Differential effect of methotrexate on the increased CCR2 density on circulating CD4 T lymphocytes and monocytes in active chronic rheumatoid arthritis, with a down regulation only on monocytes in responders.

OBJECTIVES: To evaluate the effect of orally administered methotrexate (MTX) on the density of CC chemokine receptor 2 (CCR2) and CXC chemokine receptor 3 (CXCR3) on circulating monocytes, and the coexpression of CXCR3 and CCR2 on CD4 T lymphocytes in patients with active chronic rheumatoid arthritis. METHODS: All 34 patients with rheumatoid arthritis fulfilled the 1987 American Rheumatism Association criteria and were followed for 16 weeks after starting MTX. Peripheral blood mononuclear cells were analysed for CCR2 and CXCR3 density by three-colour flow cytometry before initiation of MTX and at week 12. RESULTS: 22 (65%) patients were non-responders, 12 (35%) patients responded to MTX by American College of Rheumatology (ACR)20% criteria, and 8 (24%) of these patients responded by ACR50%. In patients with active rheumatoid arthritis before starting MTX, CCR2 density on circulating monocytes, CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes was increased compared with controls. During 12 weeks of MTX treatment, the CCR2 density on monocytes decreased significantly in the ACR50% group but not in the ACR20% and non-responder groups. The increased CCR2 density on CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes was unaffected by the reduction in disease activity measured in relation to MTX treatment. The percentage of both monocytes and CD4(+) CXCR3(+) and CD4+ CXCR3(-) T lymphocytes among the peripheral circulating mononuclear cells did not change during MTX treatment. CONCLUSIONS: Active chronic rheumatoid arthritis is characterised by enhanced CCR2 density on circulating monocytes and CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes. During MTX treatment, a decrease in CCR2 density on monocytes in the ACR50% responder group was associated with decreased disease activity. The increased CCR2 density on CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes was uninfluenced by MTX and disease activity.

Comparison of the pharmacokinetics of tacrolimus and cyclosporine at equivalent molecular doses.

Even though calcineurin inhibitors, namely Tacrolimus (FK) and Cyclosporine (CsA) share similar physicochemical properties and a common mechanism of action, their pharmacokinetics (pk) are different and unpredictable. Both drugs are metabolized by cytochrome P450-3A4 isoforms in the liver and in the mucosa of the upper gastrointestinal tract. FK in clinical practice is given in doses up to 50-fold lower than those of CsA due to its greater potency. It is often assumed that the diverse dosing contributes to the observed pharmacokinetic differences between the two drugs. The objective of the present study was to compare single-dose pk profiles of the two drugs, following oral and intravenous administration, on the basis of equivalent molecular dosing, thus ruling out the quantitative factor. Five healthy volunteers and 14 dialysis patients (7 hemodialysis, 7 peritoneal dialysis) were included in the study. Comparing the pharmacokinetic parameters obtained from the drugs, it appeared that cyclosporine has an greater primary volume of distribution and clearance rate compared to tacrolimus. No other statistically significant differences were observed regarding bioavailability, absorption rate, or elimination rate. The only significant correlation between the pk values of the drugs was in primary volume of distribution. We conclude that even at equivalent molecular doses the pk of each drug remains unique and unpredictable. Furthermore our data fail to reveal significant correlations between the bioavailability, clearance, absorption, and elimination rates of the two drugs.

Plasma concentration of topiramate correlates with cerebrospinal fluid concentration.

