Population pharmacokinetics and pharmacogenetics of once daily tacrolimus formulation in stable liver transplant recipients.

PURPOSE: The once daily formulation of tacrolimus is an important immunosuppressive drug. Interpatient variability in metabolism has been related to genetic variation in CYP3A4 and CYP3A5. However, in liver transplantation, both donor and recipient genotypes may affect pharmacokinetics. The primary objective of this study was to investigate the effect of CYP3A4*22 and CYP3A5*3 of both donor and recipient on once daily tacrolimus pharmacokinetics. The secondary objective was to develop a limited sampling model able to accurately predict exposure. METHODS: Stable liver transplant patients receiving once daily tacrolimus (N = 66) were included. Population pharmacokinetic analysis was performed with patients of whom DNA was available (N = 49), and demographic factors, CYP3A4*22 and CYP3A5*3, were tested as covariates. Moreover, a limited sampling model was developed using data of 66 patients. RESULTS: Pharmacokinetics was best described by a two-compartment model with delayed absorption. CYP3A5*1 carrying recipients engrafted with a CYP3A5*1 carrying liver had an average 1.7-fold higher clearance compared to non-carriers. CYP3A5*1 carrying recipients engrafted with a CYP3A5*1 non-carrying liver or vice versa showed an average 1.3-fold higher clearance compared with non-carriers. CYP3A4*22 was not significantly associated with once daily tacrolimus pharmacokinetics. Using 0, 2, and 3 h postdose as limited sampling model resulted in significantly improved prediction of tacrolimus exposure compared with trough concentration. CONCLUSIONS: Both donor and recipient CYP3A5 genotype significantly influences tacrolimus once daily pharmacokinetics. In contrast, CYP3A4*22 appears not suitable as biomarker. The developed limited sampling model can be used to accurately estimate tacrolimus once daily exposure.

Concentration-controlled treatment of lupus nephritis with mycophenolate mofetil.

BACKGROUND: Mycophenolate mofetil (MMF) has recently been established as a potent drug in maintenance treatment for lupus nephritis. However, there is no consensus on the optimal dosing regimen because of a high inter-individual variability of mycophenolic acid (MPA), the active metabolite of MMF. This retrospective study aimed to investigate the effect of an individualized dosing regimen through concentration-controlled treatment on MPA exposure and renal outcome in patients with lupus nephritis. METHODS: Sixteen patients with lupus nephritis and treatment with low-dose intravenous cyclophosphamide followed by MMF were included. MPA area under the plasma concentration-time curve from 0 to 12 hours (MPA-AUC(0-12)) was assessed within a month after MMF initiation. After determination of MPA-AUC(0-12), MMF doses were titrated to achieve a target MPA-AUC(0-12) of 60-90 mg*h/l. After on average six months, MPA-AUC(0-12) measures were repeated to assess the effect of dose adjustment. RESULTS: One month after introducing MMF, MPA-AUC(0-12) was low and showed a high inter-individual variability. Dose adjustment with a target MPA-AUC(0-12) of 60-90 mg*h/l resulted in individualized MMF dosing, significantly higher MPA-AUC(0-12) levels, and a non-significant reduction in variability of MPA-AUC(0-12). Adverse effects were reported by 37.5% of patients, which resulted in a switch to azathioprine in two patients. There was no significant relationship between the occurrence of adverse effects and MPA-AUC(0-12). At 12 months of follow-up 87.5% of patients had achieved either partial (18.7%) or complete (68.8%) remission. CONCLUSION: Concentration-controlled dose adjustments with a target MPA-AUC(0-12) of 60-90 mg*h/l was associated with optimized MPA exposure and an excellent renal outcome at 12 months of follow-up in a small sample of SLE patients with lupus nephritis.

The EGF receptor is an actin-binding protein.

In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996.

Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor.

