graphite tubes in combination with ammonium nitrate matrix modification. This latter treatment could not completely abolish the interferences caused by the matrix, notably in urine samples. The variability of the urinary matrices required an additional standardization procedure by solid-phase extraction on strongly acidic cation exchange cartridges. Matrix-matched samples were used for the establishment of calibration curves with the addition-calibration method. Calibration curves were linear up to 0.72 μmol/l (1.0 > r² > 0.99). The described method enables accurate measurements of trace-lithium in biological samples at concentrations down to 0.03 μmol/l with intra- and inter-day variabilities < 10%. The method was applied to the determination of trace-lithium levels in urine and plasma samples from healthy individuals enabling the calculation of its fractional excretion (Fe_Li) (median range 17.3%), a value which reflects the functional capacity of the kidney to reabsorb sodium and water at the proximal tubular portion of the nephron. This sensitive method can thus be used as an investigative and diagnostic tool in various renal pathophysiological conditions, in clinical research, and may also be applied to studies on the trace-lithium status of population in connection with psycho-affective disorders.     

Determination of methazolamide concentrations in human biological fluids using high performance liquid chromatography

Methazolamide is a carbonic anhydrase inhibitor used to treat glaucoma. In vivo, methazolamide readily distributes into red blood cells. Therefore, both blood and plasma concentration data are needed in order to characterize the pharmacokinetics of methazolamide. In the present study, an analytical method using high performance liquid chromatography was validated for determination of methazolamide concentrations in several biological fluids. Through slight modification of a previously reported method for acetazolamide, another carbonic anhydrase inhibitor, methazolamide was readily quantitated in whole blood, plasma and urine. Sample preparation involved liquid–liquid extraction with ethyl acetate followed by a washing step using phosphate buffer (pH 8.0). After back extraction into glycine buffer (pH 10.0), samples were then washed with ether and injected onto the chromatograph. Chromatography was performed using a C-18, 5 μm reverse-phase column with UV detection at a wavelength of 285 nm. Mobile phase consisted of 0.05 M sodium acetate (pH 4.0) and acetonitrile (20%). The assay was validated over two standard concentration ranges from 1 to 100 μg ml⁻¹, concentrations reflective of those expected in vivo. Calibration curves were linear for all biological fluids and coefficients of variation for interday and intraday reproducibility studies were less than 8% (range 3.1–7.9%). The method was used to measure methazolamide concentrations in blood, plasma and urine following oral administration to five human subjects.     

Comparison of 5-Fluorouracil Pharmacokinetics in Whole Blood,Plasma, and Red Blood Cells in Patients with Colorectal Cancer

Study Objective . To compare 5-fluorouracil (5-FU) pharmacokinetics in whole blood, plasma, and red blood cells in patients with colorectal cancer. Design . Prospective, unblinded observational study in consecutive patients. Setting . Large regional teaching hospital. Patients . Five patients with colorectal cancer. Interventions . Patients received folinic acid 200 mg/m² intravenously over 2 hours, followed by 5-FU 600 mg/m² intravenous bolus over 30 minutes, then 5-FU 600 mg/m² intravenous infusion over 22 hours, administered on days 1 and 2. This 48-hour cycle was repeated every 14 days. Measurements and Main Results . Concentrations of 5-FU in whole blood, plasma, and red blood cells were determined by high-performance liquid chromatography. ADAPT II was used for pharmacokinetic computations. The optimum model was determined for each matrix by calculating Akaike's information criteria values. Concentrations of 5-FU in whole blood were 106–115% of simultaneous plasma concentrations (median 112%), and packed red blood cell levels were 5–17% of plasma concentrations (median 11%). The drug's concentration-time profile was similar in the three matrices. The drug is reported to be unstable in whole blood, and red blood cell 5-FU concentrations were near the limit of detection (10 ng/ml), supporting plasma as the preferred matrix for therapeutic drug monitoring studies. Six pharmacokinetic models were fitted to the 5-FU individual data sets to determine the best curve fit. The optimal model for whole blood and plasma data sets was one compartment with both linear and nonlinear elimination models; a one-compartment model with nonlinear elimination provided the best curve fit for 5-FU in red blood cells. A two-compartment model with nonlinear elimination gave a similar degree of curve fit for plasma 5-FU as the one-compartment model with both linear and nonlinear elimination. Conclusions . These pharmacokinetic results provide the basis for further investigation into the ability to correlate 5-FU systemic exposure with clinical drug activity. (Pharmacotherapy 1997;17(5):881–886)     

