Development and assessment of immediate-release tablets containing clopidogrel bisulphate & aspirin-strategy for optimizing the combination formulation.

The main goal of the study was to improve the compliance and convenience of patients by designing and development of an immediate release (IR) fixed-dose combination (Clopidogrel bisulphate and Aspirin) tablets. The proposed combination product utilizes Clopidogrel to protect the moisture-sensitive aspirin component, enhancing its stability against atmospheric conditions. Response-surface approach (Design Expert vs. 13) was used to generate this IR tablet by calculating the right composition of independent variables such as Microcrystalline cellulose 102, pregelatinized starch and Hydroxypropyl cellulose. 32 factorial design was used to estimate the effects of these independent variables on the responses of dependent variables (disintegration & friability) and constructed a total of nine (9) formulations. Pre and Post formulation, quality control parameters were investigated as per pharmacopeia. A systematic approach was used for the optimization process and a prototype checkpoint batch (CPB) based on the better contrast of independent variables was prepared. In vitro analysis of formulations was carried out to estimate the responses. Friability was found in the range of 0.088-1.076%w/w, except F1 = 1.076 all are within limits (NMT 1.0%). Disintegration time was recorded 7.3 ± 1.20 as lower and 24.5 ± 1.63 min was the highest. The release of drugs from their dosage form was fast and rapid, for clopidogrel after 15min was 70.42-96.82% with SD ± 8.71 and aspirin was 69.88-91.49% in 15 min with SD ± 6.41, all the tablets were released more than 80% in 20 min. The stability outcomes of CPB tablets after 15 days of stress study (60 ± 2°C and 75 ± 5%) indicated good compatibility and stability of APIs with excipients. It was concluded that the direct compression method can be preferred to prepare a combination product with cost-effectiveness. It was also concluded that the proposed methodology could increase Aspirin's stability and allow for an aqueous coating system to finish the product with a film coating. By using Design Expert software, the best composition of the formulation can be selected and optimized in a short period of time with minimum trial and errors. The results also demonstrated that the use of a fixed-dose combination tablet instead of the individual is expected to be more convenient to patients and thus improves patient compliance and decreases the occurrence of adverse effects and side effects.

Pretreatment with antiplatelet drugs improves the cardiac function after myocardial infarction without reperfusion in a mouse model.

BACKGROUND: Reperfusion therapy is known to improve prognosis and limit myocardial damage after myocardial infarction (MI). The administration of antiplatelet drugs prior to percutaneous coronary intervention also proves beneficial to patients with acute MI (AMI). However, a good number of AMI patients do not receive reperfusion therapy, and it is not clear if they would benefit from antiplatelet pre-treatment. METHODS: Experimental C57BL/6 mice were randomly allocated to five groups: the sham group, control, post-treatment, pre-treatment, and pre- and post-treatment groups. Acetylsalicylic acid (15 mg/kg), clopidogrel (11 mg/kg), ticagrelor (27 mg/kg), and prasugrel (1.5 mg/kg) were intragastrically administered in the treatment groups. On day 7 post MI, cardiac function and cardiac fibrosis were evaluated using echocardiography and Masson's trichrome staining, respectively. Histopathological examinations were performed on tissue sections to grade inflammatory cell infiltration. Platelet inhibition was monitored by measuring thrombin-induced platelet aggregation. RESULTS: Left ventricular ejection fraction and fractional shortening improved significantly (p < 0.01) in the pre-treatment groups when compared to the post-treatment and control groups. A significant (p < 0.01) decrease in cardiac fibrosis was observed in the pre-treatment group, compared with the posttreatment and control groups. Inflammatory cell infiltration significantly decreased in the pre-treatment group compared with the control group (p < 0.05). Thrombin-induced platelet aggregation was significantly inhibited by antiplatelet drugs, but increased with the exposure to H2O2. CONCLUSIONS: In the absence of reperfusion therapy, pre-treatment with antiplatelet drugs successfully improved cardiac function, reduced cardiac fibrosis and inflammatory cell infiltration, and inhibited oxidative stress-induced platelet aggregation after MI in the mouse model.