In this paper we demonstrate that cytoskeletons isolated from A431 cells have associated with them high activities of several kinases involved in inositol lipid metabolism, such as phosphatidylinositol kinase, phosphatidylinositol phosphate kinase, and diacylglycerol kinase. In addition also phospholipase C activity was detected on isolated cytoskeletons. Controlled extraction of the cytoskeletons followed by in vitro polymerization of actin demonstrated an association of the kinases to the actin filament system consisting of actin and a number of actin-binding proteins. The cytoskeleton-associated lipid kinase activities were significantly increased upon treatment of intact cells with EGF. These data suggest that the association of the phosphoinositide kinases, diacylglycerol kinase, phospholipase C, and also the EGF receptor to the cytoskeleton may play a role in the efficient signal transduction induced by EGF, by providing a matrix for the various components involved in signal transduction.

Once-daily intravenous busulfan with therapeutic drug monitoring compared to conventional oral busulfan improves survival and engraftment in children undergoing allogeneic stem cell transplantation.

Because of intra- and interindividual variability, bioavailability, and pharmacokinetics of busulfan (Bu) in children, oral busulfan without therapeutic drug monitoring (TDM) is assumed to be associated with higher graft failure rates as well as higher toxicity (eg, veno-occlusive disease [VOD]). This study compares the outcome of hematopoietic stem cell transplantation (HSCT) of 2 groups: 1) 30 patients who received myeloablation with once-daily intravenous (i.v.) dose-targeted busulfan (BUdtIV) based on TDM and 2) 30 patients who received the current practice of untargeted oral busulfan (BUPO). Patients received a 3-hour infusion of Bu at a first dose of 120 mg/m(2) (age >or=1 year) or 80 mg/m(2) (<1 year), or BUPO 1 mg/kg 4 times daily. Both regimens were continued for 4 days. The target area under the curve (AUC) was defined as 17,500 microg *h/l. BUdtIV resulted in higher event-free survival (EFS) and survival rates compared to BUPO (EFS: 30% versus 83%, P < .001, survival: 53% versus 83%, P = .016). BUdtIV was associated with more cases of VOD. TDM was feasible in routine clinical practice. The results show that i.v. Bu using TDM is preferable over oral Bu in children undergoing allogeneic stem cell transplantation, especially in those at high risk for graft failure/relapse.

Ciclosporin kinetics in children after stem cell transplantation.

AIMS: To develop a limited sampling strategy to determine ciclosporin systemic exposure [area-under-the-curve(AUC)]. This is meant to be the first step in a future study of the relationship between AUC and the biological effects of ciclosporin. METHODS: The pharmacokinetics of ciclosporin was investigated prospectively following stem cell transplantation (SCT) in 17 children, aged 1.8-16.1 years. Ciclosporin was given twice daily, intravenously over a short infusion of 2 h duration during the early post-SCT period, or orally later on, when oral medication was well tolerated. Parameter estimation was performed using nonlinear mixed effect modelling as implemented in the NONMEM program. Individual empirical Bayes estimates of clearance and distribution volume were correlated with the demographic variables. RESULTS: Pharmacokinetics was described adequately with a two-compartment model with lag time (population estimates: CL = 11.3 l h(-1); V(c) = 16.5 l; V(p) = 59.9 l; t(1/2) absorption = 0.78 h, t(lag) = 0.6 h). The AUCs, determined for the combination of trough level with one time point between 2 and 3 h after dosing, correlated very well with the reference AUC (r(2) = 0.97). No correlation was found between clearance and distribution volume, and the demographic patient variables length, body weight, age and glomerular filtration rate. CONCLUSION: A two-point limited sampling strategy, in combination with a Bayesian fitting procedure using the pharmacokinetic population model described, can adequately determine the AUC of ciclosporin. Since no correlation between clearance and body weight was found, dosing ciclosporin per kg bodyweight is not supported by the results of this study. We suggest starting with a fixed dose, followed by AUC determination and dose adjustment.

