Dose individualization of intravenous busulfan in pediatric patients undergoing bone marrow transplantation: impact and in vitro evaluation of infusion lag-time.

OBJECTIVES: To apply therapeutic drug monitoring and dose-individualization of intravenous Busulfan to paediatric patients and evaluate the impact of syringe-pump induced Busulfan infusion lag-time after in vitro estimation. METHODS: 76 children and adolescents were administered 2 h intravenous Busulfan infusion every 6 h (16 doses). Busulfan plasma levels, withdrawn by an optimized sampling scheme and measured by a validated HPLC-PDA method, were used to estimate basic PK parameters, AUC, Cmax, kel, t1/2, applying Non-Compartmental Analysis. In vivo infusion lag-time was simulated in vitro and used to evaluate its impact on AUC estimation. KEY FINDINGS: Mean (%CV) Busulfan AUC, Cmax, clearance and t1/2 for pediatric population were found 962.3 µm × min (33.1), 0.95 mg/L (41.4), 0.27 L/h/kg (33.3), 2.2 h (27.8), respectively. TDM applied to 76 children revealed 6 (7.9%) being above and 25 (32.9%) below therapeutic-range (AUC: 900-1350 µm × min). After dose correction, all patients were measured below toxic levels (AUC < 1500 µm × min), no patient below 900 µm × min. Incorporation of infusion lag-time revealed lower AUCs with 17.1% more patients and 23.1% more younger patients, with body weight <16 kg, being below the therapeutic-range. CONCLUSIONS: TDM, applied successfully to 76 children, confirmed the need for Busulfan dose-individualization in paediatric patients. Infusion lag-time was proved clinically significant for younger, low body-weight patients and those close to the lower therapeutic-range limit.

The effect of athletes` hyperhydration on the urinary 'steroid profile' markers in doping control analysis.

The urinary 'steroid profile' in doping control analysis is a powerful tool aimed at detecting intra-individual deviations related to the abuse of endogenous steroids. Factors altering the steroid profile include, among others, the excessive fluid intake leading to low endogenous steroids concentrations compared to an individual's normal values. Cases report the use of hyperhydration by athletes as a masking method during anti-doping urine sample collection. Seven healthy physically active non-smoking Caucasian males were examined for a 72-hour period using water and a commercial sports drink as hyperhydration agents (20 mL/kg body weight). Urine samples were collected and analyzed according to World Anti-Doping Agency (WADA) technical documents. Although, significant differences were observed on the endogenous steroid concentrations under the studied hyperhydration conditions, specific gravity adjustment based on a reference value of 1.020 can eliminate the dilution induced effect. Adjustment methods based on creatinine and urinary flow rate were also examined; however, specific gravity was the optimum method in terms of effectiveness to adjust concentrations close to the baseline steroid profile and practicability. No significant effect on the urinary steroid ratios was observed with variability values within 30% of the mean for the majority of data. Furthermore, no masking on the detection ability of endogenous steroids was observed due to hyperhydration. It can be concluded that any deviation on the endogenous steroid concentrations due to excessive fluid intake can be compensated by the specific gravity adjustment and therefore, hyperhydration is not effective as a masking method on the detection of the abuse of endogenous steroids.

Crocus sativus L. aqueous extract reduces atherogenesis, increases atherosclerotic plaque stability and improves glucose control in diabetic atherosclerotic animals.