Develop a High-Throughput Screening Method to Identify C-P4H1 (Collagen Prolyl 4-Hydroxylase 1) Inhibitors from FDA-Approved Chemicals.

Collagen prolyl 4-hydroxylase 1 (C-P4H1) is an α-ketoglutarate (α-KG)-dependent dioxygenase that catalyzes 4-hydroxylation of proline on collagen. C-P4H1-induced prolyl hydroxylation is required for proper collagen deposition and cancer metastasis. Therefore, targeting C-P4H1 is considered a potential therapeutic strategy for collagen-related cancer progression and metastasis. However, no C-P4H1 inhibitors are available for clinical testing, and the high content assay is currently not available for C-P4H1 inhibitor screening. In the present study, we developed a high-throughput screening assay by quantifying succinate, a byproduct of C-P4H-catalyzed hydroxylation. C-P4H1 is the major isoform of collagen prolyl 4-hydroxylases (CP4Hs) that contributes the majority prolyl 4-hydroxylase activity. Using C-P4H1 tetramer purified from the eukaryotic expression system, we showed that the Succinate-GloTM Hydroxylase assay was more sensitive for measuring C-P4H1 activity compared with the hydroxyproline colorimetric assay. Next, we performed high-throughput screening with the FDA-approved drug library and identified several new C-P4H1 inhibitors, including Silodosin and Ticlopidine. Silodosin and Ticlopidine inhibited C-P4H1 activity in a dose-dependent manner and suppressed collagen secretion and tumor invasion in 3D tissue culture. These C-P4H1 inhibitors provide new agents to test clinical potential of targeting C-P4H1 in suppressing cancer progression and metastasis.

Box-Behnken design directed optimization for sensitivity assessment of anti-platelet drugs.

Optimization of electrospray ionization (ESI) parameters is routinely carried out by one factor at a time (OFAT) or auto-tune software (ATS). Design of experiments (DOE) approach has been reported to be an excellent alternative to OFAT or ATS. Box-Behnken Design (BBD) was successfully used to optimize ESI parameters like nebulizing gas flow rate, desolvation line temperature, heat block temperature, and drying gas flow rate for [M + H]+ intensity of Clopidogrel bisulfate (CLP) and Ticlopidine (TLP). BBD model was found to be significant with p < .0001 for both CLP and TLP. The predicted and optimized (OL) ESI parameters were used for chromatographic analysis and were compared against three levels of ESI parameters, i.e. low level (LL), medium level (ML), and high level (HL). The OL ESI parameters were subjected to chromatographic analysis and its mean peak area was significantly higher than mean peak area for LL, ML, and HL ESI in case of CLP and TLP (p < .001). However, no significant difference was observed between the mean peak area for ML and OL of TLP. Thus, BBD can be considered with 29 trials to optimize four mass spectrometric parameters. The liquid chromatographic parameters percentage of methanol, percentage of formic acid and flow rate were also optimized using BBD. However, the optimized method did not significantly influence the peak response over the non-optimized method.

Elucidating the interaction of ticlopidine with serum albumin and its role in bilirubin displacement in vitro.

Ticlopidine is an anti-platelet drug that functions as a P2Y12 receptor antagonist. The present study provides a detailed characterization of interaction of ticlopidine with a model transport protein, bovine serum albumin (BSA) as well as an assessment of its bilirubin displacing ability using a multi-spectroscopic approach in combination with isothermal titration calorimetry. The value of binding constant determined using ITC studies was found to be 3.03 × 103 M-1 with a binding stoichiometry of approximately 1:1. Competitive site marker experiments indicate that ticlopidine binds to Sudlow site I, located in subdomain IIA of BSA. In addition, Circular dichroism and 3D fluorescence spectroscopy indicated structural and conformational changes in BSA on interaction with ticlopidine. Thermodynamic parameters suggested that the reaction was spontaneous, exothermic, entropically driven, and involved hydrophobic interactions. These results were well supported by those obtained through molecular docking studies. Additionally, the effect of ticlopidine on bilirubin and albumin interaction was evaluated using the peroxidase method as well as through fluorescence spectroscopy. Ticlopidine was found to displace bilirubin from serum albumin. Moreover, the binding constant of bilirubin-serum albumin interaction also decreased in presence of ticlopidine. The results indicated that ticlopidine is a competitive displacer of bilirubin in vitro and may contribute to the incidences hyperbilirubinemia associated with the usage of this drug.

