Cyclosporine A area-under-time-concentration-curve in rheumatologic patients after the first dose. Which of the sparse sampling strategies will predict the best?

OBJECTIVE: The aim of the present study was to validate the limited sampling strategies (LSS:s) for prediction of AUC of cyclosporine A (CsA) after the first dose in rheumatologic patients. METHODS: 22 patients suffering from rheumathoid arthritis, systemic lupus erythematodus, ankylosing spondylitis dermato(poly)myositis or seronegative spondylarthritis were treated with Neoral® (female/male: 11/3, mean ± SD: age 49 ± 14 y, body weight 75 ± 12 kg, height 166 ± 7 cm, dose 71 ± 25 mg, dose per kg 1.0 ± 0.3 mg/kg), or Consupren® (7/1, 78 ± 36, 175 ± 8, 82 ± 22, 1.1 ± 0.3). Two patients whose C12h were missing were excluded from the AUC0-12 calculation. Whole blood levels of CsA were analyzed with HPLC. Blood samples were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after taking the first dose. Altogether 115 LSS:s obtained from the literature were validated. A linear trapezoidal rule was used as a reference method. Mean percentage prediction error (%PE) < ± 15% and maximal one value of absolute %PE > 30% were considered to be acceptable. The root mean squared error (RMSE) was evaluated for equations that passed the criteria. RESULTS: The best performance with all values of the absolute %PE < 30% was found in three LSS:s for AUC0-12 and two for AUC0-8: AUC0-12 = 123.792 + 1.165 × C1h + 3.021 × C3h + 7.33 × C8h; 97.6 + 1.27 × C1h + 3.14 × C3h + 4.06 × C6h; or 124.3 + 1.34 × C1h - 0.16 × C2h + 3.27 × C3h + 3.96 × C6h; AUC0-8 = -19.8 + 1.99 × C2h + 2.38 × C4h + 3.15 × C6h or -22.4 + 2.51 × C2h + 5.49 × C6h. Validation criteria were further fulfilled in AUC0-12 = 24 + 3.66 × C0h + 2.11 × C1.5h + 4.54 × C4h or 0.2 + 2 × C2h + 10.2 × C6h; AUC0-8 = 55.37 + 2.89 × C0h + 1.08 × C1 + 0.9 × C2h + 2.23 × C3h; and AUC0-4 = -41 + 1.17 × C1h + 1.85 × C2h. Only one equation proposed for AUC0-6 did not pass the validation criteria. CONCLUSIONS: Equations validated for prediction of AUC0-12, AUC0-8 and AUC0-4 might be used for LSS:s of CsA independently of the length of treatment, indication, dosage or galenic formulation.

The possibility of using the specific RIA method for the area-under-time-concentration curve sparse sampling calculation of cyclosporin A despite a large post-dose overestimation.

OBJECTIVE: To find limited sampling strategies (LSS) for prediction of the real AUC using the RIA analytical method. METHOD: Blood samples of 40 male renal transplant patients taken pre-dose and after 0.5, 1, 1.5, 2, 3, 5, 8, and 12 h in the steady-state were analyzed with HPLC and the specific RIA method. I. Eight equations for AUC0-12 and one for AUC0-8 obtained from the literature, that produced the mean percentage prediction error (%PE) < ± 15% and absolute %PE < 30% in 95% of predictions, were analyzed for possibility to predict the real AUC of CsA. II. Multiple regression analysis (MRA) was provided for the AUC equation proposal. Patients were divided into two groups according to the AUC0-12. Group I was used for LSS : s proposals while Group II for validation. The bias and precision were expressed as %PE, r2 and RMSE. The relationship of %PE interassay and with LSS:s was expressed as Pearson correlation r. GraphPad InStatt Software was used for MRA and Pearson r calculation. RESULTS: None of the equations described in the literature predicts AUC of CsA proprietarily. Seven equations for AUC0-12 and five for AUC0-8 were proposed with MRA for prediction of real AUC from RIA values. CONCLUSIONS: LSS:s can moderate the interassay %PE in AUC of CsA. New patients should be tested with both RIA and HPLC for the level of overestimation. The conversion factors should be calculated for patients with an overestimation higher than 90%. Our equation 251.09 + 0.5195 × C1h + 4.926 × C3h or 196.13 + 4.526 Â× C0h + 2.089 × C1.5h for AUC0-12, and 171.80 + 0.4759 × C1h + 4.132 × C3h for AUC0-8 may be used in patients with medium or low RIA and HPLC differences. Repeated analysis with HPLC is thus suggested in cases with AUC:s results close to the lower or upper margin of the therapeutic window.