Stability,tissue metabolism,tissue distribution and blood partition of azosemide

Stability of azosemide after incubation in various pH solutions, human plasma, human gastric juice, and rat liver homogenates, metabolism of azosemide after incubation in 9000g supernatant fraction of various rat tissue homogenates in the presence of NADPH, tissue distribution of azosemide and M1 after intravenous (IV) administration of azosemide, 20 mg kg^?1, to rats, and blood partition of azosemide between plasma and blood cells from rabbit blood were studied. Azosemide seemed to be stable for up to 48 h incubation in various pH solutions ranging from two to 13 at an azosemide concentration of 10 μg mL^?1; more than 93.4% of azosemide was recovered and a metabolite of azosemide, M1, was not detected. However, the drug was unstable in pH 1 solution: 75.8% of azosemide was recovered and 2.16 μg mL^?1 of M1 (expressed in terms of azosemide) was formed after 48 h incubation in pH 1 solution at an azosemide concentration of 10 μg mL^?1. Azosemide was stable in both human plasma and rat liver homogenates for up to 24 h incubation at an azosemide concentration of 1 μg mL^?1, and in human gastric juice for up to 4 h incubation at an azosemide concentration of 10 μg mL^?1. However,-all rat tissues stdied had metabolic activity for azosemide in the presence of NADPH, with heart having a considerable metabolic acitivity: approximately 22% of azosemide disappeared and 9.32 μg of M1 was formed per gram of heart (expressed in terms of azosemide) after 30 min incubation of 50 μg of azosemide in 9000g supernatant fraction of heart homogenates. The tissue to plasma ratios of azosemide (T/P) were greater than unity only in the liver (1.26) and kidney (1.74); however, M1 showed high affinity for all tissues studied except the brain and spleen when each tissue was collected at 30 min after IV administration of azosemide to rats. The equilibrium plasma to blood cell concentration ratios of azosemide were independent of azosemide blood concentrations: the values were 2.78–4.25 at azosemide blood concentrations of 1, 10, and 20 μg mL^?1 three rabbits. There was negligible ‘blood storage effect’ of azosemide, especially at low blood concentrations of azosemide, such as 1 and 10 μg mL^?1.     

Effect of ibuprofen on lithium plasma and red blood cell concentrations

The effect of ibuprofen on steady-state lithium plasma and red blood cell concentrations was studied in 11 normal volunteers. During the seven-day control phase, sustained-release lithium carbonate 450 mg was administered every 12 hours. Lithium plasma and red blood cell concentrations were determined on days 5, 6, and 7. During the treatment phase (days 7-15), ibuprofen 400 mg was administered four times a day concurrently with lithium. Lithium plasma and red blood cell concentrations were obtained on days 14, 15, and 16. Multiple blood samples were obtained over a 12-hour period on days 6 and 15. Urine samples were collected from six subjects. The mean minimum lithium concentration increased 15% when ibuprofen was added. Mean maximum lithium concentration, area under the curve, red blood cell concentrations, and the lithium red blood cell to plasma ratio were significantly higher during the treatment phase. Mean lithium total body and renal clearance values were significantly lower during the treatment with ibuprofen. The administration of ibuprofen can increase steady-state plasma lithium concentrations and decrease lithium clearance.     

Plasma levels of morphine and morphine glucuronides in the treatment of cancer pain: relationship to renal function and route of administration

Summary There is growing evidence that renally-impaired patients receiving morphine therapy are at greater risk of developing opiate toxicity, due to the accumulation of an active metabolite, morphine-6-glucuronide (M6G), which is usually excreted by the kidneys. This study examined the relationships between morphine dosage, renal function, and trough plasma concentrations of morphine and its glucuronide metabolites in 21 patients (aged mean: 68.5 years; 11 males) receiving either oral or subcutaneous morphine for terminal cancer pain. The median daily morphine dosages (mg · kg^–1) were: orally 1.87 (range 0.37–6.82) and subcutaneously 1.64 (range 0.22–3.60).The median plasma concentrations of morphine, morphine-3-glucuronide (M3G), and M6G (ng · ml^–1) were: 36.0, 1035.2, and 142.3, respectively. The plasma concentrations of morphine, M3G and M6G were each significantly related to the daily morphine dosage (n=21, Spearman r=0.79, 0.91, and 0.88 respectively). Accumulation of the morphine glucuronides was dependent on renal function. The plasma concentrations of M3G and M6G, when divided by the morphine concentration, were significantly related to the caluclated creatinine clearance of the patient. Patients receiving oral morphine had higher plasma concentration ratios of glucuronide/morphine than those receiving subcutaneous therapy, presumably due to first-pass glucuronidation.The results of this study confirm that accumulation of the pharmacologically active M6G is related to renal function, which probably explains the observation that morphine dosage requirements are generally reduced in patients with renal impairment.     