Interaction study between antiplatelet agents, anticoagulants, thyroid replacement therapy and a bioavailable formulation of curcumin (Meriva®).

OBJECTIVE: The objective of this clinical study is to evaluate possible interactions between antiplatelet agents, anticoagulants, thyroid hormone replacement therapy and a formulation of curcumin (Meriva®) that resulted effective for the complementary treatment of osteoarthritis. PATIENTS AND METHODS: Interaction between antiplatelet agents and Meriva® was evaluated by measuring anti-platelet activity with the in-vivo bleeding-time (BT) in patients assuming acetylsalicylic acid or ticlopidine or clopidogrel from at least 2 years. The BT was evaluated before and after 10 days of supplementation with Meriva®. The interaction between anticoagulants and Meriva® was evaluated in patients using warfarin or dabigatran for previous venous thrombosis. The INR level was evaluated before and after 10 days of supplementation with the curcumin formulation. Thyroid function tests in hypothyroid patients using LT4 replacement therapy (Eutirox®) were evaluated before and after 15 days of supplementation with Meriva®. Similarly, levels of glycemia and glycated hemoglobin were evaluated in diabetic patients in treatment with metformin, before and after 10 days of supplementation with the studied product. RESULTS: After 10 days of supplementation with Meriva® the average BT value was not significantly different for patients assuming acetylsalicylic acid, ticlopidine or clopidogrel at standard dosages. Similarly, after 10 days of Meriva® treatment, the INR level in the two groups of patients assuming warfarin or dabigatran was not statistically different from that observed at baseline. In the analyzed patients assuming LT4 or metformin, no interactions between the therapy and Meriva® were observed. CONCLUSIONS: Results from this non-interaction clinical study suggest that Meriva® does not interfere with the antiplatelet activity of the most common antiplatelet agents nor alters the INR values in stable patients assuming warfarin or dabigatran. Similarly, dosages of LT4 or metformin do not need to be adjusted in case of complementary treatment with Meriva®.

Enzyme Kinetics and Molecular Docking Studies on Cytochrome 2B6, 2C19, 2E1, and 3A4 Activities by Sauchinone.

Sauchinone, an active lignan isolated from the aerial parts of Saururus chinensis (Saururaceae), exhibits anti-inflammatory, anti-obesity, anti-hyperglycemic, and anti-hepatic steatosis effects. As herb-drug interaction (HDI) through cytochrome P450s (CYPs)-mediated metabolism limits clinical application of herbs and drugs in combination, this study sought to explore the enzyme kinetics of sauchinone towards CYP inhibition in in vitro human liver microsomes (HLMs) and in vivo mice studies and computational molecular docking analysis. In in vitro HLMs, sauchinone reversibly inhibited CYP2B6, 2C19, 2E1, and 3A4 activities in non-competitive modes, showing inhibition constant (Ki) values of 14.3, 16.8, 41.7, and 6.84 µM, respectively. Also, sauchinone time-dependently inhibited CYP2B6, 2E1 and 3A4 activities in vitro HLMs. Molecular docking study showed that sauchinone could be bound to a few key amino acid residues in the active site of CYP2B6, 2C19, 2E1, and 3A4. When sibutramine, clopidogrel, or chlorzoxazone was co-administered with sauchinone to mice, the systemic exposure of each drug was increased compared to that without sauchinone, because sauchinone reduced the metabolic clearance of each drug. In conclusion, when sauchinone was co-treated with drugs metabolized via CYP2B6, 2C19, 2E1, or 3A4, sauchinone-drug interactions occurred because sauchinone inhibited the CYP-mediated metabolic activities.