Quantiferon-CMV test in prediction of cytomegalovirus infection after kidney transplantation.

Infection with cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunosuppressed patients, including organ and bone marrow transplant recipients. The majority of CMV disease is caused by reactivation of alatent infection rather that by newly acquired virus. Many techniques have been currently available to aid in the diagnostics of CMV disease. In this report we performed a prospective evaluation of Quantiferon-CMV assay (Cellestis) to determine whether the test is predictive of CMV disease. CD8+ T-cell CMV-specific immunity was assessed in a longitudinal cohort of 14 kidney transplant recipients. According to our data, subjects with higher cellular immune response measured with Quantiferon test had a lower risk of manifestation of CMV infection than subjects with lower responses. Despite the small number of patients and large intra- and interindividual variability of the data in the study, we observed the Quantiferon-CMV assay to be a sensitive specific test to detect a virus-specific T-cell response. We propose that this assay in combination with viral DNA load estimates may prove to be useful to stratify patients at risk of CMV disease.

Significant discrepancy in cyclosporin A post-dose concentrations when analyzed with specific RIA and HPLC method.

C(2) or AUC sparse sampling methods are widely recommended for therapeutic monitoring of cyclosporin A (CsA). One additional reason for promoting the C(2) sampling time in place of commonly used C(0) is that the C(2) level may actually provide more accurate measurement of parent drug concentration by immunoassays, as lower portion of metabolites has been formed 2 hours post-dose than at the steady-state trough time point. HPLC and RIA whole blood levels of CsA and its main metabolites AM1, AM9 and AM4N were compared during 12 hours profile after chronic administration. 40 stable renal transplant male patients (age 49 +/- 6 years, body weight 76 +/- 7 kg) were treated with CsA (Sandimmun Neoral, Novartis s.r.o, Prague, Czech Republic) in doses 198 +/- 56 mg twice daily. Samples were collected in steady state (after 2 weeks of regular treatment regimen) as follows: pre-dose, 0.5, 1, 1.5, 2, 3, 5, 8 and 12 hours after dose. CsA concentrations were determined both specific RIA assay (Cyclo-Trac SP Whole, Dia Sorin) and HPLC method, where concentrations of metabolites AM1, AM9 and AM4N were simultaneously analyzed. The AUC(0-12) was calculated by the linear trapezoidal rule. The percentage prediction error defined as [(RIA value-HPLC value)/HPLC value] x 100 was used for estimation of differences. C(max), t(max), and C(avg) were compared using Student's t-test. RIA produced significantly higher CsA levels than HPLC method in the period of 0.5 - 5 hours after application. The greatest differences (43 - 56%) occurred between 1 and 3 hours after dose. AUC(0-12), C(max) a C(avg) calculated from RIA results were consequently significantly higher. Only t(max) remained unchanged. The ratio of metabolites/parent drug after CsA intake is decreasing but their absolute concentrations are significantly increasing. Mean levels at C(0)/C(2) were CsA-RIA 82/612, CsA-HPLC 89/425, AM1 121/179, AM9 4.1/81.4, AM4N 9.5/21.0 ng/ml. TDM using C(2) and AUC sparse sampling may cause misleading interpretation using both methods alternately for the same patient.

[Determination of valproic acid by on-line coupled capillary isotachophoresis with capillary zone electrophoresis with conductometric detection]. / Stanovení kyseliny valproové pomocí on-line spojení izotachoforézy a kapilární zónové elektroforézy s vodivostní detekcí.