The authors examined the ratio between the plasma and the cerebrospinal fluid (CSF) concentration of topiramate in 14 adults with epilepsy. Simultaneous trough samples of venous blood and CSF were collected and analyzed as total and unbound concentrations. Concomitant levels were also analyzed of lamotrigine (n = 5) and the relevant oxcarbazepine metabolite, 10-hydroxycarbazepine (n = 3). There was a close correlation between the plasma and the CSF concentration for both the total and unbound concentration of topiramate. The median CSF/plasma ratio of total topiramate was 0.85. The free topiramate concentration in plasma was not different from the free topiramate concentration in CSF. The CSF/plasma ratios showed little variation and were independent of the plasma level for both the total and the unbound levels. The unbound fraction of topiramate was 84% in plasma and 97% in CSF. The CSF concentrations of lamotrigine and 10-hydroxycarbazepine were 50% and 61% of the plasma concentrations, respectively. For topiramate, there is a close correlation between the plasma concentration and the CSF concentration. There does not seem to be a saturable carrier mechanism restricting topiramate transport across the blood-brain barrier. The concentration of topiramate in CSF is equal to the unbound proportion of topiramate in plasma, implying that the delivery of topiramate to the brain occurs via transfer from the unbound plasma pool. Plasma is thus a relevant matrix for therapeutic drug monitoring of topiramate.

Removal of 10-hydroxycarbazepine by plasmapheresis.

Removal of the oxcarbazepine metabolite 10-hydroxycarbazepine (MHD) by plasmapheresis was evaluated during a series of six plasmaphereses of a 13-year-old boy with Rasmussen encephalitis. Plasmapheresis was performed after steady-state concentrations of MHD had been achieved with a dose of 2550 mg oxcarbazepine daily. The mean amount of MHD removed per plasmapheresis was 78.9 mg (SD: 6.0 mg), representing 3% to 4% of the daily oxcarbazepine dose and approximately 5% to 6% of body stores of MHD. The mean steady-state trough MHD concentration was 33.3 mg/L (SD: 1.8 mg/L), and this was remarkably stable during the entire plasmapheresis period. The serum concentration of MHD was only mildly reduced by the procedure. The areas under the concentration curve of MHD on the first and sixth day of plasmapheresis were 99% and 94%, respectively, of the pre-plasmapheresis values. The results are in agreement with studies on other anticonvulsant medications (carbamazepine, valproic acid, phenobarbital, and phenytoin), indicating that minor fractions (2% to 10%) of body stores of these drugs are depleted during plasmapheresis. The authors conclude that it is unnecessary to adjust the oxcarbazepine dosage when performing single-volume plasma exchanges or even multiple exchanges during an extended period. It is further proposed that plasmapheresis is unlikely to be of therapeutic benefit in the treatment of an oxcarbazepine overdose.

A new quantitative RT-PCR assay for thymidylate synthase mRNA in blood leukocytes applied to cancer patients and healthy controls.

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). TS mRNA and protein levels in colorectal tumours are among the most important determinants for tumour response to 5-FU. TS mRNA levels in blood leukocytes may give information on pharmacokinetic and pharmacodynamic actions of 5-FU on TS as it has previously been shown that inhibition of TS levels by 5-FU in bone marrow leukocytes resembles the degree of TS inhibition in colorectal tumours. The aim of this study was to develop a quantitative high-throughput RT-PCR assay for TS mRNA expression in blood leukocytes (CURT-PCR). Furthermore the TS mRNA levels in blood of patients with colorectal cancer and healthy controls was compared. TS mRNA levels in 17 patients with colorectal cancer did not differ from 20 matched controls whereas a group of 14 younger controls had significantly lower TS mRNA expression than patients and matched controls. In order to investigate the sensitivity of the assay towards cellular reactions such as proliferative stimuli, isolated blood leukocytes were stimulated with phytohemagglutinin both in mitogenic and non-mitogenic concentrations and an induction of TS mRNA expression was measured in both cases. TS activity and cellular proliferation also increased but only at mitogenic concentrations, suggesting that TS mRNA expression is an early leukocyte activation marker. This new CURT-PCR assay may allow improved studies of functional kinetics of drugs with impact upon TS. Further studies are required to establish the possible clinical benefit of TS mRNA measurements in blood leukocytes.

Changes in thymidylate synthase mRNA in blood leukocytes from patients with colorectal cancer after bolus administration of 5-fluorouracil.