A Review of Analytical Methods for the determination of Clopidogrel in Pharmaceuticals and Biological Matrices.

P2Y12 belongs to a group of G protein-coupled (GPCR) purinergic receptors and is a receptor for adenosine diphosphate (ADP). The P2Y12 receptor is involved in platelet aggregation and acts as a biological target for treating thromboembolisms and other clotting disorders. The use of Clopidogrel (CLO) has improved the morbidity and mortality endpoints including cardiovascular death, recurrent myocardial infarction (MI) and stroke at 30 days after percutaneous coronary intervention (PCI). CLO is one such drug that specifically and irreversibly inhibits the P2Y12 subtype of ADP receptor. This review delivers a detail description of various analytical methods published for the estimation of CLO and its combinations in pharmaceuticals and biological matrices. The review highlights the basic as well as advanced techniques performed for estimating CLO. The most commonly used assay techniques were UV and Visible spectrophotometry, high performance liquid chromatography (HPLC), high performance thin layer chromatography (HPTLC), micellar liquid chromatography (MLC), micellar electro kinetic chromatography (MEKC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Despite other analytical methods employed for the assay of CLO, the review reveals that the technique of HPLC with UV detection was widely used.

Raman chemical imaging for spectroscopic screening and direct quantification of falsified drugs.

Falsified drugs are a threat to the health of patients. The analytical control of such products contributes to the fight against this global issue. Raman chemical imaging is a method that relies on consecutive measurements at the surface of a sample, combining spectroscopy, microscopy and chemometrics. This article explores the capabilities of this analytical technique proposing an innovative methodology with spectroscopic screening for the identification of chemical compounds and the direct quantification of the active substance (without prior calibration). Two chemometric methods were used: Multivariate Curve Analysis - Alternate Least Squares for the qualitative analysis and Direct Classical Least Squares for the quantitative analysis. The methodology was optimized with samples prepared in the laboratory and validation parameters were studied. The methodology was then applied to real (authentic and falsified) samples of Viagra® and Plavix®. Despite the presence of fluorescence emission in some samples, the methodology succeeded in the detection of active pharmaceutical ingredients, and in the discrimination of three salts of clopidogrel (in generic formulations of Plavix®). The quantitative deviation from the reference method ranged from -15% to +24% of the active substance content. This deviation may be considered to be acceptable since it is sufficient for assessing the risk to the health of patients and for quickly alerting the health authorities.

Development and validation of a sensitive and rapid UHPLC-MS/MS method for the simultaneous quantification of the common active and inactive metabolites of vicagrel and clopidogrel in human plasma.

Vicagrel, an analog of clopidogrel, is a novel thienopyridine P2Y12 antagonist in clinical development in China for the treatment of acute coronary syndromes. Vicagrel and clopidogrel are prodrugs requiring metabolic activation to produce a pharmacologically active thiol metabolite (H4) in vivo. The formation of H4 from vicagrel in humans is promising more efficient and consistent than that from clopidogrel. After oral dosing of vicagrel or clopidogrel to humans, the active thiol metabolite H4 and two inactive metabolites closely related to H4 formation (the thiol metabolite H3 and the S-methylated metabolite SM3) were observed in plasma; SM3 was the most abundant drug-related component of vicagrel in circulation. In this study, a sensitive, rapid, and reliable UHPLC-MS/MS method was developed for the simultaneous determination of derivatized H3 (MP-H3), derivatized H4 (MP-H4), and SM3 in human plasma to investigate the pharmacokinetics of vicagrel in healthy subjects compared with clopidogrel. After extracted from 50µL of plasma by simple protein precipitation, the analytes and stable isotope-labeled internal standards were separated on an Acquity UPLC BEH C18 column (100mm×2.1mm, 1.7µm). The mobile phase was acetonitrile-water-formic acid (45:55:0.0275, v/v/v) delivered at a flow rate of 0.45mL/min under isocratic elution. The total chromatographic run time was 6min. MP-H3, MP-H4, and SM3 were separated from their corresponding isomers under the chromatographic conditions. Mass spectrometric detection was conducted in multiple reaction monitoring mode on an AB Sciex Qtrap 5500 System using a positive electrospray ionization interface. The calibration curves were linear in the following ranges: 0.100-200ng/mL for MP-H3 and MP-H4 and 1.00-1000ng/mL for SM3. The intra- and inter-day accuracy and precision for each analyte at all concentrations were within acceptable limits (±15%). The validated method was successfully applied to compare the pharmacokinetics of vicagrel and clopidogrel after a single oral administration to healthy subjects in the first-in-human study of vicagrel.

