An-Najah National University Faculty of Graduate Studies Extemporaneous Compounding and Physiological Modeling of Amlodipine/Valsartan Suspension By Wafa’ Jassim Mahmoud Aabed Supervisor Dr. Asma Radwan This Thesis is Submitted in Partial Fulfillment of the Requirements for the Degree of Master in Pharmaceutical sciences, Faculty of Graduate Studies, An-Najah National University, Nablus - Palestine. 2018 III Dedication I dedicate this to Allah Almighty my guardian and my source of inspiration and wisdom. To the honest heart who prayed in the dark nights so I can follow the light and shine with the sunrise, you’ll always be the dearest gift I ever dreamed of. To any person who has ever thought highly enough of me to offer me any sort of help or advice. This is only the beginning of my journey… IV Acknowledgement My highest appreciation and feelings of gratitude are to my family… my sunshine… My mother and My father who have been very supportive, thank you for believing in me and giving me the strength to chase my dreams… My Brothers and Sisters, you've always been there when I needed you the most, having you in my life is a blessing… Special thanks to Dr. Asma Radwan for her supervision and endurance… Without her endless support and patience this work could never be achieved… My deep gratitude is to Professor Dr. Abdel Naser Zaid for his continuous help and encouragement. Special thanks for Pharmacare PLC for their help and support… To all my friends, thank you for the encouragement during my moments of crisis. You kept pushing me up towards achieving my goals, your friendship made me stronger; you'll always be in my heart. Wafa Aabed V اإلقرار :موقع الرسالة التي تحمل العنوان أنا الموقع أدناه Extemporaneous Compounding and Physiological Modeling of Amlodipine/Valsartan Suspension أقز تأى ها اشتولت عليَ الزسالح ُْ ًتاج جِذي الخاص، تاستثٌاء ها توت اإلشارج إليَ حيثوا ّرد، يقذم هي قثل لٌيل أي درجح أّ لقة علوي أّ تحثي لذٓ ّأى ُذٍ الزسالح ككل، أّ أي جزء هٌِا لن .تحثيح أخزٓح أّ أي هؤسسح تعليوي Declaration The work provide in this thesis, unless otherwise referenced, is the researcher's own work, and has not been submitted elsewhere for any other degree or qualification. Student's name: :اسم الطالة Signature: :التوقيع Date: :التاريخ VI Table of Contents No Contents Pages Dedication III Acknowledgement IV Declaration V List of Tables VIII List of Figures IX List of Abbreviations X Abstract XII Chapter One: Introduction 1 1.1 Tablet scoring 1 1.2 Extemporaneous compounding 3 1.2.1 Formulation for extemporaneous suspension 3 1.2.2 Risks associated with extemporaneous compounding 6 1.2.3 Stability of extemporaneous formulations 7 1.3 Lack of Bioequivalence/Bioavailability data 10 1.4 In vitro Dissolution Testing 11 1.5 In silico 12 1.6 Antihypertensive Medications 14 1.6.1 Amlodipine 15 1.6.2 Valsartan 16 1.7 Aims of this study 19 Chapter two: Methodology 21 2.1 Materials, Equipment and Dosage form 21 2.2 Preparation of the extemporaneous suspension 22 2.3 pH measurements 23 2.4 Viscosity measurements 23 2.5 Stability study 24 2.5.1 Chemical stability 24 2.5.2 Physical stability 24 2.5.3 Microbiological stability 24 2.6 Drug release study 25 2.6.1 Dissolution 25 2.6.2 Statistical Analysis 26 2.7 The HPLC analysis 26 2.7.1 Instruments, Solutions and Chromatographic Conditions 26 2.7.2 Standard stock solution 27 2.7.3 Sample stock solution 28 2.8 Gastrointestinal simulation 28 VII Chapter Three: Results 33 3.1 The formulation 33 3.2 Viscosity Determination 33 3.3 Drug release study 34 3.4 Stability study 38 3.4.1 Physical stability 38 3.4.2 Chemical stability 38 3.4.3 Microbial Stability 39 3.5 HPLC analysis 40 3.6 Drug absorption simulation 40 3.6.1 Gastrointestinal simulation 40 Chapter Four: Discussion 45 Conclusion 52 References 53 ب الممخص VIII List of Tables No Table Title Page 1 Types of stability studies and their storage criteria 9 2 The composition of the AML/VAL (5/80) suspension formula 23 3 Plasma concentration-time profile of AML and VAL from Valzadepine® 5/80 tablets 30 4 Simulation input data 31 5 The rheological behavior of the extemporaneous suspension over different shear rates 33 6 Dissolution of AML and VAL from Valzadepine® tablets 36 7 The percentage of AML and VAL released from the tablet and suspension formulations at pH 6.8 as recommended by FDA and USP 37 8 The mean percentage of the active ingredient in AML/VAL suspension throughout 4 weeks period at room temperature 39 9 Microbial study results 39 10 AML observed and predicted pharmacokinetic parameters with percentage of prediction error 42 11 VAL observed and predicted pharmacokinetic parameters with percentage of prediction error 43 12 Confidence interval of pharmacokinetic parameters of AML/VAL suspension 51 IX List of Figures No Figure Title Page 1 The BCS as defined by Amidon 11 2 AML Besylate chemical structure 15 3 VAL chemical structure 17 4 The rheological behavior of the extemporaneous preparation over different shear rates 34 5 Release profiles of AML from the tablet and the suspension at different pH values. 