5-fluorouracil (5-FU) is considered the standard antineoplastic drug of choice for metastatic colorectal cancer. It has been suggested that 5-FU administered as bolus infusion is cytotoxic mainly through an RNA damaging effect. We investigated the effect of i.v. bolus 5-FU 500-600 mg/m2 on the 5-FU target enzyme, thymidylate synthase (TS) mRNA, in blood leukocytes before and after courses 1 and 3 in 21 patients with colorectal cancer. TS mRNA expression was quantified using an RT-PCR assay with an internal RNA standard. Median TS mRNA expression decreased significantly 30 min after course no. 1 (p = 0.004), and both 15 min and 30 min after course 3 (p = 0.01). After course 1, the median TS mRNA expression decreased by 31% and after course 3 by 24%. Pharmacokinetic parameters were similar for individual patients during the two courses but did not correlate with the degree of TS mRNA inhibition. The present results indicate that TS mRNA in blood leukocytes may be an early indicator of an RNA damaging effect after i.v. bolus infusion of 5-FU.

The effects of low-dose methotrexate on thymidylate synthetase activity in human peripheral blood mononuclear cells.

OBJECTIVE: Methotrexate (MTX) in low doses is widely used in the treatment of rheumatoid arthritis (RA) and it is not known whether its effects are due to immunosuppressive and/or anti-inflammatory actions. High concentrations of MTX inhibit the activity of thymidylate synthetase (TS) and dihydrofolate reductase essential for DNA synthesis. This study investigated the effects of low-dose MTX on TS activity and proliferation in human peripheral blood mononuclear cells (PBMC). METHODS: The MTX concentrations in our experiments were chosen according to the plasma concentrations measured in 8 RA patients treated with MTX. The effect of MTX on TS activity and DNA synthesis were measured in stimulated normal PBMC and in PBMC obtained from 6 RA patients treated with oral MTX before and 2 hours after intake of their weekly MTX dose. The effect of MTX on the TS mRNA concentration was also investigated in order to elucidate its effect on TS production. RESULTS: Low-dose MTX significantly inhibited TS activity and the proliferation of stimulated PBMC independent of the mode of activation. Interestingly, the concentration of TS mRNA in normal PBMC was upregulated by the presence of MTX. Finally, there was no difference between TS activity measured before and after MTX intake in 6 RA patients on long-term MTX treatment. CONCLUSION: We show that low concentrations of MTX inhibit TS activity in vitro. An in vivo effect cannot, however, be proven given our study design. The role of these in vitro findings is discussed, particularly in relation to the in vivo effects of MTX.

The ability of hypoxia to modify the gene expression of thymidylate synthase in tumour cells in vivo.

PURPOSE: Hypoxic cells in tumours are resistant to 5-fluorouracil (5-FU). This in vivo study investigated the ability of hypoxia to regulate the gene expression of thymidylate synthase (TS), the target enzyme of 5-FU. MATERIALS AND METHODS: C3H mammary carcinomas, grown in the feet of female CDF1 mice, were used for all experiments. Mice were placed in a 10% oxygen environment for various time periods and the tumour oxygen status was determined with an Eppendorf oxygen electrode. The animals were then injected with BrdU (100 mg/kg, i.p.). Tumours were excised and immediately frozen (-80 degrees C) until isolation of total RNA. The mRNA was reversibly transcribed to complementary DNA and the resulting cDNA amplified in a multiplex PCR reaction, with beta-actin as the internal reference gene. RESULTS: One hour of low oxygen breathing made tumours significantly more hypoxic. This increase was maintained for a maximum incubation period of 48 h. In the same tumours, no change in TS gene expression was seen with up to 3 h of low oxygen breathing. At longer times it decreased, reaching significance at 12-24 h and remaining at this lower level for up to 48 h. BrdU labelling was significantly reduced after breathing low O2 for 24 h (p = 0.001). CONCLUSION: Hypoxia-induced down-regulation of TS gene expression was observed. This would be expected to make hypoxic tumour cells more sensitive to 5-FU. Other mechanisms must be responsible for the previously reported resistance to this drug.

