Tawfiq Arafat

Enzyme linked immunosorbent assay for determination of amlodipine in plasma.

Amlodipine is a calcium channel antagonist of the dihydropyridine group. It is effective for treating hypertension, chronic stable angina, and vasospastic angina. However, it is difficult clinically to pinpoint the maximum dosage for antihypertensive activity of the drug without having parallel data on the plasma drug concentrations. The methods for assaying amlodipine are either gas chromatography with electron capture detector or liquid chromatography coupled with tandem mass spectrometry (or with an electrochemical detector), which needs tedious derivatization, and is expensive and time consuming. Therefore, in this study we developed an enzyme immunoassay for determining amlodipine in plasma. Anti‐amlodipine antibodies were produced following immunization of bovine serum albumin‐amlodipine conjugate. These specific antibodies were used in a competitive biotin–avidin‐based enzyme‐linked immunosorbent assay to measure amlodipine in plasma. Biotin was linked to the antibodies in order to enhance the sensitivity of the assay. The assay was specific for the free form of amlodipine with a detection limit of 0.1 ng/ml and the intra‐ and interassay coefficient of variation ranged from 1.6–10.2%. This immunoassay provides a sensitive, reliable, rapid, and accurate method for determination of amlodipine in plasma, which can be used in therapeutic drug monitoring pharmacokinetic studies and pharmaceutical analysis. J. Clin. Lab. Anal. 15:47–53, 2001.

Assessment of a controlled release hydrophilic matrix formulation for metoclopramide HCl.

Metoclopramide HCl showed controlled release behavior when embedded in a hydrophilic matrix of chitosan and sodium alginate. The in vitro release data was found to be first order according to the Higuchi mechanism. An in vivo evaluation of the metoclopramide controlled release matrix on six male volunteers was carried out. The plasma samples were analyzed using a high-performance liquid chromatography (HPLC) method using a mobile phase of acetonitrile:acetic acid (30:70), with the pH adjusted to 4.7, a reverse phase Hypersil BDS Phenyl column (4 microm, 250 x 4 mm) and the detection was performed at 305 nm. The controlled release formula was found to be effective in delaying absorption (t(max) 4.5h as compared to 1.2h), reducing the peak plasma concentrations (C(max) 63.4 ng/ml as compared to 95.9 ng/ml) and maintaining higher concentrations during the elimination phase when compared to the immediate release formula. This proves the suitability of the suggested system for further studies.

Simultaneous determination of amlodipine and atorvastatin with its metabolites; ortho and para hydroxy atorvastatin; in human plasma by LC–MS/MS

A simple liquid chromatography/ion trap mass spectrometry method for the quantification of amlodipine and atorvastatin with its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma was developed. Analytes with internal standard were extracted by protein direct precipitation with acetonitrile. Adequate chromatographic separation was achieved using Phenomenex Synergi 4u polar-RP 80A (150mm×4.6mm, 4μm) column in the isocratic elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of 0.50mL/min. Standard solutions for the analytes were prepared using amlodipine besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt, para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard calibration levels were prepared by pooled human plasma to attain final dynamic range of 0.2-20.0ng/mL for amlodipine, 1.5-150ng/mL for atorvastatin, 1.0-100.0ng/mL for ortho-hydroxy atorvastatin and 0.2-20.0ng/mL for para-hydroxy atorvastatin. Clinical bioequivalence study was successfully investigated by the application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products 10mg amlodipine/80mg atorvastatin in a single dose. In this study, 29 healthy volunteers were participated in randomized, two periods, double blend, open label cross over design. Pharmacokinetic parameters of C(max), AUC(0-t) and AUC(0-∞) were calculated to compare a test product with CADUET(®) reference product.

Plasma concentrations of 25-hydroxyvitamin D among Jordanians: Effect of biological and habitual factors on vitamin D status

BackgroundVitamin D is cutaneously synthesized following sun exposure (vitamin D3) as well as it is derived from dietary intake (vitamin D3 and D2). Vitamin D2 and D3 are metabolized in the liver to 25-hydroxyvitamin D (25(OH)D). This metabolite is considered the functional indicator of vitamin D stores in humans. Since Jordan latitude is 31°N, cutaneous synthesis of vitamin D3 should be sufficient all year round. However, many indications reveal that it is not the case. Thus, this study was conducted to determine the 25(OH)D status among Jordanians.MethodsThree hundred healthy volunteers were enrolled in a cross sectional study; 201 females and 99 males. 25(OH)D and calcium concentrations were measured by enzyme linked immunosorbent assay and spectroscopy techniques, respectively. All participants filled a study questionnaire that covered age, sex, height, weight, diet, and dress style for females. Females were divided according to their dress style: Western style, Hijab (all body parts are covered except the face and hands), and Niqab (all body parts are covered including face and hands).ResultsThe average plasma 25(OH)D levels in males and females were 44.5 ± 10.0 nmol/l and 31.1 ± 12.0 nmol/l, respectively. However, when female 25(OH)D levels were categorized according to dress styles, the averages became 40.3, 31.3 and 28.5 nmol/l for the Western style, Hijab and Niqab groups, respectively. These 25(OH)D levels were significantly less than those of males (p < 0.05, 0.001, 0.001, respectively). In addition, the plasma 25(OH)D levels of the Western style group was significantly higher than those of Hijab and Niqab groups (p < 0.001). Furthermore, dairy consumption in males was a positive significant factor in vitamin D status. Even though calcium concentrations were within the reference range, the Hijab and Niqab-dressed females have significantly less plasma calcium levels than males (p < 0.01).ConclusionsVery low plasma 25(OH)D levels in females wearing Hijab or Niqab are highly attributed to low sunlight or UVB exposure. In addition, most of males (76%) and Western style dressed females (90%) have 25(OH)D concentrations below the international recommended values (50 nmol/l), suggesting that although sun exposure should be enough, other factors do play a role in these low concentrations. These findings emphasize the importance of vitamin D supplementation especially among conservatively dressed females, and determining if single nucleotide polymorphisms of the genes involved in vitamin D metabolism do exist among Jordanians.