Developments in the synthesis of the antiplatelet and antithrombotic drug (S)-clopidogrel.

S-(+)-Methyl 2-(2-chlorophenyl)-2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate, also known as (S)-clopidogrel, is marketed under the trade names Plavix and Iscover. It is a potent thienopyridine-class of antithrombotic and antiplatelet drug (antiaggregant). Among the two available stereoisomers of clopidogrel, for pharmaceutical activities only the S-enantiomer is applicable, as no antithrombotic activity is observed in the R-enantiomer and causes political upheavals and social turmoil in animal experiments. Worldwide sales of Plavix amounted to $6.4 billion yearly, which ranks second. Attributed to the increased demand of (S)-clopidogrel drug, it provoked the synthetic community to devise facile synthetic approaches. This review aims to summarize the synthetic methods of (S)-clopidogrel drug reported in the literature. The present review discusses the pros and cons of each synthetic methodology, which would be beneficial to the scientific community for further developments in the synthetic methodologies for (S)-clopidogrel. In addition, the compilation approach of literature-reported synthetic strategies of (S)-clopidogrel in one platform is advantageous, supportive, and crucial for the synthetic community to elect the best synthetic methodology of (S)-clopidogrel and to create new synthesis ideas.

Overcoming Clopidogrel Resistance: Three Promising Novel Antiplatelet Drugs Developed in China.

Clopidogrel is one of the most frequently prescribed drugs worldwide; however, the presence of clopidogrel resistance and high susceptibility to genetic variations and drug interactions are facilitating the development of other antiplatelet drugs. To overcome clopidogrel resistance, several promising clopidogrel analogues have been developed in China, such as vicagrel (and its deuterated analogues), PLD-301, and W1. These novel chemical analogues are all characterized by much faster and more efficient bioconversion to clopidogrel thiolactone (or 2-oxo-clopidogrel, the precursor of clopidogrel active metabolite) in the intestine than clopidogrel itself through bypassing the first-step P450-mediated oxidation of clopidogrel in the liver. Of them, metabolic conversion of vicagrel and PLD-301 to 2-oxo-clopidogrel is catalyzed by intestinal carboxylesterase 2 and alkaline phosphatase, respectively. In this review article, we summarized all evidence on highly efficient bioconversion to their shared precursor of clopidogrel active metabolite and the mechanisms underlying such a pronounced improvement. These drugs in the pipeline would be promising antiplatelet drugs that could be superior to clopidogrel in future patient care.

Effect of directly compressible excipient and treated agar on drug release of clopidogrel oral disintegrating tablets.

AIM: In the present investigation, oral dissolving/dispersible/disintegrating tablets (ODTs) of clopidogrel were designed with a view to enhance the bioavailability and patient compliance by two different methods, namely, direct compression and effervescent methods using directly compressible excipient and treated agar (TAG). MATERIALS & METHODS: In the direct compression method, TAG was used as a disintegrant and another method used a mixture of sodium bicarbonate and tartaric acid along with TAG as disintegrants. RESULTS: Among the directly compressed tablets, treated agar formulation 3 and among the effervescent tablets, treated agar and effervescent formulation 4 was found to be promising. CONCLUSION: Treated agar formulation 3 prepared by direct compression method emerged as an overall best formulation based on the in vitro drug release characteristics.