Effect of intrapulmonary tetrahydrocannabinol administration in humans.

This randomised, double-blind, placebo-controlled, cross-over study was designed to identify which pharmacodynamic parameters most accurately quantify the effects of delta-9-Tetrahydrocannabinol (THC), the predominantly psychoactive component of cannabis. In addition, we investigated the acceptability and usefulness of a novel mode of intrapulmonary THC administration using a Volcano vaporizer and pure THC instead of cannabis. Rising doses of THC (2, 4, 6 and 8 mg) or vehicle were administered with 90 minutes intervals to twelve healthy males using a Volcano vaporizer. Very low between-subject variability was observed in THC plasma concentrations, characterising the Volcano vaporizer as a suitable method for the administration of THC. Heart rate showed a sharp increase and rapid decline after each THC administration (8 mg: 19.4 bpm: 95% CI 13.2, 25.5). By contrast, dose dependent effects of body sway (8 mg: 108.5%: 95% CI 72.2%, 152.4%) and different subjective parameters did not return to baseline between doses (Visual Analogue Scales of 'alertness' (8 mg: -33.6 mm: 95% CI -41.6, -25.7), 'feeling high' (8 mg: 1.09 U: 95% CI 0.85, 1.33), 'external perception' (8 mg: 0.62 U: 95% CI 0.37, 0.86)). PK/PD-modeling of heart rate displayed a relatively short equilibration half-life of 7.68 min. CNS parameters showed equilibration half-lives ranging between 39.4 - 84.2 min. Some EEG-frequency bands, and pupil size showed small changes following the highest dose of THC. No changes were seen in saccadic eye movements, smooth pursuit and adaptive tracking performance. These results may be applicable in the development of novel cannabinoid agonists and antagonists, and in studies of the pharmacology and physiology of cannabinoid systems in humans.

Rifampin levels in daily practice: the accuracy of a single measurement.

BACKGROUND: Measurement of rifampin levels is not part of routine practice. However, low levels are associated with failure of tuberculosis treatment. The clinical relevance of serum levels in daily practice is unclear. The objective was to evaluate rifampin serum concentrations and factors associated with insufficient concentrations. METHODS: Patients with at least one rifampin concentration drawn 3 hours after intake (C3) between 2005 and 2014 were included. Data on demographic and clinical characteristics were collected, including side effects and dose adjustments. Two different criteria were used to define adequate concentrations (criterion 1: C3 a nd C 6 ≥ 3 mg/l; criterion 2: C3 or C6 ≥ 5 mg/l). RESULTS: Of 63 patients, 66% and 76% had a sufficient level according to criterion 1 or 2, respectively. C3 exceeded C6 in most patients, while a late maximum was significantly associated with diabetes mellitus (p = 0.003). A dose adjustment was made in 19% of cases, more frequently in patients with insufficient levels (p = 0.02) or with ≥ 2 side effects (p = 0.03). CONCLUSION: Rifampin levels varied but were mostly adequate and a single measurement at 3 hours after intake provided the required information in most cases, indicating that full AUC0-24 measurements could be limited to specific situations.

Individualized population pharmacokinetic model with limited sampling for cyclosporine monitoring after liver transplantation in clinical practice.