35 6 Release profiles of VAL from the tablet and the suspension at different pH values. 36 7 Standard peaks of AML and VAL as eluted in HPLC analysis. 40 8 The simulated plasma profile of AML from Valzadepine® IR tablet 41 9 The simulated plasma profile of AML suspension 42 10 The simulated plasma profile of VAL from Valzadepine® IR tablet 43 11 The simulated plasma profile of VAL suspension 44 X List of Abbreviations ACAT Advanced Compartmental Absorption and Transit model ACN Acetonitrile ADME Absorption, Distribution, Metabolism, Excretion AML Amlodipine ARB Angiotensin Receptor Blocker AUC Area Under the Curve BCS Biopharmaceutical Classification System BE Bioequivalence BP Blood Pressure CACO-2 Human colon carcinoma cell line CFU Colony Forming Unit cm Centimeter cm 2 /s Centimeter square per second Cmax Maximum serum concentration CNS Central nervous system ºC Degree celsius Cp Viscosity f1 Difference factor f2 Similarity factor FDA Food and Drug Administration GI Gastrointestinal g/gm Gram H2 blocker Histamine 2 blocker Hr/s Hour/s HCl Hydrochloric acid HPLC High performance liquid chromatography IR Immediate Release IV Intravenous IVIVC In vitro-In vivo Correlation Kg Kilogram L Liter M1 Metabolite1 MeOH Methanol mg Milligram min Minute ml Milliliter XI µL Micro liter Mm Mellimeter µm Micro meter N Normality n Number of samples ng Nano gram nm Nanometer PDA Photo diode array PE Prediction Error Peff Effective permeability pH Potential of Hydrogen PK Pharmacokinetic pka Acid dissociation constant PTFE Polytetrafluro ethylene R1 Percentage of drug dissolved at each time point of the reference drug RH Relative Humidity RLD Reference Listed Drug rpm Round per minute SD Standard deviation sec Second SUPAC Scale Up and Post Approval Changes T1 Percentage of drug dissolved at each time point of the test drug tmax Time to maximal concentration USP United States Pharmacopeia UV Ultraviolet VAL Valsartan Vd Volume of distribution S. aureus Staphylococcus aureus P. aeroginosa Pseudomonas aeroginosa C. albicans Candida albican XII Extemporaneous Compounding and Physiological Modeling of Amlodipine/Valsartan Suspension By Wafa’ Jassim Mahmoud Aabed Supervisor Dr. AsmaRadwan Abstract Background: In case of absent liquid dosage form, crushing a tablet or dispersing a capsule would be the most convenient option for using these drugs in patients with dysphagia difficulties. However, no bioequivalence or stability studies are conducted for these extemporaneous preparations, which leads to confusion regarding its efficacy and safety. In silico and in vitro tools have proven to be useful in predicting the in vivo performance of drugs depending on its physicochemical properties and it’s in vitro dissolution profiles. No liquid formulation of combination Amlodipine and Valsartan is available in the pharmaceutical market for use in pediatric population with hypertension. Purpose: The aim of the present study was to prepare an extemporaneous suspension of Amlodipine and Valsartan from available commercial tablets, and to evaluate the stability and dissolution properties of the compounded suspension. Method: Amlodipine/Valsartan extemporaneous suspension was prepared from available commercial tablets Valzadepine®. The dissolution profiles XIII for the extemporaneous preparation and the commercial tablet was determined in different pH media. The physical, chemical and microbial stability of the compounded formulation was evaluated over one month period at room temperature. Moreover, In silico modeling using GastroPlus TM software was used to build absorption models for both drugs based on the in vitro dissolution data. The simulated plasma profile for both active ingredients were compared with the in vivo plasma profile to examine the similarity of the extemporaneous suspension and the commercial tablets. Results: The Amlodipine/Valsartan extemporaneous suspension was successfully prepared with acceptable organoleptic properties. The suspension was stable for four weeks period preserving its physical and chemical features. The release profiles of valsartan and Amlodepine from the suspension were similar to that from source tablet Valzadepine®. In silico modeling predicted similarity of the extemporaneous suspension and the commercial tablets. Conclusion: Amlodipine/Valsartan extemporaneous suspension could be prepared from available commercial tablets. Moreover, GastroPlus TM can be applied along with the in vitro dissolution in order to affirm similarity in extemporaneous compounding situations. 