Bioadhesive Controlled Metronidazole Release Matrix Based on Chitosan and Xanthan Gum

Metronidazole, a common antibacterial drug, was incorporated into a hydrophilic polymer matrix composed of chitosan xanthan gum mixture. Hydrogel formation of this binary chitosan-xanthan gum combination was tested for its ability to control the release of metronidazole as a drug model. This preparation (MZ-CR) was characterized by in vitro, ex vivo bioadhesion and in vivo bioavailability study. For comparison purposes a commercial extended release formulation of metronidazole (CMZ) was used as a reference. The in vitro drug-release profiles of metronidazole preparation and CMZ were similar in 0.1 M HCl and phosphate buffer pH 6.8. Moreover, metronidazole preparation and CMZ showed a similar detachment force to sheep stomach mucosa, while the bioadhesion of the metronidazole preparation was higher three times than CMZ to sheep duodenum. The results of in vivo study indicated that the absorption of metronidazole from the preparation was faster than that of CMZ. Also, MZ-CR leads to higher metronidazole Cmax and AUC relative to that of the CMZ. This increase in bioavailability might be explained by the bioadhesion of the preparation at the upper part of the small intestine that could result in an increase in the overall intestinal transit time. As a conclusion, formulating chitosan-xanthan gum mixture as a hydrophilic polymer matrix resulted in a superior pharmacokinetic parameters translated by better rate and extent of absorption of metronidazole.

Saliva versus plasma pharmacokinetics: theory and application of a salivary excretion classification system.

The aims of this work were to study pharmacokinetics of randomly selected drugs in plasma and saliva samples in healthy human volunteers, and to introduce a Salivary Excretion Classification System. Saliva and plasma samples were collected for 3-5 half-life values of sitagliptin, cinacalcet, metformin, montelukast, tolterodine, hydrochlorothiazide (HCT), lornoxicam, azithromycin, diacerhein, rosuvastatin, cloxacillin, losartan and tamsulosin after oral dosing. Saliva and plasma pharmacokinetic parameters were calculated by noncompartmental analysis using the Kinetica program. Effective intestinal permeability (Peff) values were estimated by the Nelder-Mead algorithm of the Parameter Estimation module using the SimCYP program. Peff values were optimized to predict the actual average plasma profile of each drug. All other physicochemical factors were kept constant during the minimization processes. Sitagliptin, cinacalcet, metformin, tolterodine, HCT, azithromycin, rosuvastatin and cloxacillin had salivary excretion with correlation coefficients of 0.59-0.99 between saliva and plasma concentrations. On the other hand, montelukast, lornoxicam, diacerhein, losartan and tamsulosin showed no salivary excretion. Estimated Peff ranged 0.16-44.16 × 10(-4) cm/s, while reported fraction unbound to plasma proteins (fu) ranged 0.01-0.99 for the drugs under investigation. Saliva/plasma concentrations ratios ranged 0.11-13.4, in agreement with drug protein binding and permeability. A Salivary Excretion Classification System (SECS) was suggested based on drug high (H)/low (L) permeability and high (H)/low (L) fraction unbound to plasma proteins, which classifies drugs into 4 classes. Drugs that fall into class I (H/H), II (L/H) or III (H/L) are subjected to salivary excretion, while those falling into class IV (L/L) are not. Additional data from literature was also analyzed, and all results were in agreement with the suggested SECS. Moreover, a polynomial relationship with correlation coefficient of 0.99 is obtained between S* and C*, where S* and C* are saliva and concentration dimensionless numbers respectively. The proposed Salivary Excretion Classification System (SECS) can be used as a guide for drug salivary excretion. Future work is planned to test these initial findings, and demonstrate SECS robustness across a range of carefully selected (based on physicochemical properties) drugs that fall into classes I, II or III.

Pharmacokinetics and pharmacodynamics profiles of enalapril maleate in healthy volunteers following determination of enalapril and enalaprilat by two specific enzyme immunoassays

Background and objectives:  Most of the pharmacokinetic (PK) parameters for enalapril and enalaprilat were established following determination of the drug and its metabolite, using angiotensin converting enzyme (ACE) inhibition assays. In these methods, enalapril has to be hydrolysed to enalaprilat first and then assayed. The purpose of this study was to re‐estimate the PK parameters of enalapril and enalaprilat in healthy volunteers using two specific enzyme immunoassays for enalapril and enalaprilat.