BACKGROUND: We recently developed and validated limited sampling models (LSMs) for cyclosporine monitoring after orthotopic liver transplantation based on individualized population pharmacokinetic models with Bayesian modelling. Aim To evaluate LSM in practice, and to seek optimal balance between benefit and discomfort. METHODS: In 30 stable patients, more than 6 months after orthotopic liver transplantation, previously switched from trough- to 2 h post-dose (C2)-monitoring, we switched to 3-monthly LSM 0,1,2,3 h-monitoring. During 18 months we evaluated dose, creatinine clearance, calculated area under the curve, intra-patient pharmacokinetic variability and ability to assess systemic exposure by several previously validated LSMs. RESULTS: Within patients, there was variability of cyclosporine-area under the curve with the same dose (CV of 15%). Compared to C2-monitoring, there was no significant difference in dose (P = 0.237), creatinine clearance (P = 0.071) and number of rejections. Some models showed excellent correlation and precision with LSM 0,1,2,3 h comparing area under the curves (0,2 h: r(2) = 0.88; 0,1,3 h: r(2) = 0.91; 0,2,3 h: r(2) = 0.92, all P < 0.001) with no difference in advised dose. CONCLUSIONS: The limited sampling model, with only trough- and 2-h sampling, yields excellent accuracy and assesses systemic exposure much better than C2 with less bias and greater precision. Considering the calculated intra-patient variability, more precision is redundant, so LSM 0,2 h seems the optimal way of cyclosporine-monitoring.

[Treatment of falciparum malaria with artemether-lumefantrine according to a 5-day schedule: results of a study in 21 patients and recommendations]. / Behandeling van malaria tropica met artemether-lumefantrine volgens een 5-daags schema: resultaten van onderzoek bij 21 patiënten en aanbevelingen.

OBJECTIVE: To describe the outcome of 21 successive patients with falciparum malaria treated with artemether-lumefantrine in 6 oral doses of 80-480 mg in 96 hrs (5-day schedule). DESIGN: Retrospective cohort study. METHOD: The data on all successive patients with falciparum malaria that were treated with artemether-lumefantrine in the Leiden University Medical Centre (n = 15) and the Bronovo Hospital (n = 6), the Netherlands, during the period from August 2003 to February 2006 were evaluated. RESULTS: Ten patients with uncomplicated falciparum malaria were treated exclusively with artemether-lumefantrine; time to parasitological clearance was less than 48 hrs in 5 out of 6 tested patients. The mean admission time was 1.3 days (95% CI: 0.7-1.9). 6 patients with non-severe falciparum malaria were initially treated with intravenous quinine because of vomiting; artemether-lumefantrine was started after an average of 0.9 days. Parasite clearance required more than 48 hrs in all patients. The mean admission time was 3.8 days (95% CI: 2.7-4.9). 5 patients had severe falciparum malaria. They received artemether-lumefantrine 4-24 hrs after starting quinine therapy. The parasite count was < 0.1% after an average of 1.5 days (95% CI: 0.3-2.7). The mean admission time was 4.2 days (95% CI: 2.2-5.2). The serum level of lumefantrine was > or = 500 microg/l (target value) in 9 out of 10 tested patients. CONCLUSION: Treatment of falciparum malaria with artemether-lumefantrine according to a 5-day schedule resulted in a swift recovery and short admissions. No relapses were reported.

Pharmacokinetics of mitomycin C in humans.

This paper describes an investigation into the pharmacokinetic behavior of mitomycin C (MMC) in 36 patients receiving either single-agent chemotherapy (10 to 20 mg/sq m), or a combination regimen including MMC (5 to 10 mg/sq m). A high-performance liquid chromatographic assay of MMC was applied for the analysis of plasma, urine, bile, and ascites fluid samples. The detection limit is 1 ng/ml sample. Most patients were given short-term i.v. infusion, although other methods of administration were used as well. Most plasma concentration-time curves fit a two-compartment model. Pharmacokinetic parameters revealed large interindividual variations. Median terminal half-lives in single-agent chemotherapy and combination chemotherapy were 50 and 42 min, respectively. The apparent volume of the central compartment was 7 liters/sq m in both groups. The volume of distribution was 22 liters/sq m in single-agent chemotherapy, and 25 litres/sq m in combination chemotherapy. Linear regression analysis of the area under the plasma concentration-time curve versus the dose did not produce any evidence that the pharmacokinetics was dose dependent. However, differences were observed between patients receiving MMC alone and those on combination chemotherapy, in particular with regard to the total body clearance: 18 liters/hr/sq m for single-agent chemotherapy and 28 liters/hr/sq m for combination chemotherapy. Urinary recovery was limited to a maximum of 15% of the administered dose. In one patient studied, MMC was found to be present in the bile. There is evidence for enterohepatic circulation of MMC, and MMC was also found to penetrate into the ascites fluid.