BACKGROUND AND AIMS: We aimed to evaluate a possible atheroprotective effect of saffron aqueous extract (SFE), and its potential anti-inflammatory mechanisms, in apoE knockout (ApoE-/-) mice. METHODS: Fifty male, ApoE-/- mice, fed a high-fat diet (HFD) for 12 weeks, were randomized into 5 groups: (1) baseline group, euthanatized, without intervention, (2) three saffron groups, receiving HFD and 30,60,90 mg/kg/day of SFE, respectively, for four weeks, per os through gavage, after reconstitution in water for injection (WFI), (3) control group (COG), receiving daily HFD and the same volume of WFI (four weeks). After blood sampling and euthanasia, aortic roots were excised and analyzed for gene expression and/or percentage of aortic stenosis, relative content of macrophages, smooth muscle cells (SMCs), connective tissue, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases-2,-3,-9 (MMP-2,-3,-9) and their inhibitor (TIMP-2) and IL-6. SFE doses were determined by a pilot serum pharmacokinetic study in C57BL/6J wild-type mice. RESULTS: SFE did not affect body weight and total cholesterol levels (p > 0.05), while high SFE dose significantly ameliorated glucose and triglycerides profiles compared to other groups (p < 0.05). SFE considerably decreased aortic stenosis in a dose-dependent manner (p < 0.05). Furthermore, increasing SFE doses proportionally reduced macrophages content and increased within plaques content of collagen, elastin, and SMCs, promoting more stable plaque phenotype compared to COG (p < 0.05). Those effects seemed to be associated with a considerable reduction (>30%) in IL-6, TNF-α, MCP-1, MMP-2,-3,-9 (p < 0.05) and MMP-2/TIMP-2 ratio. CONCLUSIONS: SFE exerted dose-dependent anti-atherosclerotic and plaque-stabilizing effects in Apo-E-/- mice, probably mediated by a favorable modification of inflammatory mechanisms, which requires further investigation.

Saffron (Crocus sativus) intake provides nutritional preconditioning against myocardial ischemia-reperfusion injury in Wild Type and ApoE(-/-) mice: Involvement of Nrf2 activation.

BACKGROUND AND AIMS: Saffron is an antioxidant herbal derivative; however, its efficacy as a nutritional cardioprotective agent has not been fully elucidated. We investigated the cardioprotective properties of a standardized saffron aqueous extract (SFE) against ischemia/reperfusion (I/R) injury in Wild-Type (WT) and ApoE(-/-) mice and the underlying molecular mechanisms. METHODS AND RESULTS: WT and ApoE(-/-) mice were subjected to 30 min I and 2 h R, with the following per os interventions for 4 weeks: 1) WT Control Group, receiving Water for Injection (WFI); 2) WT Crocus Group, receiving SFE at a dose of 60 mg/kg/day; 3) WT Crocus + Wort group, receiving SFE as described above and wortmannin at a dose of 60 µg/kg bolus 15 min before R; 4) ApoE(-/-) Control Group, receiving WFI; 5) ApoE(-/-) Crocus Group, receiving SFE at a dose of 60 mg/kg/day and 6) ApoE(-/-) Crocus + Wort: receiving SFE as described above and wortmannin at a dose of 60 µg/kg bolus, 15 min before R. Ischemic area/area at risk (I/R%) ratio was measured. Blood samples and ischemic myocardial tissue were collected at the 10th min of reperfusion for assessment of troponin I, malondialdehyde (MDA), nitrotyrosine (NT), p-eNOS, eNOS, p-Akt, Akt, p-p42/p-p44, p-GSK3ß, GSK3ß, IL-6, Nrf2, HO-1 and MnSOD expression. The effect of SFE on Nrf2 expression was also evaluated in vitro. SFE reduced infarct size in WT (16.15 ± 3.7% vs 41.57 ± 2.48%, ***p < 0.001) and in ApoE(-/-) mice (16.14 ± 1.47% vs 45.57 ± 1.73%, ***p < 0.001). The administration of wortmannin resulted in partial inhibition of the infarct size limitation efficacy of SFE (in both WT and Apo-E(-/-) mice). Mice receiving SFE showed increased levels of eNOS, p-Akt, p-ERK1/2, p-44/p-42 and p-GSK3ß-Ser9 and reduced expression of IL-6 and iNOS; furthermore, SFE reduced the levels of MDA and NT. SFE induced Nrf2 expression and its downstream targets, HO-1 and MnSOD in the myocardium of the treated animals, and induced Nrf2 expression in vitro in a dose-dependent manner. CONCLUSIONS: SFE limits myocardial infarction in Wild-Type and ApoE(-/-) mice in a multifaceted manner including activation of Akt/eNOS/ERK1/2/GSK3-ß and through Nrf2 pathway, bestowing antioxidant protection against I/R.