The paper aimed to test suitable conditions for the determination of VPA in serum using the ITP-CZE method with conductometric detection. It attempted to find whether for the method described by Olvecká et al. it is possible to use a similar procedure of sample preparation as for the gas chromatographic method which is routinely employed for the assessment of VPA in serum. A 200 rl sample of serum was extracted with 200 rl of acetone and after centrifugation 200 rl of the upper layer was withdrawn and supplemented with demineralized water to make 10 ml. When a microsyringe is used for dosing, consumptions of serum can be reduced up to 5 rl, which is advantageous mainly for therapeutic drug monitoring in children. The length of analysis was 20 minutes. The method was linear within the range of 20-400 mg/l, which includes the therapeutic range of VPA (50-100 mg/l). The detection and quantification limits were 2.5 and 8.5 mg/l, respectively. The variation coefficients were below 10% in all three concentrations (30, 70 and 120 mg/l). The new method was compared with the method of gas chromatography by means of the Passing-Bablok regression analysis and both methods were found to give identical results.

Differences between prescribed daily doses and defined daily doses of antiepileptics--therapeutic drug monitoring as a marker of the quality of the treatment.

OBJECTIVE: Prescribed daily doses (PDDs) of antiepileptics (N03A ATC group) were recorded for drugs used in monotherapy or in combination therapy in the University Hospital in Ostrava, Czechia. Plasma levels were used as an indicator of the quality of treatment. METHOD: Request and reply forms for therapeutic drug monitoring (TDM) were used as a source of PDDs and plasma levels. The study included 1,144 in-patients examined in the period 1993 - 2004. The differences in PDD were tested by Mann-Whitney-U-test. ATC/DDD index 2005 was used. Doses given in mono- and polytherapy were compared. RESULTS: Median PDDs in samples within the therapeutic range (in mg) in mono-/polytherapy were as follows (DDDs in parenthesis): carbamazepine 600/800 (1,000), clonazepam 2.0/2.0 (8), phenytoin 300/300 (300), ethosuximide -/1000 (1,250), lamotrigine 250/200 (300), phenobarbital -/200 (100), primidone 500/625 (1,250), topiramate -/300 (300), valproic acid 750/1,000 (1,500). Median PDDs in polytherapy with antiepileptics not analyzed for TDM were: gabapentin 900 (1,800), levetiracetam 1,500 (1,500), vigabatrin 1,500 (2,000). CONCLUSIONS: PDDs in monotherapy were similar or slightly lower than in combination therapy with an exception for lamotrigine, NS. The differences were significant in carbamazepine, p < 0.0001, and valproic acid, p < 0.001. Patients with plasma levels within the therapeutic range were usually treated with similar or slightly higher doses than the remainder. In polytherapy the PDDs were similar to DDDs in carbamazepine, ethosuximide, phenytoin, and topiramate in samples within the therapeutic range when difference +/- 20 per cent was considered as acceptable PDD of levetiracetam was also similar to actual DDD. In general plasma levels tended to be below the therapeutic range. The differences between PDD and DDD of antiepileptics have to be taken into account especially when utilization of different drugs is compared.

[Therapeutic monitoring of cyclosporine A]. / Terapeutické monitorování cyklosporinu A.

Cyclosporine A (CyA) is a drug with a specific influence on the immune system and it is used both to prevent tissue rejection of transplanted organs and to treat autoimmune diseases. The properties, metabolism, and methods of therapeutic drug monitoring (TDM) in patients under immunosuppressive therapy are described. TDM of CyA reflects the clinical condition of the patient during immunosuppression and may include pharmacokinetic and pharmacodynamic data obtained either from measuring CyA levels in blood, or from determining some other parameters, which are modulated by CyA (II-2). Although TDM is mainly based on analyzing trough levels of CyA, the determination of the whole biological exposition calculated as AUC enables better correlation with the clinical state of patients. Pharmacodynamic parameters have not been measured routinely yet.

[Determination of cyclosporine A using high-performance liquid chromatography]. / Stanovení cyklosporinu A metodou vysoce úcinné kapalinové chromatografie (HPLC).

Recent analytical possibilities of therapeutic drug monitoring (TDM) of cyclosporine A (CyA) both in solid organ transplanted patients and in patients with autoimmune diseases are described. The standard method for determination of CyA in blood is a validated HPLC method. HPLC methods were developed which make it possible to determine not only the parent drug, but also the main metabolites of CyA: AMI (M17), AM9 (M1), and AM4N (M21). Preparation of blood samples, their extractions and purifications are discussed. Chromatography is usually carried on C18 or CN columns by isocratic elution under high temperature (70 degrees C). CyD or CyC is used as the internal standard HPLC-MS method enables unambiguous identification of CyA metabolites after their separation on a chromatographic column and it is used mostly for research purposes only.