Everolimus pharmacokinetics and its exposure-toxicity relationship in patients with thyroid cancer.

BACKGROUND: Everolimus is a mTOR inhibitor used for the treatment of different solid malignancies. Many patients treated with the registered fixed 10 mg dose once daily are in need of dose interruptions, reductions or treatment discontinuation due to severe adverse events. This study determined the correlation between systemic everolimus exposure and toxicity. Additionally, the effect of different covariates on everolimus pharmacokinetics (PK) was explored. METHODS: Forty-two patients with advanced thyroid carcinoma were treated with 10 mg everolimus once daily. Serial pharmacokinetic sampling was performed on days 1 and 15. Subsequently, a population PK model was developed using NONMEM to estimate individual PK values used for analysis of an exposure-toxicity relationship. Furthermore, this model was used to investigate the influence of patient characteristics and genetic polymorphisms in genes coding for enzymes relevant in everolimus PK. RESULTS: Patients who required a dose reduction (n = 18) due to toxicity at any time during treatment had significant higher everolimus exposures [mean AUC0-24 (SD) 600 (274) vs. 395 (129) µg h/L, P = 0.008] than patients without a dose reduction (n = 22). A significant association between everolimus exposure and stomatitis was found in the four-level ordered logistic regression analysis (P = 0.047). The presence of at least one TTT haplotype in the ABCB1 gene was associated with a 21 % decrease in everolimus exposure. CONCLUSION: The current study showed that dose reductions and everolimus-induced stomatitis were strongly associated with systemic everolimus drug exposure in patients with cancer. Our findings confirm observations from another study in patients with cancer and show us that everolimus is a good candidate for individualized dosing in patients with cancer. CLINICALTRIAL. GOV NUMBER: NCT01118065.

Cholesterol and phosphoinositides increase affinity of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGF-R) has been purified from human epidermoid carcinoma A431 cells by affinity chromatography in a single step using a monoclonal antibody (528) which competes with EGF for receptor binding. The purified EGF-R exhibits EGF inducible tyrosine kinase and autophosphorylation activity. Steady-state binding of EGF to the purified receptor revealed the presence of one class of binding sites exhibiting an apparent dissociation constant (Kd) of approx. 2 nM. When Angiotensin II was used as a receptor tyrosine kinase substrate the specific activity of the EGF induced kinase was 87 nmol/min per mg and the Km of the reaction was about 2 mM. Reconstitution of the EGF receptors into lipid vesicles was achieved by octylglucoside dialysis. Reconstitution of the receptor into pure dioleoylphosphatidylcholine (DOPC) vesicles had no effect on the EGF-binding properties in comparison to receptors in Triton X-100 micelles. Binding of EGF to the reconstituted receptor with ATP and Angiotensin II incorporated into the vesicles resulted in a five fold stimulation of the receptor kinase activity. The introduction of cholesterol, ranging from 10% to 50% (w/w), into DOPC vesicles resulted in an increase of the affinity of the receptor for its ligand. The Kd for EGF decreased from 1.8 nM in pure DOPC vesicles to 0.3 mM in DOPC/cholesterol (1:1 (w/w)) vesicles. With the introduction of small amounts (2% (w/w)) of phosphatidylinositol lipids into DOPC vesicles the Kd changed from 1.8 nM to 0.2 nM with phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-biphosphate (PtdIns4,5-P2) and to 0.1 nM in the case of phosphatidylinositol 4 phosphate (PtdIns4-P). No change in affinity was found when equal amounts of phosphatidylserine (PS) or phosphatidic acid (PA) were used.