Pharmacokinetics of doripenem in CSF of patients with non-inflamed meninges.

OBJECTIVES: To investigate intact blood-brain barrier (BBB) penetration by doripenem and characterize doripenem pharmacokinetics in CSF using a pharmacokinetic model. PATIENTS AND METHODS: Thirty-eight neurological patients with no active neurological disease or CNS infection received a single 500 mg doripenem dose before pump implantation surgery, or lumbar puncture, for intrathecal baclofen administration. In most cases single CSF and blood samples were collected per patient and analysed for doripenem with HPLC. A two-stage pharmacokinetic analysis was performed to estimate: (i) empirical Bayesian estimates (EBEs) of individual doripenem plasma pharmacokinetic parameters, using plasma doripenem concentrations and literature population priors for a two-compartment model; and (ii) doripenem CSF pharmacokinetic parameters using simulated plasma concentrations from stage (i) as a forcing function. The mean values of the structural model parameters, k(CSF) (distribution rate constant) and PC (CSF/plasma partition coefficient), and the residual variability were estimated. RESULTS: The mean estimates of the parameters were k(CSF)= 0.105 h(-1) and PC= 0.053, corresponding to mean steady-state doripenem CSF concentrations of 0.20 mg/L and 0.40 mg/L for regimens of 3 × 500 mg daily and 3 × 1000 mg daily, respectively, and a mean equilibrium half-life of 6.6 h. The model was validated internally using a visual predictive check (VPC) and bootstrap. Simulating two dosing scenarios gave doripenem levels in the CSF above or close to the literature MIC values. CONCLUSIONS: The present NONMEM software analysis shows that doripenem crosses intact BBB significantly and suggests that the drug should be further evaluated as a candidate to treat certain CNS infections, since drug penetration through BBB is enhanced by meningeal inflammation.

Computational-Regulatory Developments in the Prediction of Oral Drug Absorption.

Early prediction of human intestinal absorption is important in selection of potential orally administered drugs. Various computational models for prediction of the fraction of dose absorbed, Fa, have been developed. In 1989, a sigmoidal relationship between Fa and drug absorption potential was shown. Since then various physicochemical descriptors of molecules (lipophilicity, polar surface area, hydrogen bond descriptors) have been found to correlate with human intestinal absorption and various attempts in estimating Fa have been reported. Most studies rely on the presupposition that Fa is mainly dependent on drug's solubility, which drives the dissolution rate in the gastrointestinal (GI) fluids, and the rate of passive drug transport across the intestinal membrane. In the same vein, the biopharmaceutics classification system (BCS) and the relevant FDA guideline classify drugs in four categories according to their aqueous solubility and permeability. However, the biopharmaceutics drug disposition classification system (BDDCS) revealed the poor predictability of permeability estimates for Fa and the major role of transporters for GI uptake of drugs. The role of solubility in the reaction limited model of dissolution and the ubiquitous presence of supersaturated solubility-dissolution phenomena in the GI lumen, call for a more physiologically relevant consideration of GI absorption.

Development of a reaction-limited model of dissolution: application to official dissolution tests experiments.

A reaction-limited model for drug dissolution is developed assuming that the reaction at the solid-liquid interface is controlling the rate of dissolution. The dissolution process is considered as a bidirectional chemical reaction of the undissolved drug species with the free solvent molecules, yielding the dissolved species of drug complex with solvent. This reaction was considered in either sink conditions, where it corresponds to the unidirectional case and the entire amount of the drug is dissolved, or reaching chemical equilibrium, which corresponds to saturation of the solution. The model equation was fitted successfully to dissolution data sets of naproxen and nitrofurantoin formulations measured in the paddle and basket apparatuses, respectively, under various experimental conditions. For comparative purposes these data were also analyzed using three functions based on the diffusion layer model. All functions failed to reveal the governing role of saturation solubility in the dissolution process associated with the diffusion layer model when the conditions for the valid estimation of saturation solubility, established theoretically in this study, were met by the experimental set up employed. Overall, the model developed provides an interesting alternative to the classic approaches of drug dissolution modeling, quantifying the case of reaction-limited dissolution of drugs.