2 1 Chapter One Introduction Among all pharmaceutical formulations; oral preparations are still the most popular and convenient. When considering pediatrics and geriatrics with swallowing difficulty, liquid preparations are the most preferred formulations due to the ease of administration, flexibility of the administered doses. In case of absence of liquid preparation of an active ingredient; health care providers tend to split or crush the oral solid dosage form ignoring its safety and efficacy to get access to the required dose [1]. However, this may be associated with the risks of loss of effectiveness, safety, and stability problems, since, these extemporaneous preparations are not generally assessed for their safety, stability, efficacy and bioavailability. Therefore, there is an urgent need to develop efficient and stable extemporaneous liquid dosage forms starting from the commercially available solid pharmaceutical products such as tablets and capsules. 1.1 Tablet scoring There are different reasons for splitting a tablet into halves or quarters, for example healthcare providers tend to split a tablet to get access to smaller doses that are not available and still needed, for tapering or titrating a dose, or to ease the administration of large tablets especially in children and elderly patients with swallowing difficulties. Cost saving is another common reason for tablet splitting especially for patients with chronic conditions [2, 3]. 2 FDA recommends that the generic product must follow the RLD regarding scoring manner, tablet scoring is considered as a sign for patients and healthcare providers for splitting a tablet in order to fraction a dose assuming content uniformity of the fractions Nevertheless, there are many limitations in splitting a tablet; unsuitable dosage form such as controlled release, sustained release and film coated tablets, another is loss of fragments due to the poor techniques used, tablets tend to shatter when split, then weight uniformity and accordingly, content uniformity of the subdivided tablets cannot be guaranteed all the time even for scored tablets. Moreover, elderly patients with weak muscles and vision and poor focus find it difficult to cut tablets into halves even for scored ones [4].This attempt may lead to the administration of the incorrect doses, especially when there are many available commercial tablets have failed the weight uniformity test which leads to serious complications especially in case of narrow therapeutic window drugs [5]. On the other hand, many of available medications are not stable in liquid vehicles, and for pharmaceutical companies to produce such preparations it is considered economically ineffective, especially when liquid preparations have a shelf-life of two years from the date of production, this time is mostly lost in distribution system and waiting on the shelves for the time of administration. Another reason for the lack of pediatric preparations is the small size of the targeted population of children that make it financially unattractive for pharmaceutical companies to produce a liquid preparation of each medication [6]. Moreover such formulations require adequate 3 studies on pediatric patients concerning its safety and efficacy in such population which means additional costs and increased liability concerns [7]. 1.2 Extemporaneous compounding Extemporaneous compounding is the art of remediation of drugs and excipients into new doses or dosage forms that are not available in the market in order to match up with specific individual needs [8]. For neonatal and unconscious patients who cannot swallow even halves or quarters of a tablet, health care providers go after the off-label medications by preparing extemporaneous suspensions from available commercial solid dosage form [9]. Extemporaneous preparations are referred to as off-label medications because they are used out of the license limits that is approved by Food and Drug Administration (FDA), while registered medications follow the internationally recommended standards of good manufacturing practices, extemporaneous preparations are compounded manually with the traditional techniques that are lacking any of these standards [8]. 