Indomethacin and the role of prostaglandins in adipose tissue.

Indomethacin (2 or 10 μg/ml) or meclofenamic acid (4 μg/ml), two potent inhibitors of prostaglandin biosynthesis, did not affect basal or noradrenaline (10^?6 M) stimulated lipolysis when added to isolated rat fat cells. Indomethacin (5 μg/ml blood) similarly was without effect on blood flow, on lipolysis or on ³H-noradrenaline overflow before, during and after nerve stimulation (4 Hz) in perfused canine subcutaneous adipose tissue in situ. Indomethacin given to rats 5 × 5 mg/kg at 10–14 hr intervals p.o. had no effect on arterial glycerol concentration, but caused a significant hypoglycemia. Fat cells or fat pads extracted from such rats had an unchanged basal lipolytic rate but a lowered responsiveness to high concentrations of noradrenaline (4 × 10^?7-2 × 10^?6 M), compared with controls. However, 10^?7 M noradpresumbaly caused a higher lipolytic response in fat cells from indomethacin-treated rats than from controls, presumably because the former caused a smaller degradation of naradrenaline during the incubation period. Indomethacin (0·2–20 μg/ml) had no effect on cAMP binding to protein kinase, but apparently caused membrane stabilization, since erythrocytes from indomethacin-treated rats were more resistant to hypotonic lysis than red cells from control animals. Our results suggest that indomethacin has a multitude of biological effects, some of which may be unrelated to inhibition of prostaglandin biosynthesis. When considered together with previous results the findings also suggest that endogenous prostaglandins are of minor importance as feed back inhibitors of lipolysis in adipose tissue.     

Biotransformation of 4-dimethylaminophenol in the dog

4-Dimethylaminophenol (DMAP), after an i.v. injection, quickly forms ferrihemoglobin by catalytic transfer of electrons from ferrohemoglobin to oxygen. This reaction is rapidly terminated by covalent binding of oxidized DMAP to the reactive SH-groups of hemoglobin and to reduced glutathione within the red cells, and by conjugation with glucuronic or sulfuric acid presumably in the liver. Fifteen min after i.v. injection of DMAP, 3.25 $\text{mg}\text{kg}$, ¹⁴C-labeled in the ring, no intact DMAP was detected in the blood. The concentrations of metabolites in the blood were as follows: 33 μM DMAP covalently bound to hemoglobin. 30 μM S,S,S-(2-dimethylamino-5-hydroxy-1,3,4-phenylene)-Tris-glutathione (Tris-IGS)-DMAP) 90 per cent of it located within the red cells, 5 μM DMAP-glucuronide, and 22 μM DMAP-sulfate. Within 3 days, 90 per cent of the radioactivity was excreted in the urine, 4 per cent in the faeces. In the 24 hr urine, 25 per cent of the DMAP injected was excreted as DMAP-sulfate, 15 per cent as DMAP-glucuronide, and 23 per cent as DMAP-thioethers, mainly as S,S,S-(dimethylamino-5-hydroxy-1,3,4-phenylene)-Tris-cysteine. When DMAP, ¹⁴C-labeled in the methyl groups, was administered 11 per cent of the radioactivity was excreted in the urine as dimethylamine. It is concluded that most of the thioethers found in the urine derived from Tris-(GS)-DMAP which had been produced within the red cells indicating an important role of the red cells on biotransformation of DMAP.     

Simplified reversed-phase HPLC method with spectrophotometric detection for the assay of verapamil in rat plasma

A high-performance liquid chromatographic (HPLC) method was developed for the assay of verapamil in rat plasma. After deproteinization of the plasma sample with an acetonitrile-perchloric acid (8:2) mixture containing dextromethorphan, the internal standard, an aliquot of the supernatant was directly analyzed on a cyanopropylsilane column with methanol-acetonitrile-triethylamine acetate buffer (10:30:60) as the mobile phase and detection at 235 mm. At a flow rate of 1.5 ml min⁻¹, a complete analysis was completed in less than 6 min. The method was linear for verapamil concentrations in the range 0.5–10 μg ml⁻¹ (r=0.9999). Recoveries for the same drug concentrations from spiked rat plasma ranged from 85.6-93.0% (n=8). The mean RSD values for intraday and interday assay reproducibility (n=3) were, in both cases, less than 0.9%. The limit of detectability was about 0.1 μg ml⁻¹. The method was found useful to monitor the plasma levels of verapamil in rats that had received this drug by the nasal, oral and intravenous routes of administration.     