Modelling and simulation in drug absorption processes.

Drug absorption is a complex process dependent upon drug properties such as solubility and permeability, formulation factors, and physiological variables including regional permeability differences, pH, luminal and mucosal enzymes, and intestinal motility, among others. Despite this complexity, various qualitative and quantitative approaches have been proposed for the estimation of oral drug absorption. These approaches are reviewed in this article with particular emphasis on drug dissolution modelling, dynamic systems for oral absorption and absorption models based on structure. The regulatory aspects of oral drug absorption and in particular the biopharmaceutic classification of drugs are also discussed. Models for drug dissolution and release describe adequately the in vitro data, and models for oral drug absorption provide reasonable results. The development of in vitro-in vivo correlations based on the official compendia specifications are facilitated using commercial computer packages.

In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique.

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.

The power law can describe the 'entire' drug release curve from HPMC-based matrix tablets: a hypothesis.

The purposes of this study were to (i) re-examine the relevance of Higuchi equation and the power law using both simulated and experimental release data in conjunction with the linearized, in terms of t(1/2), percent of drug release plots, (ii) demonstrate that the power law describes the entire drug release profile of several experimental data, and (iii) point out a physically based hypothesis for the successful use of power law in describing the entire drug release profile. Simulated data generated from the equation of power law were further analyzed using linear regression analysis in accord with the Higuchi equation. The analysis revealed that data generated from the equation of power law can be misinterpreted as obeying the Higuchi equation. The use of power law in describing the entire drug release curve from HPMC-based matrix tablets is validated by direct fit of power law equation to published data of other authors. A hypothesis based on the nonclassical diffusion of the solutes in the HPMC matrices is used to interpret the successful use of the power law in describing the entire release profile.

Non-linear regression analysis with errors in both variables: estimation of co-operative binding parameters.

Four different parameter estimation criteria, the geometric mean functional relationship (GMFR), the maximum likelihood (ML), the perpendicular least-squares (PLS) and the non-linear weighted least squares (WLS), were used to fit a model to the observed data when both regression variables were subject to error. Performances of these criteria were evaluated by fitting the co-operative drug-protein binding Hill model on simulated data containing errors in both variables. Six types of data were simulated with known variances. Comparison of the criteria was done by evaluating the bias, the relative standard deviation (S.D.) and the root-mean-squared error (RMSE), between estimated and true parameter values. Results show that (1) for data with correlated errors, all criteria perform poorly; in particular, the GMFR and ML criteria. For data with uncorrelated errors, all criteria perform equally well with regard to the RMSE. (2) Use of GMFR and ML lead to lower values for S.D. but higher biases compared with WLS and PLS. (3) WLS performs less well when equal dispersion is applied to the two observed variables.

Modeling of supersaturated dissolution data.

A recursion equation which relies on the population growth model of dissolution is used for the analysis of supersaturated dissolution data. The concentration-time data of dissolution experiments are initially transformed to fractions of dose dissolved-generations by adopting an appropriate time interval as the time step of the recursion equation. A computer program is used to derive estimates for the maximum fraction of dose dissolved and the fraction of dose remaining in solution at steady state. Good fittings were observed when this equation was applied to phenytoin and nifedipine supersaturated dissolution data obtained from literature.

Studies on the interaction of diflunisal ion with cyclodextrins using ion-selective electrode potentiometry.