1.2.1 Formulation for extemporaneous suspension Extemporaneously prepared suspensions range from simply crushing a tablet or opening a capsule then the addition of water or any other liquid to its complex formulations with the addition of preservatives and organoleptic enhancers. 4 A successful formulation of an extemporaneous suspension usually starts with crushing available commercial tablets or opening a capsule to be suspended in a vehicle. However, most of available medications are not soluble in water and hence suspending agents such as methylcellulose or others are needed. Anti-oxidants is another component to be added to improve the stability and ensure that the extemporaneous suspension is safe and effective during the treatment period. Sweeteners, colors and flavoring agents could be added as well to enhance the palatability and organoleptic properties and accordingly the compliance, preservatives, to prohibit microbial growth in the suspension [10]. As a result, the final suspension must be rapidly dispersed upon brief shaking in order to get the accurate doses upon administration. Furthermore, it must be palatable with acceptable taste and odor. It has to be stable over the intended period of treatment, and easy to prepare and store, taking into consideration that filtration has to be avoided to prevent loss of active ingredient [7]. Most of drugs are poorly soluble in water, although intravenous preparations could be an alternative option to the crushed tablets but limitations like high cost and poor oral bioavailability of some drugs restrict such option, moreover, intravenous preparations contain excipients such as propylene glycol or others that are not preferred to be administered in large amounts or for long periods [10]. 5 A large number of medications that are not available in liquid dosage forms are prepared by unprofessional caregivers through crushing the tablets and mixing them with food or beverages at time of administration, this action may include errors in the preparation and incomplete administered doses. Therefore, the pharmacist or any other professional healthcare provider is preferred to prepare an extemporaneous suspension suitable to cover an extended period of time by containing multiple doses to meet patient’s needs [7]. There were several attempts to prepare extemporaneous oral liquid dosage forms from commercially available products [11-13]. An extemporaneous suspension containing Amlodepine (AML) was prepared and a comparative bioavailability study was conducted in which bioequivalence was proved between the tablet and the extemporaneous preparation [14].Another attempt was to prepare valsartan (VAL) extemporaneous suspension; which was successfully prepared from available commercial tablets without hindering its chemical stability or dissolution profile [15, 16] but no bioavailability study was conducted. However, there was no effort done for preparing the combined (amlodipine/valsartan) suspension. Generally, for most of extemporaneous preparations no bioequivalence studies are conducted, which leads to further confusion whether these crushed tablets preserve its efficacy or this action may lead to serious complications. 6 1.2.2 Risks associated with extemporaneous compounding The risks associated with extemporaneous compounding cannot be underestimated, some are due to weighing and calculation errors, other risks are related to mistakes in selecting the appropriate formula and the right excipients. In extreme cases, these errors may lead to the death of the patient [8, 17].According to a prospective study conducted in a children’s hospital, pediatric patients were identified as the most vulnerable population in suffering adverse reactions in such situations [18]. Moreover, those extemporaneous suspensions do not follow any stability or bioavailability testing. The pharmacokinetics of these preparations may vary because of the different behavior of the different dosage forms and the type of excipients used. Accordingly adverse reactions or toxicity might occur [19]. In a study involving preparing an alcohol-free extemporaneous suspension of spironolactone for pediatric use, different excipients were used to come up with four different formulations. Those formulations were investigated regarding their