Effect of omeprazole and feeding on plasma gastrin in patients with achlorhydria

Background: The mechanism of hypergastrinaemia during omeprazole therapy is unclear, but is generally assumed to be entirely a consequence of acid suppression. However, direct stimulation of G cells by omeprazole could also be a factor. In order to further investigate the mechanism of omeprazole-induced hypergastrinaemia, we have studied the effects of the drug on plasma gastrin in patients with achlorhydria, in whom altered acid secretion cannot play a role. Methods: We estimated fasting and peptone meal stimulated plasma gastrin in nine patients (seven female) with pernicious anaemia and achlorhydria, before and on the final day of 4 weeks’dosing with omeprazole 40 mg daily. Results: Despite the high fasting gastrin concentrations, the peptone meal produced a further elevation in plasma gastrin concentrations, median gastrin concentrations rising from 1500 ng/L (range 225–10875 ng/L) to 3750 ng/L (range 585–15600 ng/L) post-prandially (P = 0.004). The median post-prandial rise in plasma gastrin at this initial visit was 44% (3–260%), and the median time interval until plasma gastrin concentrations returned to fasting levels was 120 min (range 10- > 150 min). There was a significant negative correlation between fasting plasma gastrin concentrations and the percentage increase in plasma gastrin levels in response to meal stimulation (Spearman correlation coefficient -0.79, P= 0.01). Fasting plasma gastrin concentrations were similar pre-omeprazole (median 1950 ng/L, range 240–16500 ng/L) and postomeprazole (median 1500 ng/L, range 315–7650 ng/L). Likewise, peak plasma gastrin concentrations were also similar pre-omeprazole (median 2700 ng/L, range 585–16500 ng/L) and post omeprazole (median 3420 ng/L, range 720–11250 ng/L). Conclusions: (i) The hyperplastic G cell mass in patients with pernicious anaemia can be further stimulated by a peptone meal, which causes a prolonged rise in plasma gastrin concentrations. (ii) There is a negative correlation between fasting plasma gastrin concentrations and the percentage increase in plasma gastrin levels in response to meal stimulation. (iii) Omeprazole has no effect on plasma gastrin in achlorhydric patients, which is consistent with its hypergastrinaemic effect being entirely secondary to acid inhibition.     

Novel and potent BK channel openers: CGS 7181 and its analogs

This article discloses the identification of a novel series of the opener of the large-conductance Ca²⁺-activated K⁺ (BK) channel, CGS 7181, and its analogs, CGS 7184, CGS 7590, and CGS 7725. The stimulatory effects of these compounds on the channels were investigated at whole-cell and single channel levels using the patch-clamp technique in single smooth muscle cells from vascular (coronary artery) and non-vascular (bladder detrusor) tissues of several animal species. With a threshold of submicromolar concentration, extracellularly applied CGS 7181 and CGS 7184 (0.5–50 μM) induced a concentration-dependent stimulation of the whole-cell BK current (I_BK) and concomitant membrane hyperpolarization in porcine coronary artery cells. The activation was prevented and reversed by TEA, but unaffected by nifedipine, suggesting that the effect was not subsequent to Ca²⁺ entry. CGS 7184, CGS 7590, and CGS 7725 (0.1–50 μM) produced augmentation of I_BK in a similar manner in cells from bladder detrusor of guinea pig, rat, and dog. The onset and offset of the drug actions were slow compared to the known BK channel opener NS004. The effects of compounds applied intracellularly were assessed on single BK channels in inside-out patches. With threshold concentrations ranging between 0.01 and 0.1 μM, all compounds caused a drastic and reversible increase in channel open-state probability. The onset and washout of drug actions were considerably faster in inside-out patches than in whole cells. It is concluded that CGS 7181 and its analogs directly open BK channels from either side of the membrane with a combination of potency and efficacy superior to any known BK channel openers, and an internal site of action best accounts for the results of our studies. Drug Dev. Res. 41:10–21, 1997. © 1997 Wiley-Liss, Inc.     