The interaction of diflunisal ion (DF) with beta-cyclodextrin (betaCD), gamma-cyclodextrin (gammaCD), and hydroxypropyl-beta-cyclodextrin (HPbetaCD) was studied in phosphate buffer, pH 7.4, at 5-37 degrees C and various CD concentrations using a home-made diflunisal ion-selective electrode. Typical direct binding plots and Scatchard plots were obtained with HPbetaCD. The Scatchard model for one class of binding sites was used for the estimation of binding parameters for the DF/HPbetaCD interaction. The estimates for n (number of binding sites per CD molecule) were in all cases very close to unity, indicating 1:1 complexation. The association constant (K) estimates decrease with increasing temperature. Sigmoidal direct binding plots and concave-downwards Scatchard plots were obtained with various betaCD or gammaCD concentrations. The Hill model was used for the estimation of the binding parameters for the DF/betaCD and DF/gammaCD interactions. Both the Hill coefficients and the binding constants were markedly dependent on the CD concentration. These findings indicate the cooperative character of DF/betaCD and DF/gammaCD interactions. The free energy change, DeltaG, and the thermodynamic parameters, DeltaH and DeltaS, were estimated for each of the interactions studied using the Van't Hoff equation.

A displacement approach for competitive drug-protein binding studies using the potentiometric 1-anilino-8-naphthalene-sulfonate probe technique.

A displacement approach for competitive binding studies was developed. The method utilizes the potentiometric 1-anilino-8-naphthalene-sulfonate (ANS) probe technique and is applied to the binding study of several non-steroidal anti-inflammatory drugs (NSADs) to bovine serum albumin (BSA). A home-made ANS electrode was used to monitor the displaced free ANS probe from its binding sites on the protein molecule by the stepwise addition of the studied drug. To assess and compare quantitatively the displacing ability of the various drugs, the 'ANS Displacement Index' is used. The possible interference of 19 ionizable drugs (NSADs, sulfonamides, etc.) to the ANS selective electrode at pH 7.4 was studied and their potentiometric selectivity coefficients (K(pot)(ANS,D)) were determined. Correction procedures for the determination of the free ANS concentration are proposed in the case of interfering ionic drugs. A blank binding experiment in conjunction with the incorporation of K(pot)(ANS,D) values in the 'general competitive site oriented model' allows one to derive estimates for the drug binding parameters, i.e. the number of binding sites and association constants.

General treatment of competitive binding as applied to the potentiometric ion probe technique: application to the interaction of nonsteroidal anti-inflammatory drugs with bovine serum albumin.

The binding of naproxen, ketoprofen, phenylbutazone, salicylic acid, azapropazone, and indobufen to bovine serum albumin was studied by applying the potentiometric ion probe technique. An ion-selective electrode for the ion probe 1-anilino-8-naphthalene-sulfonate was utilized for the purposes of this study. A modified site-oriented competitive binding model was used for the estimation of the drugs' binding parameters, considering different number of binding sites on the competing binding class(es) for the probe and the drug. Calculations were based exclusively on the concentration data of the free probe. The model's ability for accurate estimations of binding parameters was evaluated by simulation studies. The following values of binding parameters were found at 25 degrees C for the drugs under study; naproxen, n1 = 9.1, k1 = 9.4 x 10(5) M-1; ketoprofen, n1 = 8.8, k1 = 10.8 x 10(5) M-1; phenylbutazone, n1 = 3.2, k1 = 1.4 x 10(5) M-1; salicylic acid, n1 = 2.6, k1 = 1.8 x 10(5) M-1, n2 = 21.5, k2 = 1.0 x 10(4) M-1; azapropazone, n1 = 0.5, k1 = 7.8 x 10(5) M-1, n2 = 26.3, k2 = 1.9 x 10(4) M-1; indobufen, n1 = 5.8, k1 = 5.8 x 10(5) M-1, n2 = 19.9, k2 = 3.8 x 10(5) M-1, where ni the number of binding sites of the i class and ki the corresponding association constant.

Automated flow-injection technique for use in dissolution studies of sustained-release formulations: application to iron(II) formulations.

The application of flow-injection analysis (FIA) to automated dissolution studies of sustained-release formulations is described. The long-term stability of the dissolution-FIA analyser was checked during unattended operation for 42 h. The construction of multiple calibration curves with the so-called electronic dilution FIA procedure was used to extend the linear range of the determination. The computer-controlled FIA system and the principles of associated software are described and applied to dissolution studies of sustained-release formulations of iron(II) using its sensitive reaction with the colour reagent, ferrozine. The extended linear range of the determination is 1-130 ppm iron(II) and the precision (RSD) better than 3% (n = 3).