dissolution profiles and their physical, chemical and microbiological stability; only one of the four extemporaneous suspensions preserved the optimum conditions required for a safe and effective dosage form [20]. The bioavailability of an H2 blocker; Nizatidine was investigated in two different extemporaneous solutions and compared with that of a commercially available oral syrup and Nizatidine capsule. The two extemporaneous solutions were prepared one in infant formula and the 7 other in an apple juice. The study showed that the bioavailability of Nizatidine in apple juice was markedly retarded, whereas; for Nizatidine in infant formula and the commercially prepared solution they were bioequivalent to the capsule [21] 1.2.3 Stability of extemporaneous formulations Stability of an extemporaneous suspension is another challenge. A medication is considered to be stable over a specific period of time when this product retains its particular specifications of identity, quality and purity over a specific period of time (shelf-life) [22]. Regarding the stability of a medication, there are different aspects to consider: physical, chemical, microbiological and therapeutic stability. -Chemical Stability A pharmaceutical product is considered chemically stable when its active ingredient preserves its chemical integrity and labeled potency over a specified period of time (shelf life). Usually, the shelf life of a drug product can be considered as the time taken for the drug concentration to be reduced to 90% of the original concentration. The shelf life of a formulation needs to be determined at the realistic storage temperature, normally at room temperature or in a refrigerator. 8 - Physical Stability Physical stability is confirmed by retaining the original properties such as, appearance, palatability, uniformity and suspendability over the shelf-life. Physical instability in suspensions is expressed as caking of sediment or particle growth - Microbiological Stability Microbiological stability refers to the absence of bacterial growth in the formulation during the specified period. Microbial instability may lead to spoilage in the product’s appearance and change in its organoleptic properties. Furthermore, the presence of microorganisms in the formulation may render it ineffective or even toxic. - Therapeutic stability Therapeutic stability means that the product should remain effective during the shelf-life period [22]. Stability testing is the study of the effect of different environmental conditions (temperature, humidity, light) on the quality of a drug product over time, as well as to set a re-testing period for the active ingredient or a shelf life for a drug product including the recommended storage conditions for each starting material and drug product [23].Stability testing is a legal requirement before the registration of a new drug product, in order to ensure that the medication remains within acceptable limits of safety efficacy and good quality until a patient consumes the last dose [24]. 9 There are different types of stability testing; long term stability testing, intermediate stability testing and accelerated condition stability testing. They are described in table 1. Table 1: Types of stability studies and their storage criteria [23]. Stability Study Storage Criteria Time Period* Long term (25+2) ºC / (60+5) % RH Or:(30+2) ºC / (65+5) % RH ** 12 months Intermediate (30+2) ºC / (65+5) % RH 6 months Accelerated (40+2) ºC / (75+5) % RH 6 months  * Minimum time required to be covered by data at submission.  ** If (30+2) ºC / (65+5) % RH is the long term condition, then there is no intermediate condition When a drug product fails to meet its specifications it is considered as a significant change, if such a condition occurred in the accelerated stability study and the long term testing was conducted at (25+2) ºC / (60+5) % RH, then intermediate stability testing should be carried out [23]. Stability of extemporaneous preparations is an add on challenge, it’s important to consider the stability of the entire formulation than the active ingredient alone, where some extemporaneously prepared suspensions have increased stability due to the introduction of antioxidants into its formulations, however there are some cases where stability of these suspensions is inversely affected as a result of interaction between the active ingredient and the excipients used more than the degradation of the drug substance by oxidation or hydrolysis means [19]. 