Inhibitory Effects of Trapping Agents of Sulfur Drug Reactive Intermediates against Major Human Cytochrome P450 Isoforms.

In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM) fortified with ß-nicotinamide adenine dinucleotide phosphate (NADPH), resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP) oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A). Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM)). Three trapping agents (dimedone, N-acetyl-lysine and arsenite) did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine) and trapping agent (NEM) were chosen for further studies. For the same amount of ticlopidine (1 µM), increasing concentrations of the trapping agent NEM (0.007-40 mM) resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM), due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.

Preventing graft restenosis after coronary artery bypass grafting with tissue-type plasminogen activator.

OBJECTIVE: To explore the feasibility and safety of using tissue-type plasminogen activator (t-PA) to prevent graft restenosis after coronary artery bypass grafting (CABG). METHODS: In this prospective observational study, 37 patients underwent CABG between June 2009 and May 2013. These patients were grouped according to the anti-coagulation strategy after surgery: t-PA (n = 12) and conventional treatments (n = 25). In the t-PA group, the patients received acetylsalicylic acid (ASA) and clopidogrel plus intravenous infusion of t-PA (0.25 mg/kg/day) starting at 24 h after surgery and that lasted for 3 days. In the conventional group, the patients received only ASA and clopidogrel. 64-row spiral computed tomographic coronary angiography was performed at 1 week, 1, and 3 months after surgery to evaluate the patency of the graft vessel. RESULTS: The mean stenosis severity of the saphenous vein grafts was lower in the t-PA group compared with the conventional group at 3 months after surgery (p < 0.05), but there was no significant difference at 1 week and 1 month (p > 0.05). The patency rate of the grafts was not significantly different between the two groups at 1 week, 1, and 3 months after surgery (p > 0.05). CONCLUSION: Early application of t-PA after CABG was feasible and safe, and might help prevent early restenosis of SV grafts. Additional clinical randomized trials are necessary to address this issue.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA.

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103M-1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Design and Development of Clopidogrel Bisulfate Gastroretentive Osmotic Formulation Using Quality by Design Tools.

Clopidogrel bisulfate (CBS) is antiplatelet drug and it is becoming a drug of choice in the treatment and management of prevention of heart attacks and strokes. CBS is stable and soluble in acidic pH; therefore, retention in stomach for prolonged period appears to be beneficial for controlling the bioavailability. The gastroretentive osmotic system (GROS) facilitates prolonged retention of drug in stomach and provides zero-order drug release. A complex formulation like GROS poses many challenges, and QbD tools can help in designing robust formulation which takes all aspects of product and process development in order to deliver a robust product. The GROS was formulated in three steps: core tablet, osmotic tablet, and gastroretentive osmotic tablet. The design of experiment was used for screening and optimization of formulation and process-related parameters. The dissolution study was carried out to analyze the release pattern of tablet. The optimized batch O-4 showed cumulative drug release of 19.43, 30.49, 64.41, and 85.11% at 2, 4, 8, and 12 h which is in the range of QTPP predictions. The novel technique of GROS was implemented successfully which demonstrates robust design giving consistent and desired results.

DEVELOPMENT OF COMPRESSED COATED POLYPILL WITH MUCOADHESIVE CORE COMPRISING OF ATORVASTATIN/CLOPIDOGREL/ASPIRIN USING COMPRESSION COATING TECHNIQUE.

The study was conducted to formulate and assess a novel polypill comprising of atorvastatin calci- um (ATVC), clopidogrel bisulfate (CLB) and aspirin (ASP) which, after in vivo correlation, can be intended for use in hyperlipidemic chronic heart disease patients. Polypill was made by the compression coating technique (CCT) with multiple active ingredients along with different concentrations of mucoadhesive and sustained release polymers, i.e., Carbopol 934 (CAB), Methocel k15 (MTH) and sodium carboxymethyl cellulose (NaCMC). The effect of different concentration of polymers on physical properties, wash off time, mucoadhe- sion strength, swelling behavior, surface pH and drug release kinetics were investigated. In vitro drug release studies showed that combination of CAB-NaCMC (1 : 1) retarded drug release up to 96.7 ± 1.15%, while com- bination of CAB-MTH and MTH-NaCMC retarded drug release up to 81.9 ± 1.5% and 101.4 ± 1.3%, respec- tively, at the same polymer concentration. Core enteric coated tablet of ATVC (K 11) was compressed over with CLB and ASP granules with the help of CCT and produced the desired results with zero order release rate thus indicating successful formulation of proposed polypill.