COMPARISON OF PHARMACOKINETICS OF M1, M2, M3, AND M4 AFTER INTRAVENOUS ADMINISTRATION OF DA-125 OR ME2303 TO MICE AND RATS. NEW ADRIAMYCIN ANALOGUES CONTAINING FLUORINE

The pharmacokinetics of M1, M2, M3, and/or M4 were compared after intravenous (iv) administration of DA-125 and/or ME2303 to mice (25 mg kg⁻¹) and rats (5, 10, 20, 30, and 40 mg kg⁻¹). The mean plasma concentrations of M1 were detected up to 8 h after iv administration of both DA-125 and ME2303 to mice, and were significantly higher for DA-125 than ME2303; this resulted in a considerably greater AUC (303 against 148 μg min mL⁻¹) and a considerably slower CL of M1 (69·3 against 136 mL min⁻¹ kg⁻¹) after iv administration of DA-125. The MRT (371 against 189 min) and CL_NR of M1 (68·7 against 136 mL min⁻¹ kg⁻¹) were considerably greater and slower, respectively, after iv administration of DA-125. The mean plasma concentrations of M2 were detected up to 8 and 4 h after iv administration of DA-125 and ME2303, respectively, to mice and were significantly higher for DA-125 than ME2303, resulting in a considerably greater AUC of M2 (148 against 27·1 μg min mL⁻¹) after iv administration of DA-125. The mean plasma concentrations of M3, being the lowest among M1–M4, were detected only up to 15 min after iv administration of both DA-125 and ME2303 to mice, and were comparable after iv adminstration of DA-125 and ME2303 to mice. The mean plasma concentrations of M4 were detected up to 8 h after iv administration of both DA-125 and ME2303 to mice, and were higher after iv administration of DA-125 than ME2303, resulting in a considerably greater AUC of M4 (197 against 61·9 μg min mL⁻¹) after iv administration of DA-125. Similar results on M1 and M2 were also obtained from rats: the mean plasma concentrations of both M1 and M2 were significantly higher after iv administration of DA-125, 10 mg kg⁻¹, than after ME2303. The plasma concentrations of M1, M2, and M4, and hence their AUCs, were significantly higher after iv administration of DA-125, 5, 10, 20, 30, and 40 mg kg⁻¹, to rats than after ME2303: the mean plasma concentrations of M2, approximately 0·1–0·4 μg mL⁻¹, were maintained from 30 min to 8–10 h after iv administration of DA-125, 20, 30, and 40 mg kg⁻¹, to rats; the plasma concentrations of M3 were the lowest among M1–M4 at all DA-125 doses; and those of M1 and M4 were maintained for a long period of time. However, after iv administration of M2, 5 mg kg⁻¹, to rats, the mean plasma concentrations of M2 were detected up to 60 min with a mean terminal half-life of only 38·8 min, and the concentrations of M3 were negligible. After iv administration of M3, 5 mg kg⁻¹, to rats, the mean plasma concentrations of M3 were detected up to 15 min; the plasma concentrations of M4, reaching their peak at 5 min, decayed more slowly and were higher than those of M3. The AUC of M4 after iv administration of M3, 5 mg kg⁻¹, was comparable to that after iv administration of M4, 5 mg kg⁻¹, to rats, suggesting that M4 is formed fast and almost completely from M3. M1 was not detected in plasma after iv administration of either M2 or M3 to rats. After iv administration of M4, 5 mg kg⁻¹, to rats, the mean plasma concentrations of M4 decayed fast with a mean terminal half-life of 43·9 min and neither M2 nor M3 were detected in plasma. The following disposition mechanisms for M1, M2, M3, and M4 after iv administration of DA-125 to rats could be obtained from the above data: (i) the maintenance of plasma concentrations of M2 for a longer period of time after iv administration of DA-125 than those after iv administration of M2 could be due to the continuous formation of M2 from M1; (ii) the lowest plasma concentrations of M3 among M1–M4 after iv administration of DA-125 could be due to the fast and almost complete formation of M4 from M3 as soon as M3 is formed from M1, and not due to the fast renal excretion of unchanged M3; (iii) M4 was exclusively and continuously formed from M3 and the formation of M4 from M2 was negligible; and (iv) reversible metabolism among M1–M4 did not take place. The following results could also be obtained after iv administration of DA-125 or ME2303 to mice and rats: (i) the lower plasma concentrations of M1 after iv administration of ME2303 than of DA-125 could be due to the greater biliary excretion of unchanged ME2303 (approximately 30% of iv dose) than unchanged DA-125 and (ii) the lower plasma concentrations of M2 and M4 after iv administration of ME2303 than after DA-125 could be due to lower plasma concentrations of M1 and hence less formation of both M2 and M4 from M1. Liver showed the highest metabolic activity for M1 and a considerable amount of M1 was also metabolized in the kidney after 30 min incubation of 50 μg of DA-125 in 9000 g supernatant fraction of rat tissue homogenates. The mean amount of M1 remaining per gram of tissue, the total amount of M1 remaining in whole tissue, and the tissue to plasma ratio of M1 were significantly higher in the heart, lung, large intestine, and kidney at 15 min after iv administration of DA-125, 25 mg kg⁻¹, to mice than after ME2303. M1, the active antineoplastic moiety of DA-125, had higher affinity for the lung after iv administration of DA-125 to mice than after ME2303, indicating that lung tumours could be subjected to a greater exposure to M1 after iv administration of DA-125 than ME2303. The 24 h biliary excretion of M1 was significantly greater after the iv administration of ME2303 than after DA-125 (344 against 79·3 μg). However, reversed results were obtained for M2 (267 against 467 μg). M3 and M4 were under the detection limit in the bile sample after iv administration of either DA-125 or ME2303.     