Influence of P-glycoprotein on the transplacental passage of cyclosporine.

The transfer kinetics of cyclosporine across the dually perfused rat placenta in the maternal to fetal direction and a possible involvement of P-glycoprotein were investigated. The transplacental clearance of cyclosporine in the materno-fetal direction was found to be dependent on the maternal inflow concentration of cyclosporine. Coadministration of cyclosporine with an excess of quinidine or chlorpromazine into the maternal compartment revealed 1.7- and 1.9-fold increase in cyclosporine concentration in the fetal compartment. In the experiments where quinidine was present both in the maternal and fetal compartments, cyclosporine appeared in the fetal compartment significantly faster, and its amount was three times higher when compared with controls. Conversely, quinidine or chlorpromazine did not affect the transplacental passage of L-[(3)H]-glucose. The interference of quinidine with the metabolism of cyclosporine in the placenta was excluded because only traces of M-1 and M-17 metabolites were found in the fetal solutions. Sodium azide, a mitochondrial respiratory inhibitor, was found to double the rate of cyclosporine, but not L-[(3)H]-glucose, passage across the placenta. Our findings indicate that P-glycoprotein pumps cyclosporine out of the trophoblast cells of the rat placenta in the ATP-dependent manner and restricts the passage of cyclosporine across the placental barrier.

Evaluation and comparison of therapeutic monitoring of whole-blood levels of cyclosporin A and its metabolites in renal transplantation by HPLC and RIA methods.

BACKGROUND: The aim of the work was to evaluate the possibility to estimate the level of cyclosporin A (CyA) metabolites as the difference of radioimmunoassay (RIA) non-specific and RIA specific methods. METHODS: Blood samples of renal transplant patients were analyzed by three different methods: RIA specific method (CYCLO-Trac, DiaSorin, USA) (RIA(SP)), RIA non-specific method (Immunotech, Czech Republic) (RIA(NS)), and high performance liquid chromatography (HPLC) method. RESULTS: Although values obtained by RIA(SP) correlated well those obtained by HPLC (RIA(SP)=0.995.HPLC+9.68; r(2)=0.962, n=448), the results of HPLC methods were lower by 8%. The values obtained by RIA(NS) were 2.57 times higher than the values obtained by RIA(SP) (RIA(SP)=0.356RIA(NS); r(2)=0.713, n=448). The ratio (CyA+CyA metabolites)/(CyA) calculated as the ratio RIA(NS)/RIA(SP) values for 42 renal transplant patients was relatively stable for each particular patient. The sum of selected CyA metabolites (M1+M17+M21) measured by HPLC correlated well with that estimated from the difference of RIA(NS)-RIA(SP): HPLC(metab)=0.921.(RIA(NS)-RIA(SP))+21.3; (r(2)=0.746, n=448). CONCLUSION: The combination of both the specific and non-specific methods for the determination of CyA presents an improved means for the TDM of CyA and CyA metabolites in renal transplant patients. Moreover, a combination of both methods can help to elucidate some unexpected events, such as the persistence of high cyclosporin blood levels.

High-performance liquid chromatographic method for therapeutic drug monitoring of cyclosporine A and its two metabolites in renal transplant patients.

A novel fast HPLC method was developed for the determination of cyclosporine A (CyA) and its two metabolites M17 (AM1) and M21 (AM4N) in blood. Whole blood was precipitated with zinc sulphate, extracted with diethyl ether, evaporated, dissolved in aqueous methanol and partitioned twice with n-hexane. Chromatography was carried out using a microbore RP-column under isocratic elution with acetonitrile-methanol-water (200:80:140, v/v/v) at 70 degrees C and a detector set at 205 nm. Linearity for all three compounds was tested in the range of 1-1000 ng/ml. Recovery was 97-109%, and a coefficient of variation was 1.6-8.8% depending on the particular compound and its concentration. The method was used for a group of renal transplant patients having an inadequate response to CyA therapy in order to evaluate the possible role of CyA and its metabolites on the occurrence of hypertension and other toxicological events.