10 1.3 Lack of Bioequivalence/Bioavailability data Bioequivalence means that there is no significant difference in rate and extent of absorption between the test and the reference listed product. Bioequivalence is usually assessed in terms of peak plasma concentration (Cmax), time to reach Cmax (tmax) and area under the concentration time curve (AUC). In terms of regulatory guidance, two formulations can be considered bioequivalent, if the 90% confidence interval for either Cmax or AUC falls within the limits of 80–125%. For licensed medicine, it relies on the manufacturer to prove that anew generic drug product is bioequivalent to the listed drug product, however, few bioequivalence data are available in the literature for the extemporaneous preparations compared to the licensed product. In some cases, the extemporaneous formulations were not bioequivalent to the reference medications. Bioequivalence studies are cost expensive and time consuming. The development of the in vitro and in silico methods can help in the prediction of the in vivo absorption profiles of drugs and adoption of in vitro in vivo correlation (IVIVC). As a result ,several regulations were put to waive the need for bioequivalence studies for a large number of drugs when specific criteria are met. (Biowaiver) which is based on the biopharmaceutical classification system BCS [25], classifies the drugs into four groups considering their solubility and permeability (Figure 1); with the high solubility and permeability are combined in BCS1, and the lowest are combined in BCS 4, BCS 2 lacks in solubility, and BCS 3 lacks in permeability [26]. In silico and in vitro methods can help in predicting the bioequivalence of extemporaneously 11 prepared formulations without the need of expensive and time consuming bioequivalence studies. Figure 1: The BCS as defined by Amidon [26]. 1.4 In vitro Dissolution Testing For a pharmaceutical drug product, the rate and extent of absorption is primarily controlled by its dissolution behavior from its specific dosage form. Accordingly, for a drug to be effective, it must be released from the dosage form and dissolved in the gastrointestinal fluids as a first and essential step before being absorbed into blood circulation [27]. Differences in dissolution behavior among drugs have a great impact on their bioavailability, which leads to different therapeutic responses that ranges from toxicity to sub therapeutic levels [28]. In vitro dissolution testing is a distinctive tool that illustrates the release behavior of a drug by reducing human exposure without abandoning product quality. For pharmaceutical drug products, dissolution testing is 12 routinely performed for quality control and quality assurance purposes. It is used in the drug development stages and for commercial pharmaceutical manufacturing as well [29, 30]. In vitro dissolution testing is a regulatory requirement in the development and assessment of new pharmaceutical formulations; it ensures batch to batch consistency, helps in evaluating stability of the drug product during its shelf life period, and to confirm product quality in scale up post approval changes (SUPAC) for means of bioequivalence studies [31]. In addition, in vitro dissolution is appreciated as time and money saving method since it is considered an FDA-approved surrogate for in vivo studies (Biowaiver), [30].The dissolution characteristics of a pharmaceutical dosage form can affect its bioavailability. Formulations with different release rates can produce different pharmacokinetic (PK) profiles of the same drug substance, potentially resulting in bioavailability differences. Evaluation of the dissolution behavior of an extemporaneous preparation is an important quality-control parameter. 1.5 In silico With the evolution of combinatorial chemistry, a large series of related chemical compounds are prepared with the same reaction and a variety of reagents. However these compounds have to run through high throughput screening and only few of them are chosen to complete with for further reactions and testing. To keep up with such dramatic increase in chemical compounds capacity, there is an insistent need of developing new methods 13 to facilitate the screening of absorption, distribution, metabolism, excretion (ADME), and so the need of new tools and equipment [32]. Recently, in silico modeling play an important role in the prediction of in vivo behavior based on in vitro data [33], by the estimation of specific parameters. The computational simulation technology has proven