Minimal Toxicity to Myeloid Progenitor Cells of Weekly24‐Hr Infusion of High‐Dose 5‐Fluorouracil: Direct Evidence from Colony Forming Unit‐Granulocyte andMonocyte (CFU‐GM) Clonogenic Assay

Although very high doses of 5‐fluorouracil was used in the weekly 24‐h infusion, high‐dose 5‐fluorouracil (2600 mg/m²/week) and leucovorin (500 mg/m²/week) protocol, myelosuppression was surprisingly low. The current study was conducted to investigate the possible mechanism underlying the low myelosuppression. To mimic the clinical situation, peripheral blood progenitor cells collected from 12 patients were used for colony forming unit‐granulocyte and monocyte clonogenic assay; and 2 representative modes of 5‐fluorouracil exposure (30 min. versus 24 hr) were examined for cytotoxic effects on human myeloid progenitor cells. Previous pharmacokinetic studies have estimated the concentrations of 5‐fluorouracil in the bone marrow to be 200–400 μM and 1–2 μM for the 30 min. infusion (600–900 mg/m²) and the 24 hr‐infusion (1000–2000 mg/m²) regimens, respectively. The results of our colony‐forming unit‐granulocyte and monocyte clonogenic assay showed that 24‐hr exposure to 5‐fluorouracil (2 μM) and 30 min. exposure to 5‐fluorouracil (100 μM) resulted in 27.2% and 78.2% inhibition of the colony formation, respectively. Our data provided direct evidence which may explain why myelotoxicity is significantly less in weekly 24 hr infusion of fluorouracil than in the conventional bolus regimens.     

Nociceptin/Orphanin FQ Suppresses Adaptive Immune Responses In Vivo and at Picomolar Levels In Vitro

Nociceptin/orphanin FQ (N/OFQ), added in vitro to murine spleen cells in the picomolar range, suppressed antibody formation to sheep red blood cells in a primary and a secondary plaque-forming cell assay. The activity of the peptide was maximal at 10^?12 M, with an asymmetric U-shaped dose–response curve that extended activity to 10^?14 M. Suppression was not blocked by pretreatment with naloxone. Specificity of the suppressive response was shown using affinity-purified rabbit antibodies against two N/OFQ peptides and with a pharmacological antagonist. Antisera against both peptides were active, in a dose-related manner, in neutralizing N/OFQ-mediated immunosuppression, when the peptide was used at concentrations from 10^?12.3 to 10^?11.6 M. In addition, nociceptin given in vivo by osmotic pump for 48 h suppressed the capacity of spleen cells placed ex vivo to make an anti-sheep red blood cell response. These studies show that nociceptin directly inhibits an adaptive immune response, i.e., antibody formation, both in vitro and in vivo.     

The binding of paracetamol to plasma proteins of man and pig

The binding of N-acetyl-4-aminophenol (paracetamol) to human and porcine plasma at both toxic and therapeutic concentrations was investigated by ultrafiltration and equilibrium dialysis over the range 50–300 μg ml^?1. Plasma protein binding occurred at paracetamol concentrations greater than 60 μg ml^?1. The extent of protein binding at a plasma concentration of 280 μg ml^?1 of the drug is between 15 and 21% for both pig and man. There is no appreciable binding to erythrocytes in either species over the whole concentration range studied.     