Use of 1-anilino-8-napthalenesulphonate as an ion probe for the potentiometric study of the binding of sulphonamides to bovine serum albumin and plasma.

The binding of sulphafurazole, sulphamethizole and sulphamethoxazole to bovine serum albumin (BSA) and human plasma has been studied in-vitro by potentiometry using an electrode selective for the ion probe 1-anilino-8-napthalenesulphonate (ANS). The method requires two separate potentiometric titrations of the binder solution with ANS in the absence and in the presence of sulphonamides. ANS displaced sulphonamides from the first-class of binding sites of both binders. The binding constants for sulphonamide-BSA interactions were higher than those for sulphonamide-human plasma. The expansion of the least linear limit of the response curve of the electrode down to 10(-7) M in the presence of BSA was also demonstrated. The reverse reaction, i.e. the displacement of the probe from the binding sites induced by the sulphonamides, was also explored. The proposed method is suitable for studying competitive binding interactions in biological specimens.

Determination of association constants in cyclodextrin/drug complexation using the Scatchard plot: application to beta-cyclodextrin-anilinonaphthalenesulfonates.

The appropriate Scatchard equation was developed for a system involving the formation of 1:1 and 1:2 substrate:cyclodextrin complexes. Simulation of this system was performed under the most common experimental conditions encountered in this type of study. The use of the equation allows for nonlinear least-squares estimation of the association constants. The interaction of the model compounds 1-anilino-8-naphthalenesulfonate (1,8-ANS) and 2(p-toluidinyl)-6-naphthalenesulfonate (2,6-TNS) with beta-cyclodextrin (beta-CD) was used to evaluate the theoretical model. Binding experiments were performed using either potentiometric titration or fluorimetric detection. The experimental data for 1,8-ANS/beta-CD fit well to the 1:1 binding model, with an association constant of 87 +/- 1 M-1. The association constants of the 1:1 and 1:2 2,6-TNS/beta-CD complexes utilizing direct potentiometry were 3737 +/- 6 and 149 +/- 2 M-1. It is shown that fluorimetry can give biased estimates for the association constants of the complexation 2,6-TNS/beta-CD, since the assumption of an equivalent quantum yield of bound species is not valid.

Complexation studies of cyclodextrins with tricyclic antidepressants using ion-selective electrodes.

The complexation of six tricyclic antidepressant drugs [amitriptylin (AMN), nortriptylin (NRN), imipramin (IMN), doxepin (DXN), protriptylin (PTN), and maprotilin (MPN)] with alpha- and beta-cyclodextrins (CDs) using ion-selective electrodes (ISEs) as drug ion sensors is described. Binding parameters were calculated by nonlinear fitting of the model described by the Scatchard equation, to the experimental data of a titration of a CD solution with the ion of interest. One binding site (the CD cavity) was found in all cases with both CDs. The calculated association constants at 25 degrees C using CD concentrations in the range of 0.0100-0.0010 M, varied from 4.81 x 10(3) M-1 (MPN) to 23.9 x 10(3) M-1 (AMN) in the case of beta-CD and from 50 M-1 (DXN) to 123 M-1 (MPN) in the case of alpha-CD. The precision for the estimation of the binding parameters was 0.1-5.0% (within-run RSD%) and 8-10% (between-run RSD%; n = 3). The complexation of the drugs with beta-CD was also examined as a function of temperature in the range of 5-37 degrees C; it was found to decrease by increasing temperature. Van't Hoff analysis gave good correlations (r greater than or equal to 0.989) for all drug ions studied. The estimates of the thermodynamic parameters indicate that the formation of inclusion complexes is enthalpy driven. A compensation plot based on the thermodynamic parameters delta H and delta S resulted in a linear relationship, which is indicative of a common type of force involved in the complexation of drugs to beta-CD.