its usefulness in their ability of predicting the rate and extent of drug absorption using the properties predicted from the chemical structure alone. This method give pharmaceutical companies invaluable opportunity to estimate and assess the capacity of absorption before compounds being actually synthesized [34]. Nowadays, several commercial software for in silico simulations of oral drug absorption are available. GastroPlus™ software is an example, which employs the Advanced Compartmental Absorption and Transit (ACAT) model and the BCS principles to establish IVIVC, assess biowaiver studies and facilitates the evolution of new formulations and dosage forms through which saving time and budget of pharmaceutical companies [33]. GastroPlus TM simulate the pharmacokinetics of the drug and its absorption in gastro intestinal tract. ACAT model consists of nine compartments (stomach, duodenum, jejunum 1, jejunum 2, ileum 1, ileum 2, ileum 3, caecum, and ascending colon) to mimic the human GI tract. Beside human physiology, models for rat, cat, rabbit or dog are available as well, taking into consideration the physicochemical properties of the drug such as solubility, pKa, lipophilicity, and permeability, beside formulation characteristics in addition to pharmacokinetic properties. GastroPlus TM was 14 proved for its powerful efficiency in predicting plasma concentration profile for many drugs [35]. In a study on calcium channel blocker agent (Nifedipine); In silico modeling was coupled with in vitro dissolution for the prediction of in vivo behavior of the drug [36]. For another study, GastroPlus TM was applied to predict oral bioavailability of newly developed high permeability low solubility CNS drug followed by in vivo study on beagle dogs in order to build a preclinical formulation through which a simple oral dosage form gave the acceptable pharmacokinetic parameters without the need of complex formulations and hence considerable budget saving was achieved [37]. Ajay Saxena and others have established an In vitro- In silico- In vivo (IVISIV) correlation using GastroPlus TM to predict the absorption of weak basic drugs that undergo pH dependent solubility, thus growing liability assessment in early drug development stages [38]. 1.6 Antihypertensive Medications Globally; cardiovascular diseases are one of the leading causes of mortality[39]. Hypertension, also known as high or elevated blood pressure is one of the risk key factors of cardiovascular diseases [40]. Nowadays, there is an increasing interest in developing new formulations of marketed agents to keep up with the market need. Anti-hypertension medications are among the most common drugs that pharmaceutical market 15 still of continuous need especially with the lack of liquid preparations of these agents. 1.6.1 Amlodipine AML as besylate is a long acting, 3 rd generation dihydropyridine derivative of calcium channel blockers group, amlodipine chemically: C26H31ClN2O8S (Figure2). Figure 2: AML Besylate chemical structure. AML blocks the influx of calcium through the “slow” channels in both coronary and peripheral blood arteries causing them to dilate and subsequently reducing blood pressure [41]. AML either 5 or 10 mg tablets have proved its efficacy as an anti- hypertensive agent either as a monotherapy or combined with other classes of medications [42]. It’s used for the treatment of hypertension and of angina as well [41]. 16 AML has a partition coefficient of 2.66 at pH 7.4.It is a basic drug with a pKa value of 8.7 ,which keeps AML in its ionized form at physiological pH [43]. According to BCS, AML is considered as class 1 [44], with high solubility of 0.774 mg/ml and high permeability with 0.0743 ×10 -4 cm/sec (caco-2) [45]. AML is 98% bound to plasma proteins and has a volume of distribution (Vd) of 21 L/Kg and a bioavailability of 60-80%and a clearance is 7 ml/min/kg [46, 47]. AML was 62% recovered from urine and 23% from feces after IV administration [48]. Although AML is extensively metabolized in the liver, this process is considered relatively slow with retarded elimination rate that results in prolonged elimination half- life (40-60) hrs, such properties make AML substantially unique drug if compared to other calcium channel blockers of dihydropyridines and non dihydropyridines [49, 50]. 1.6.2 Valsartan VAL chemically:C24H29N5O3 (Figure 3) is angiotensin II receptor blocker (ARB). 