Easy-to-use, accurate and flexible individualized Bayesian limited sampling method without fixed time points for ciclosporin monitoring after liver transplantation.

BACKGROUND: New methods to estimate the systemic exposure to ciclosporin such as the level 2 h after dosing and limited sampling formulas may lead to improved clinical outcome after orthotopic liver transplantation. However, most strategies are characterized by rigid sampling times. AIM: To develop and validate a flexible individualized population-pharmacokinetic model for ciclosporin monitoring in orthotopic liver transplantation. METHODS: A total of 62 curves obtained from 31 patients at least 0.5 year after orthotopic liver transplantation were divided into two equal groups. From 31 curves, relatively simple limited sampling formulas were derived using multiple regression analysis, while using pharmacokinetic software a two-compartment population-pharmacokinetic model was derived from these same data. We then tested the ability to estimate the AUC by the limited sampling formulas and a different approach using several limited sampling strategies on the other 31 curves. The new approach consists of individualizing the mean a priori population-pharmacokinetic parameters of the two-compartment population-pharmacokinetic model by means of maximum a posteriori Bayesian fitting with individual data leading to an individualized population-pharmacokinetic limited sampling model. From the individualized pharmacokinetic parameters, AUC(0-12h) was calculated for each combination of measured blood concentrations. The calculated AUC(0-12h) both from the limited-sampling formulas and the limited-sampling model were compared with the gold standard AUC(0-12h) (trapezoidal rule) by Pearson's correlation coefficient and prediction precision and bias were calculated. RESULTS: The AUC(0-12h) value calculated by individualizing the population-pharmacokinetic model using several combinations of measured blood concentrations: 0 + 2 h (r(2) = 0.94), 0 + 1 + 2 h (r(2) = 0.94), 0 + 1 + 3 h (r(2) = 0.92), 0 + 2 + 3 h (r(2) = 0.92) and 0 + 1 + 2 + 3 h (r(2) = 0.96) had excellent correlation with AUC(0-12h), better than limited sampling formulas with less than three sampling time points. Even trough level with limited sampling method (r(2) = 0.86) correlated better than the level after 2 h of dosing (r(2) = 0.75) or trough level (r(2) = 0.64) as single values without limited sampling method. Moreover, the individualized population-pharmacokinetic model had a low prediction bias and excellent precision. CONCLUSION: Multiple rigid sampling time points limit the use of limited sampling formulas. The major advantage of the Bayesian estimation approach presented here, is that blood sampling time points are not fixed, as long as sampling time is known. The predictive performance of this new approach is superior to trough level and that after 2 h of dosing and at least as good as limited sampling formulas. It is of clear advantage in busy out-patient clinics.

Intravenous busulfan in children prior to stem cell transplantation: study of pharmacokinetics in association with early clinical outcome and toxicity.

We studied the pharmacokinetics of intravenous busulfan (Bu) in children in order to further optimize intravenous Bu dosing in relation to toxicity and survival. A total of 31 children undergoing Bu-based conditioning for allogeneic SCT were enrolled in a study. The starting dose was 1.0 mg/kg (age < 4 years) and 0.8 mg/kg (age > or =4 years), four doses per day during 4 days. Dose adjustment was allowed up to a maximum dose of 1.0 mg/kg per dose if the target area under the serum concentration-time curve (AUC) was not reached. Pharmacokinetic studies were performed after the first dose. Donor engraftment was established in 28 out of 31 patients. The average AUC after the first dose was the same in children < 4 years as in children > or =4 years. Mean clearance was higher in children < 4 years than in children > or =4 years. In 35% of all patients, total AUC was within the target AUC. The other children's AUCs were below the target range. No relationships were found between systemic exposure to Bu and toxicity or clinical outcome. We concluded that, in accordance with previous data, within the observed AUCs no clear relationship was observed between Bu AUC and outcome with respect to toxicity, engraftment and relapse.