UDP-glucuronosyltransferase 1A4 (UGT1A4) polymorphisms in a Jordanian population

Glucuronidation is one of the most important phase II metabolic pathways. It is catalyzed by a family of UDP-glucuronosyltransferase enzymes (UGTs). One of the subfamilies is UGT1A. Allele frequencies in UGT1A4 differ among ethnic groups. The aim of this study was to determine the allelic frequency of two most common defective alleles: UGT1A4*2 and UGT1A4*3 in a Jordanian population. A total of 216 healthy Jordanian Volunteers (165 males and 51 females) were included in this study. Genotyping for UGT1A4*1, UGT1A4*2 and UGT1A4*3 was done using a well established polymerase chain reaction–restriction fragment length polymorphism test. Among 216 random individuals studied for UGT1A4*2 mutation there were 26 individuals who were heterozygous, giving a prevalence of 12% and an allele frequency of 6.5%. Only one individual was homozygous for UGT1A4*2. The UGT1A4*3 mutation was detected as heterozygous in 9 of 216 individuals indicating a prevalence of 4.2% and allele frequency of 3.5%. Three individuals were homozygous for the UGT1A4*3 indicating a prevalence of 1.4%. The prevalence of UGT1A4*2 is similar to the Caucasians but different from other populations whilst the UGT1A4*3 prevalence in the Jordanian population is distinct from other populations. Our results provide useful information for the Jordanian population and for future genotyping of Arab populations in general.

Simultaneous determination of triprolidine and pseudoephedrine in human plasma by liquid chromatography-ion trap mass spectrometry

A highly efficient, selective and specific method for simultaneous quantitation of triprolidine and pseudoephedrine in human plasma by liquid chromatography-ion trap-tandem mass spectrometry coupled with electro spray ionization (LC-ESI-ion trap-tandem MS) has been validated and successfully applied to a clinical pharmacokinetic study. Both targeted compounds together with the internal standard (gabapentin) were extracted from the plasma by direct protein precipitation. Chromatographic separation was achieved on a C(18) ACE((R)) column (50.0mmx2.1mm, 5mum, Advance Chromatography Technologies, Aberdeen, UK), using an isocratic mobile phase, consisting of water, methanol and formic acid (55:45:0.5, v/v/v), at a flow-rate of 0.3mL/min. The transition monitored (positive mode) was m/z 279.1-->m/z 208.1 for triprolidine, m/z 165.9-->m/z 148.0 for pseudoephedrine and m/z 172.0-->m/z 154.0 for gabapentin (IS). This method had a chromatographic run time of 5.0min and a linear calibration curves ranged from 0.2 to 20.0ng/mL for triprolidine and 5.0-500.0ng/mL for pseudoephedrine. The within- and between-batch accuracy and precision (expressed as coefficient of variation, %C.V.) evaluated at four quality control levels were within 94.3-106.3% and 1.0-9.6% respectively. The mean recoveries of triprolidine, pseudoephedrine and gabapentin were 93.6, 76.3 and 82.0% respectively. Stability of triprolidine and pseudoephedrine was assessed under different storage conditions. The validated method was successfully employed for the bioequivalence study of triprolidine and pseudoephedrine formulation in twenty six volunteers under fasting conditions.

Everolimus (RAD001) sensitizes prostate cancer cells to docetaxel by down-regulation of HIF-1α and sphingosine kinase 1

Resistance to docetaxel is a key problem in current prostate cancer management. Sphingosine kinase 1 (SK1) and phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways have been implicated in prostate cancer chemoresistance. Here we investigated whether their combined targeting may re-sensitize prostate cancer cells to docetaxel. In hormone-insensitive PC-3 and DU145 prostate cancer cells the mTOR inhibitor everolimus (RAD001) alone did not lead to significant cell death, however, it strongly sensitized cells to low levels (5 nM) of docetaxel. We show that mTOR inhibition has led to a decrease in hypoxia-inducible factor-1α (HIF-1α) protein levels and SK1 mRNA. HIF-1α accumulation induced by CoCl2 has led to a partial chemoresistance to RAD001/docetaxel combination. SK1 overexpression has completely protected prostate cancer cells from RAD001/docetaxel effects. Using gene knockdown and CoCl2 treatment we showed that SK1 mRNA expression is downstream of HIF-1α. In a human xenograft model in nude mice single RAD001 and docetaxel therapies induced 23% and 15% reduction in prostate tumor volume, respectively, while their combination led to a 58% reduction. RAD001 alone or in combination with docetaxel has suppressed intratumoral mTOR and SK1 signaling, however as evidenced by tumor size, it required docetaxel for clinical efficacy. Combination therapy was well tolerated and had similar levels of toxicity to docetaxel alone. Overall, our data demonstrate a new mechanism of docetaxel sensitization in prostate cancer. This provides a mechanistic basis for further clinical application of RAD001/docetaxel combination in prostate cancer therapy.