HCO3-dependent pHi regulation in tracheal epithelial cells.

Regulation of intracellular pH (pHi) was studied in cultured bovine tracheal epithelial cells using microspectrofluorimetry of the fluorescent indicator 2',7'-biscarboxyethyl- 5(6)-carboxyfluorescein (BCECF). The cells, which were grown on coverslips and superfused in a chamber on the stage of a microscope, were acidified by NH4Cl-prepulses, and pHi recovery was measured (in DeltapH/min) at approximately pHi 6.7. In HCO3-free solutions the recovery rate was 0.14 pH/min, and addition of amiloride or Na-free solution reduced this rate to 0.02-0.03 pH/min. In HCO3/CO2-buffered Ringer's, the rate of recovery was 0.32 pH/min, and amiloride or Na-free reduced the rate to 0.08-0.10 pH/min. This residual Na-independent and HCO3-dependent pHi recovery was studied by using inhibitors of HCO3 and H transporters. Bafilomycin (inhibits H-ATPases) at 100 nM did not significantly affect pHi recovery, while 100 microM SCH28080 (inhibits H,K-ATPase) had a variable inhibitory effect (25-75%), indicating that a gastric-like H, K-ATPase, but not electrogenic H pump, may contribute in a minor way to the recovery from acidification. Cl-free solution and 500 microM H2DIDS (dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, blocks anion exchange and the outwardly rectifying Cl channel, ORCC), both blocked apparent anion exchange activity, but had no effect on the recovery; 100 microM DNDS (4-4''-dinitro-2-2'-stilbenedisulfonate blocks the ORCC but not the cystic fibrosis transmembrane conductance regulator, CFTR) had no effect on pHi recovery; DPC (diphenylamine carboxylate, blocks the CFTR and the ORCC) caused a complete and reversible inhibition of the recovery. When [K] was increased ten fold to depolarize the cell's membrane potential, the magnitude of the pHi recovery (though not the rate) was enhanced. Thus, the HCO3-dependent, Na- and Cl-independent, DPC-blockable pHi recovery may be largely due to an influx of HCO3 via CFTR Cl channels. Under physiological conditions, when the electrochemical gradient for HCO3 is likely to be outwardly rather than inwardly directed, the CFTR (or another HCO3-permeable channel) may mediate HCO3 secretion and contribute to regulation of pH of the periciliary fluid.

Evaluation of clinical assays for measuring high-dose methotrexate in plasma.

Four routine assays commonly used for monitoring plasma methotrexate (MTX) during high-dose therapy were validated by HPLC as the comparison method. MTX and its main metabolite, 7-hydroxymethotrexate (7-OHMTX), were analyzed by HPLC with postcolumn derivatization and fluorometric detection. About 200 clinical plasma samples from 13 children with acute lymphoblastic leukemia who received 5-8 g/m2 MTX as 24-h infusions were analyzed. The fraction of measured concentrations of MTX that were within 75-125% of the values obtained by HPLC were 64.5% for enzyme inhibition assay, 56.4% for fluorescence polarization immunoassay with polyclonal antibodies (FPIA1; Abbott), 58.9% for FPIA2 (with monoclonal antibodies; Abbott), and 46.4% for enzyme-multiplied immunoassay (Emit; Syva). All nonchromatographic procedures were subject to interferences from MTX plasma metabolites or endogenous substances. The interference from 7-OHMTX was, however, somewhat less pronounced for FPIA2 (monoclonal) than for FPIA1 (polyclonal).

Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays.

The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA inhibition studies, immunoblotting and HPLC analyses confirmed the presence of substantial amounts of KS, probably as a large proteoglycan (> 120 kDa). Commercial and heterogenic glycosaminoglycan preparations therefore must be used with great caution in immunological analyses. On the other hand the shark cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate correlated well (r = 0.90) with the concentrations obtained with a traditional KS-ELISA that uses purified aggrecan as standard and coating antigen, and KS in both serum and synovial fluid could be measured with sufficient linearity.

Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels.

A method for detection of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels was developed by a combination of (i) initial periodic acid oxidation/Alcian blue staining and (ii) subsequent staining with silver nitrate. The procedure allowed detection of as little as 1.6 ng of alpha 1-acid glycoprotein and 8-40 ng of a polydisperse mucin sample, which is at least 10 times more sensitive than previously published methods. The method should be very useful for assessment of sample purity and detection of glycoproteins in dilute mixed samples.

Quantitative estimation of the area of luminal and basolateral membranes of rat parotid acinar cells: some physiological applications.

Knowledge of luminal and basolateral acinar cell membrane areas of the secretory endpieces is a prerequisite for a detailed quantitative analysis of the ion transport involved in secretion of the primary saliva. In the present study, these areas were estimated in rat parotid acinar cells using standard stereological methods. A total of 480 micrographs--obtained by random sampling from eight glands from four rats--were analysed at a final magnification of 40000x. Expressed per unit cell volume, the area of the luminal acinar cell membrane was: 0.125 micron 2.micron-3 (SEM = 0.027 micron 2.micron-3, n = 4 animals) and the area of the basolateral membrane was: 1.54 microns 2.micron-3 (SEM = 0.085 micron 2.micron-3, n = 4 animals). These figures make it possible to perform a synthesis based upon different categories of experimental data, e.g. on ion fluxes, membrane potentials and single-channel conductances. Thus, we have estimated the density of open, low-conductance Cl- channels in the luminal membrane--which are not readily accessible for direct, patch-clamp analysis--to be approximately 18 channels per microns 2 in the stimulated state.

The effect of iron overload and iron reductive treatment on the serum concentration of carbohydrate-deficient transferrin.

The concentration of carbohydrate-deficient transferrin in serum (CDT) has been used as a reliable indicator of recent alcohol consumption. We have investigated the utility of this laboratory test in 20 patients with hereditary haemochromatosis (HH) by simultaneous evaluation of serum concentrations of liver transaminases, gamma-glutamyl transpeptidase, iron, transferrin and assessment of the liver iron concentration by magnetic resonance imaging. 11 patients were re-examined during iron depletion with phlebotomies. In all 11 patients intensive but not maintenance iron removal was associated with an increase in serum CDT, in three patients even to levels above the reference range. The mean serum CDT increased from 8.5 (SD 2.2) U/l to 16.6 (SD 7.2) U/l (P < 0.001). Iron mobilization from the liver was found particularly responsible for the increase in serum CDT. Independent of this finding we found a significant semi-logarithmic correlation (r = -0.77, P = 0.009) between the MRI determined liver iron concentration and serum CDT in the patients not on iron depletion. Our findings indicate that the utility of serum CDT as a measure of alcohol consumption in patients with HH may be compromised, especially during intensive iron depletion.

Effect of flunarizine and calcium on serotonin uptake in human and rat blood platelets and rat synaptosomes.

Calcium and the calcium overload blocker flunarizine exert profound effects on mood. We therefore studied the effect of calcium and flunarizine on serotonin uptake in human and rat blood platelets and in rat synaptosomes. Calcium (1.3 mmol/L) had a weak inhibiting effect on serotonin uptake in blood platelets, whereas no effect was observed in synaptosomes. Flunarizine inhibited serotonin uptake in a concentration dependent manner with an IC50 value of 1 mumol/L in blood platelets and 5 mumol/L in synaptosomes. The inhibition did not depend on the presence of extracellular calcium indicating that the effect is not coupled to a blockade of cellular calcium influx. In human blood platelets, the inhibition was of the noncompetitive type. These results indicate that flunarizine interacts directly with the 5-HT uptake site. The relatively high concentration of flunarizine required to inhibit 5-HT uptake may question the clinical importance of this effect.

Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel regulated by protein kinase A. The most common mutation in cystic fibrosis (CF), deletion of Phe-508 (delta F508-CFTR), reduces Cl- secretion, but the fatal consequences of CF have been difficult to rationalize solely in terms of this defect. The aim of this study was to determine the role of CFTR in HCO3- transport across cell membranes. HCO3- permeability was assessed from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein] and of channel activity (patch clamp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or delta F508-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4Cl) and incubated in Na(+)-free (N-methyl-D-glucamine substitution) solutions (to block Na(+)-dependent pHi regulatory mechanisms), pHi remained acidic (pH approximately 6.5) until the cells were treated with 20 microM forskolin (increases cellular [cAMP]); pHi then increased toward (but not completely to) control level (pHi 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in delta F508 and mock-transfected cells. This Na(+)-independent, forskolin-dependent pHi recovery was not observed in HCO3-/CO2-free medium. Forskolin-treated WT-CFTR-transfected (but not delta F508-CFTR or mock-transfected) cells in Cl(-)-containing, HCO3(-)-free solutions showed Cl- channels with a linear I/V relationship and a conductance of 10.4 +/- 0.5 pS in symmetrical 150 mM Cl-. When channels were incubated with different [Cl-] and [HCO3-] on the inside and outside, the Cl-/HCO3- permeability ratio (determined from reversal potentials of I/V curves) was 3.8 +/- 1.0 (mean +/- SEM; n = 9); the ratio of conductances was 3.9 +/- 0.5 (at 150 mM Cl- and 127 mM HCO3-. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMP-dependent influx of HCO3- through channels that conduct HCO3- about one-quarter as efficiently as it conducts Cl-. Under physiological conditions, the electrochemical gradients for both Cl- and HCO3- are directed outward, so CFTR likely contributes to the epithelial secretion of both ions. HCO3- secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO3- may lead to decreased pH of the luminal fluid.

Peptide-evoked release of amylase from isolated acini of the rat parotid gland.

Investigations of the effects of the neuropeptides, substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), gastrin releasing peptide (GRP), calcitonin gene related peptide (CGRP) and vasoactive intestinal peptide (VIP), and of acetylcholine on amylase secretion have been carried out on isolated acini of the rat parotid gland. Furthermore, the occurrence and location of the peptides in the gland was studied. Finally, binding of 125I-BH-SP to isolated acini were studied in order to characterize their tachykinin receptor(s) and their binding kinetics. Only SP, NKA, NPK and VIP stimulated amylase release. VIP, however, with a rather low potency (EC50 at 155 nmol/l). Simultaneous stimulation with two compounds elicited additive responses, except for VIP and acetylcholine which elicited an effect significantly above additive response. Only SP, NKA, VIP and CGRP could be identified in extracts of the gland. The immunoreactivity of these peptides could be located to varicose nerve fibers in the gland. Binding of labeled SP to the isolated acini exhibited the characteristics of a genuine agonist/receptor interaction, and the rank order of displacement potencies indicated the presence of NK1-receptors. Thus, the results of the present study support previous suggestions that the tachykinins and VIP are likely to be involved in amylase secretion in the rat parotid gland.

Fluctuation of serum zuclopenthixol concentrations in patients treated with zuclopenthixol decanoate in viscoleo.

Zuclopenthixol serum concentrations were measured in 58 psychiatric patients referred for routine therapeutic drug monitoring (TDM). Patients were treated for prolonged time with zuclopenthixol decanoate in viscoleo in doses of 50-500 mg, administered intramuscularly at 14-day intervals. The serum concentration was determined at days 7 (C7) and 14 (C14) following injection. The mean ratio C7/C14 was 2.0 and was independent of the dosage given. In 14 patients, additional blood samples were drawn at day 3 (C3) following injection. The mean ratio C3/C14 of this group was 3.2. An almost log-linear decline of the serum concentration from day 3 to 14 appeared, which corresponds to an apparent half-life of zuclopenthixol in this dosage form of 7.4 days. The marked fluctuations of serum concentrations of zuclopenthixol from peak to trough levels in patients given fortnightly injections of the depot preparation indicate that shorter intervals between injections should be considered in many cases in order to diminish side effects.