CHARACTERIZATION OF BINDING SITES OF CLOPIDOGREL AND INTERFERENCE OF LINOLEIC ACID AT THE BINDING SITE ON BOVINE SERUM ALBUMIN.

Binding of clopidogrel to serum albumin has been characterized in the presence and absence of linoleic acid by equilibrium dialysis method where ranitidine and diazepam were used as specific probes. Our findings suggested two binding sites for clopidogrel: a high affinity site (k1 = 11.5 x 105 M⁻¹) with low capaci- ty (n1 = 1.2) and low affinity site (k2 = 2.1 x 105 M⁻¹) with high capacity (n. = 9.3). Interaction of linoleic acid with clopidogrel in the presence of ranitidine shows an increment of clopidogrel from 71 to 85.5% at concen- tration of (1 x 105 M) to (6 x 105 M). However, interaction of linoleic acid with clopidogrel in the presence of diazepam exhibits significant rise in free fraction of clopidogrel from 93 to 116% at concentration of (0 x 10' M) to (4 x 105 M). At higher concentrations, linoleic acid displaced clopidogrel from its binding sites on serum albumin. This may cause escalation of free drug in the blood, which alters pharmacokinetic properties of clopi- dogrel taken with high fat diet.

W1, a Novel Oral Antiplatelet Agent With Less Resistance Than Clopidogrel.

Clopidogrel (CLO) is a clinical antiplatelet agent, about which there are major concerns because its antiplatelet efficiency decreases with insufficient metabolic activation, leading to "clopidogrel resistance." We aimed to determine the antiplatelet effects of W1, a novel molecule composed of 2-O-clopidogrel and aspirin (1:1 ratio), on platelet aggregation ex vivo and thrombus formation in vivo, and its susceptibility to CLO resistance in combination with other therapies in rats. Platelets were prepared, and an arteriovenous shunt thrombosis model was established using Wistar rats to measure platelet aggregation and thrombus formation, respectively. W1 markedly inhibited adenosine 5'-diphosphate (ADP)-induced platelet aggregation and thrombus formation dose dependently (0.3, 1, and 3 mg/kg). W1 (3 mg/kg) acted rapidly at 0.5 hours and lasted for 72 hours. W1 prolonged bleeding and clotting times in mice, confirming its antithrombotic properties. Compared with CLO 10 mg/kg, the positive control, W1 3 mg/kg exerted equivalent effects on the above specifications. In addition, cyclic adenosine monophosphate levels, measured in rat platelets, increased rapidly after prostaglandin E1 (alprostadil) stimulation of the vehicle control (0.5% methyl cellulose suspension) and W1 (3 mg/kg)-treated groups. ADP (50 µm) reduced the control levels more remarkably than W1 did (P < 0.05 in 3 minutes or P < 0.001 at 5 minutes), suggesting that W1 suppressed ADP-induced cyclic adenosine monophosphate reduction. This was associated with a significant platelet reactivity inhibition measured using the vasodilator-stimulated phosphoprotein assay. CLO or W1 coadministration with or without omeprazole and amlodipine to rats to investigate the pharmacodynamic interactions revealed that W1 exhibited more stable and potent antithrombotic effects than CLO did. In conclusion, both W1 and CLO showed antiplatelet and antithrombotic effects, while the former exhibited less CLO resistance in combination with omeprazole or amlodipine, 2 drugs that inhibit CLO metabolism. Therefore, this study implies that W1 may be a promising oral antiplatelet agent for reducing CLO resistance after percutaneous coronary intervention.