Studies on the inhibitory effect of apomorphine and bromocryptine on basal and TRH induced level of TSH and PRL in hypothyroid rats under pentobarbiturate anesthesia.

Groups of male rats were inserted with polyethylene tubings into femoral artery and vein under pentobarbiturate anesthesia and small blood samples were frequently taken for the estimation of TSH and PRL under maintaining isovolemia. After a single injection of apomorphine (12 mg kg-1) or bromocryptine (20 mg kg-1) much more expressed effect of these drugs on a decrease of PRL level in plasma was found than that on a decrease of TSH level and similar observation was made with the use of continuous i.v. infusion of apomorphine (50 micrograms in 20 microliter per min for 180 min). Finally, under the above dose of infused apomorphine, the effect of TRH on the increase of TSH level was depressed at the 30th min as compared to that 0 and 120th min of infusion. In addition, at 120 min of infusion the effect of TRH was significantly higher than that at 0 min. These results suggest that the effect of apomorphine may take place at the pituitary level.

In vivo study of iodothyronine deiodination in rat liver: effect of salicylate on biliary excretion of several iodothyronines.

Biliary excretion of total (i.e. conjugated plus unconjugated) thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,5-diiodothyronine (3,5-T2), 3,3'-diiodothyronine (3,3'-T2) and 3',5'-diiodothyronine (3',5'-T2) was measured with the aid of specific radioimmunoassay in 17 control rats and in 31 rats injected sodium salicylate i.v. (200 mg kg-1). The animals were anesthetized with pentobarbiturate and the samples of bile were taken in subsequent 2 h periods from a drained bile duct. In controls a gradual decrease of excretion of all compounds measured was found during 10 h observation period. In contrast, after salicylate injection a transient increase of total bile volume and of T4, T3 and 3,5-T2 excretion was observed followed by a remarkable decrease, while a multifold and prolonged increase of the excretion of rT3, 3,3'-T2 and 3',5'-T2 was found. These data suggest an acute and remarkable effect of salicylate on T4 deiodinating pathway in the liver.

Direct quantitative estimation of several iodothyronines in rat bile by radioimmunoassay and basal data on their biliary excretion.

A method was developed for the hydrolysis of conjugated iodothyronines in bile with the aid of beta-glucuronidase/arylsulfatase and for subsequent direct estimation of total and free iodothyronines with the aid of specific radioimmunoassay. The amount of conjugated fraction could then be calculated from the difference. Thus, basal biliary excretion of several iodothyronines was measured in 31 normal, fed rats in which the bile duct was drained with polyethylene tubing under pentobarbiturate anesthesia and the bile was collected for 2 h. The free fraction of thyroxine, 3,5,3'-triiodothyronine and 3,3'-diiodothyronine was approx. 30% of total content, while that of 3,3',5'-triiodothyronine and 3,5-diiodothyronine was approx. 20% and that of 3',5'-diiodothyronine was less than 10%. This suggests some considerable differences in the conjugation of individual iodothyronines in the liver. The concentration of T4 in bile was about the same as in plasma, while that of other iodothyronines was about 3-8 times higher than in plasma. This shows close interrelations between the iodothyronine deiodinating pathway in liver cells in vivo and the spectrum of iodothyronine in bile. The average ratio of T3/rT3 as found in bile was about 4.

Immediate opposite effect of salicylate on thyroxine and 3,5,3'-triiodothyronine versus 3,3',5'-triiodothyronine level in plasma in rats.

In a total of 46 male rats polyethylene tubings were introduced into femoral artery and vein under pentobarbiturate anesthesia. Then heparin (300 U kg-1) was injected at 60-90 min after pentobarbiturate and two control blood samples were subsequently taken. After that sodium salicylate (200 mg kg-1) was injected i.v. and blood samples were taken at 30-420 min later. An immediate decrease of the thyroxine (T4) level in plasma to about 20% of original level and that of 3,5,3'-triiodothyronine (T3) to about 60% of that was found, while the level of 3,3',5'-triiodothyronine (rT3) was increased 20%. It was concluded that the administration of salicylate results in an immediate displacement of T4 and T3 from plasma protein binding and possibly inhibits the conversion of T4 to T3 and of rT3 to 3,3'-diiodothyronine which results in an increase of rT3 level in plasma. This might by partially prevented by an inhibiting effect of salicylate on the binding of rT3 to plasma proteins.