Melphalan concentration dependent plasma protein binding in healthy humans and rats

Summary The binding of melphalan to plasma proteins from four healthy humans and from rats was measured by centrifugal ultrafiltration. Melphalan concentrations were determined by HPLC and by measuring ¹⁴C-melphalan activity. In whole blood, melphalan was distributed preferentially in plasma. However, a constant fraction, 37%, which was independent of the total melphalan concentration in whole blood, was present within the red blood cells. The binding of melphalan to plasma proteins from humans was less than that from rats. In both, however, the fraction bound was constant throughout the concentration range (0.1 to 9.0 µM) that is achieved during standard-dose melphalan therapy. Albumin was the primary binding protein. At concentrations equal to or in excess of 33 µM, which have been achieved during high-dose melphalan therapy, free plasma melphalan concentrations were no longer linearly related to total drug concentrations, and the plasma protein binding of melphalan in the human became concentration dependent. This occurred at concentrations of 70 µM in the rat. Scatchard analysis of the data indicated the presence of 2 groups of binding sites. Class I sites had 0.03 and 0.4 binding sites per albumin molecule in humans and rats, with respective association constants of 4.43 × 10⁴M^–1 and 1.92 × 10⁴M^–1. Class II sites had 5.18 and 2.60 binding sites per molecule, with repective association constants of 3.82 × 10²M^–1 and 2.01 × 10²M^–1.     

Investigation of ifosfamide and chloroacetaldehyde renal toxicity through integration of in vitro liver–kidney microfluidic data and pharmacokinetic‐system biology models

We have integrated in vitro and in silico data to describe the toxicity of chloroacetaldehyde (CAA) on renal cells via its production from the metabolism of ifosfamide (IFO) by hepatic cells. A pharmacokinetic (PK) model described the production of CAA by the hepatocytes and its transport to the renal cells. A system biology model was coupled to the PK model to describe the production of reactive oxygen species (ROS) induced by CAA in the renal cells. In response to the ROS production, the metabolism of glutathione (GSH) and its depletion were modeled by the action of an NFE2L2 gene‐dependent pathway. The model parameters were estimated in a Bayesian context via Markov Chain Monte Carlo (MCMC) simulations based on microfluidic experiments and literature in vitro data. Hepatic IFO and CAA in vitro intrinsic clearances were estimated to be 1.85 x 10^‐9 μL s^–1 cell^–1 and 0.185 x 10^‐9 μL s^–1 cell^–1,respectively (corresponding to an in vivo intrinsic IFO clearance estimate of 1.23 l h^–1, to be compared to IFO published values ranging from 3 to 10 l h^–1). After model calibration, simulations made at therapeutic doses of IFO showed CAA renal intracellular concentrations ranging from 11 to 131 μM. Intracellular CAA concentrations above 70 μM induced intense ROS production and GSH depletion. Those responses were time and dose dependent, showing transient and non‐linear kinetics. Those results are in agreement with literature data reporting that intracellular CAA toxic concentrations range from 35 to 320 μM, after therapeutic ifosfamide dosing. The results were also consistent with in vitro CAA renal cytotoxicity data. Copyright © 2015 John Wiley & Sons, Ltd.     

Pharmacokinetics of ticlopidine during chronic oral administration to healthy volunteers and its effects on antipyrine pharmacokinetics

1. The pharmacokinetics of ticlopidine, a novel antithrombotic agent, have been investigated in 10 healthy volunteers dosed orally with the drug (250?mg 12 hourly for 21 days), to determine the basic pharmacokinetic parameters in humans, to investigate its accumulation during repeated administration, and to assess its effects on hepatic drug-metabolizing enzymes.

2. After the first dose, peak plasma concentrations (median 0.31, range 0.08–0.80?mg/l) were generally found at 2?h. The levels decreased rapidly to a median concentration of 0.087?mg/l by 4?h then declined to 0.022 (range<0.005–0.128)?mg/l at 12?h after administration, with apparent half-lives of approx. 4?h. The median AUC value for this first dosage interval (AUC_τ) was 0.97 (range 0.41–3.49) mg?h l^?1.

3. Pre-dose plasma concentrations indicated that steady state was reached after 5–10 days, and then remained essentially unchanged through to the end of the study. From 30?h after the final dose, drug levels declined exponentially with a median half-life of 28.8 (range≤20–50)?h.