Population Pharmacokinetic Model Characterizing 24-Hour Variation in the Pharmacokinetics of Oral and Intravenous Midazolam in Healthy Volunteers.

Daily rhythms in physiology may affect the pharmacokinetics of a drug. The aim of this study was to evaluate 24-hour variation in the pharmacokinetics of the CYP3A substrate midazolam. Oral (2 mg) and intravenous (1 mg) midazolam was administered at six timepoints throughout the 24-hour period in 12 healthy volunteers. Oral bioavailability (population mean value [RSE%] of 0.28 (7.1%)) showed 24-hour variation that was best parameterized as a cosine function with an amplitude of 0.04 (17.3%) and a peak at 12:14 in the afternoon. The absorption rate constant was 1.41 (4.7%) times increased after drug administration at 14:00. Clearance (0.38 L/min (4.8%)) showed a minor 24-hour variation with an amplitude of 0.03 (14.8%) L/min and a peak at 18:50. Simulations show that dosing time minimally affects the concentration time profiles after intravenous administration, while concentrations are higher during the day compared to the night after oral dosing, reflecting considerable variation in intestinal processes.

The effects of nitrogen-containing bisphosphonates on human epithelial (Caco-2) cells, an in vitro model for intestinal epithelium.

Nitrogen-containing bisphosphonates (N-PCP) are bisphosphonates with an increased antiresorptive potency. Aminobisphosphonates, N-PCPs with an amino group, can cause nonspecific gastrointestinal complaints. It is not known whether these side effects are specific for these bisphosphonates or for the whole class of N-PCPs. In this study, we investigated the effects of two aminobisphosphonates (pamidronate and alendronate) and a structurally similar N-PCP (olpadronate) and their three respective calcium complexes on the viability and the intracellular calcium concentration ([Ca2+]i) of cultured Caco-2 cells a model for intestinal epithelium. These cells were also examined for apoptosis or necrosis. In the presence of calcium, pamidronate and alendronate were toxic to the cells, with pamidronate being more toxic than alendronate. Olpadronate induced toxicity only at concentrations more than ten times higher than the toxic concentrations of pamidronate. In the absence of calcium definite signs of toxicity were observed only with pamidronate at clinically relevant concentrations. The complexes of pamidronate and alendronate with calcium were considerably less soluble than the olpadronate calcium complex. There were no signs of apoptosis. [Ca2+]i was transiently raised after treatment with the N-PCPs. Doses at which responses were seen were, respectively, 0.02 mM (pamidronate), 0.3 mM (alendronate), and 2 mM (olpadronate). The peak of response was slightly greater after pamidronate treatment than after alendronate or olpadronate, respectively. In conclusion pamidronate, either as an ion or as a calcium complex, is the most toxic of the bisphosphonates tested for Caco-2 cells. Alendronate was less toxic while olpadronate was the least toxic in presence of calcium. The solubility of the bisphosphonate complexes with calcium may account for these differences in toxicity.

Tinnitus suppression by intravenous lidocaine in relation to its plasma concentration.

In a double-blind controlled trial in nine patients with tinnitus we measured the lidocaine plasma concentrations during and after intravenous administration of lidocaine or placebo and scored the level of tinnitus on a visual analog scale. No patient showed any effect during the placebo infusion. Administration of lidocaine resulted in total suppression or suppression to a non-annoying level of tinnitus in five patients, slight suppression but still annoying tinnitus in two patients, and worsening tinnitus in one patient. No effect of lidocaine was observed in one patient. Most relief was obtained at plasma concentrations between 1.5 and 2.5 micrograms/ml. In this concentration range a significant (p < 0.05) effect of lidocaine on tinnitus was observed. However, notable side effects were observed at plasma concentrations greater than 2.0 micrograms/ml. The effect persisted until plasma levels of about 0.5 microgram/ml were reached. A large variability in the effects existed because of variations in lidocaine kinetics and because of the presumed psychologic components of tinnitus.