In vitro effect of TRH on adenylate cyclase and cAMP in rat anterior pituitary and on TSH and PRL release into incubation medium.

Rat anterior pituitaries were incubated for 20 min with 1, 5 or 20 nmol TRH ml-1 and cAMP was measured in the homogenate of pituitary tissue after incubation, while TSH and PRL release into the medium was estimated with the aid of specific radioimmunoassay. In a similar experiment the activity of adenylate cyclase in pituitary homogenates was measured after the incubation with 0.5, 1.0 or 5.0 nmol TRH ml-1. Significant increase of TSH and PRL in the medium was found after 5 and 20 nmol TRH ml-1, while cAMP was significantly increased after 20 nmol TRH ml-1 only. The activity of adenylate cyclase was significantly increased after all doses of TRH used. In general, these findings are consistent with current views on the effect of hypothalamic hormones via adenylate cyclase - cAMP system and show that the responsiveness and sensitivity of an in vitro system using whole pituitaries appears to be less that of in vivo system.

Studies on the effect of pentobarbiturate, ether and heparin on plasma thyroxine level in rats.

In groups of 9 to 20 adult male rats decapitated at 0, 15 or 60 min after i.p. injection of saline or at the same intervals after 2 min exposure to ether no differences in plasma thyroxine level were found. In groups of 5-6 animals a significant decrease of thyroxine level in plasma was observed at 30 min after i.p. injection of pentobarbiturate (40 mg kg-1) and unsignificant decrease after 800 and 2400 U heparin i.p. per animal. Finally, in 58 animals a linear decrease of plasma thyroxine level was found during 480 min beginning from about 60th to 90th min after the onset of pentobarbiturate anaesthesia (40 mg kg-1 followed by 20 mg kg-1 every 60-90 min). It was concluded that the blood clotting preventive dose of heparin (i.e. 300 U per 400 g animal) presumably does not interfere with the level of thyroxine in plasma, while pentobarbiturate apparently results in a considerable decrease of that.

Changes in adenylate cyclase activity in rat pituitary after TRH and T3 injection in vivo.

Increased activity of adenylate cyclase in whole anterior pituitary was repeatedly found in groups of 3-6 adult male rats 5 min after iv injection of 10, 100 and 200 nmol of TRH kg-1. After the administration of 100 nmol TRH kg-1 the activity of adenylate cyclase was significantly increased at 2, 5, 10 and 30 min with a peak level at 5 min, while at 60 min the values did not differ from controls. The level of TSH in serum was significantly increased in all TRH injected groups with a peak value at 10 min, while no dose-response relationship was found at 5 min. Finally, the activity of adenylate cyclase was significantly decreased in animals injected with 20 nmol of L-triiodothyronine kg-1 180 min before sacrifice, the decrease being not overcome by 100 nmol TRH kg-1 injected 5 min before sacrifice.

Acute effect of salicylate on T4 and T3 levels in plasma and their excretion by bile in rats.

Male Wistar rats weighing about 400 g were anaesthetized with pentobarbiturate (40 mg kg-1) i.p. and thin polyethylene tubings were inserted in left femoral artery and vein as well as in a bile duct. Blood samples were taken at 30-60 min intervals for 6 h. The total bile was collected to pre-weighed glass vials. With the aid of radioimmunoassay the level of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) was estimated in plasma and the total excretion of these compounds by bile was measured after the incubation of bile aliquots with beta-glucuronidase-arylsulphatase. After i.v. injection of sodium salicylate (S; 80 mg kg-1) a significant decrease of T4 and T3 in plasma was found compared either to the initial level found in the same animals or to that found in controls not injected with S. Total excretion of bile was increased within 2 h after the administration of S. Under these conditions, the excretion of T4 was increased, while that of T3 was decreased. It is suggested that the effect of salicylate under the experimental conditions used consists of: 1. displacement of iodothyronines from plasma protein binding; 2. increased bile excretion (during the initial period after the administration of S) presumably due to the increased blood flow through the liver.