4. Following the final dose, the median peak concentration and AUC_τ were 0.99 (range 0.22–2.12)mg/l and 4.06 (range 0.90–15.2)mg?h l^?1 respectively. Based on AUC values, the mean accumulation factor±SD was 3.73±1.14.

5. The metabolic status of subjects was assessed by administration of single doses of antipyrine (700?mg orally) 7 days before the first dose of ticlopidine and 2 days after the final dose. Treatment with ticlopidine decreased antipyrine clearance, demonstrating that it inhibited drug-metabolizing enzymes. Significant correlations (r² = 0.84, p<0.01) were found between the AUC values for ticlopidine and antipyrine, indicating that the inter-individual variation in the pharmacokinetics of ticlopidine are explained by differences in metabolic clearance.     

Relationship between plasma,atrial and ventricular perhexiline concentrations in humans: insights into factors affecting myocardial uptake

AIM

Little is known regarding the steady-state uptake of drugs into the human myocardium. Perhexiline is a prophylactic anti-anginal drug which is increasingly also used in the treatment of heart failure and hypertrophic cardiomyopathy. We explored the relationship between plasma perhexiline concentrations and its uptake into the myocardium.

METHODS

Blood, right atrium ± left ventricle biopsies were obtained from patients treated with perhexiline for a median of 8.5 days before undergoing coronary surgery in the perhexiline arm of a randomized controlled trial. Perhexiline concentrations in plasma and heart tissue were determined by HPLC.

RESULTS

Atrial biopsies were obtained from 94 patients and ventricular biopsies from 28 patients. The median plasma perhexiline concentration was within the therapeutic range at 0.24 mg l⁻¹ (IQR 0.12–0.44), the median atrial concentration was 6.02 mg kg^–1 (IQR 2.70–9.06) and median ventricular concentration was 10.0 mg kg^–1 (IQR 5.76–13.1). Atrial (r² = 0.76) and ventricular (r² = 0.73) perhexiline concentrations were closely and directly correlated with plasma concentrations (both P < 0.001). The median atrial : plasma ratio was 21.5 (IQR 18.1–27.1), ventricular : plasma ratio was 34.9 (IQR 24.5–55.2) and ventricular : atrial ratio was 1.67 (IQR 1.39–2.22). Using multiple regression, the best model for predicting steady-state atrial concentration included plasma perhexiline, heart rate and age (r² = 0.83). Ventricular concentrations were directly correlated with plasma perhexiline concentration and length of therapy (r² = 0.84).

CONCLUSIONS

This study demonstrates that plasma perhexiline concentrations are predictive of myocardial drug concentrations, a major determinant of drug effect. However, net myocardial perhexiline uptake is significantly modulated by patient age, potentially via alteration of myocardial:extracardiac drug uptake.     

Effect of 5-benzylacyclouridine,a potent inhibitor of uridine phosphorylase,on the metabolism of circulating uridine by the isolated rat liver

5-Benzylacyclouridine (BAU) is a specific inhibitor of uridine phosphorylase, the first enzyme in the catabolism of uridine. It was found that 20 and 100 μM BAU dramatically reduced the rapid clearance of trace amounts of either [¹⁴C]uridine or hyperphysiologic concentrations of non-labeled uridine by the isolated rat liver perfused with an artificial oxygen carrier. In the absence of exogenously added uridine, non-treated livers maintained circulating concentrations of 1–2 μM uridine. In the presence of 20 μM BAU, these concentrations were increased 2- to 3-fold higher than physiologic levels (1.4±0.6 μM) and remained elevated for the duration of the experiment (120–160 min). In the presence of 100μM BAU, uridine concentrations rose continuously at rates of between 80 and ISOnmoles per hr per g of liver, and the clearance of a single radioactive spike of uridine was reduced extensively. The half-life of a uridine spike was extended 2-fold in the presence of 20 μM BAU and 5- to 6-fold in the presence of 100 μM BAU. Exogenously added uridine (15 and 40 μM) was cleared rapidly by nontreated livers, with a half-life of approximately 10 min. However, BAU at a concentration of 20 μM increased the half-life of 15 or 40 μM uridine added to the perfusate by approximately 10-fold. A 100 μM concentration of BAU inhibited the removal of 40 μM circulating uridine, but with 15 μM uridine there was a continuous increase in the circulating concentration similar to that seen in the absence of added uridine. We conclude that extensive inhibition of uridine phosphorylase occurs at 100 μM BAU and partial inhibition at 20 μM BAU. These data indicate independent catabolic and excretory functions of the rat liver with respect to uridine.