17 Figure 3: VAL chemical structure. VAL is available in different strength 40, 80, 160 and 320 mg tablets, it acts by preventing angiotensin from binding to angiotensin receptors, and because angiotensin is known by its ability to constrict blood vessels; then blocking these receptors leading blood pressure to be reduced [51]. VAL has a distribution co efficient of -0.34 at pH 7 [52]. VAL is a weak acid that has pH dependent solubility. It has 2 pKa values (3.9 and 4.73) [53, 54], while solubility of VAL is limited below pH 3.VALsolubility increases with increasing pH whereas permeability decreases at the same range. Accordingly, some papers assign it as BCS class 2 and others consider VAL as a BCS class 3 drug with high solubility of 16.8 mg/ml at pH 8 and low permeability of 0.262×10 -4 cm/sec (in rat) [26, 55] VAL has a bioavailability of 39%, elimination half-life 9.5 hrs and a Vd of 16.9 L [56].The main dose of VAL is excreted unchanged through faecal 18 route and to a lower extent in urine, about 9% of VAL is recovered as inactive metabolite M1 [57]. AML and VAL are considered as a safe and effective combination; as it is well tolerated in most patients with minimum adverse reactions and reduced peripheral oedema incidence [58, 59]. Furthermore, the combination therapy of AML and VAL was significantly more effective in lowering BP than using AML or VAL alone [60, 61]. Provided that both AML and VAL are safe and effective in treatment of HTN in children from 1 years and older [62-64]; this affords that liquid formulation (amlodipine/valsartan) will provide additional value for this group of patients as well. AML and VAL as a combination is available in the pharmaceutical market as a film coated tablets. However, no liquid formulation of this combination of active ingredients is available. Therefore, crushing of the tablet is the only choice for using these drugs in patients with swallowing difficulties. There were several attempts to make VAL extemporaneous suspensions [65, 66], moreover, and AML extemporaneous suspension from available commercial tablets [14], but there was no efforts done for preparing the combined (AML/VAL) suspension. No bioequivalence studies are conducted in such situations, which leads to further confusion whether these crushed tablets preserve its efficacy or this action may lead to serious complications. 19 1.7 Aims of this study The main aim of this study was to develop an extemporaneous suspension of AML and VAL as a combination using crushed commercial tablets (Valzadepine® 5/80) for use in patients with swallowing difficulties. The Specific goals were to: 1. To formulate an oral liquid dosage form of both (AML and VAL) from commercially available tablets (Valzadepine® 5/80). 2. To evaluate the chemical, physical and microbial stability of this extemporaneous suspension. 3. To determine the in vitro release behavior of this combination from the different formulations (the extemporaneous suspension and the film coated tablets). 4. To ensure the bioequivalence of the extemporaneous suspension obtained from crushed tablet with the tablet swallowed as whole, using simulation technology to predict the in vivo behavior of this formulation and compare it with the observed profile of the whole tablet based on the in vitro dissolution data. The objectives of this study In this thesis, an extemporaneous preparation of an oral suspension (AML and VAL)was developed for use in pediatric population with hypertension. In the literature review, the extemporaneous suspension and the stability 20 aspect of these preparations are discussed. In the experimental part of this work, an extemporaneous suspension containing a combination of AML and VAL was prepared from crushed oral dosage form. The stability and the in vitro dissolution properties were investigated. Furthermore, Simulation technology was used to predict the in vivo behavior of this extemporaneous suspension. 21 Chapter two Methodology 2.1 Materials, Equipment and Dosage form. Valzadepine® film coated tablet, containing 5 mg AML and 80 mg VAL, was used in this study (Pharmacare PLC, Palestine,Batch 036B16; Expiry date 02\2018). AML and VAL United States pharmacopeia (USP) reference standards, and all the excipients and materials (aspartame, mannitol, tri-sodium-citrate, guar gum, potassium dihydrogen phosphate, sodium hydroxide, glacial acetic acid); were kindly donated by Pharmacare PLC, Ramallah, Palestine. All chemicals and reagents that used were of analytical grade and no further purification was needed. HPLC grade solvents; acetonitrile (ACN) (Sigma-Aldrich), methanol (MeOH) (LAB-SCAN, Ireland), triethyl Amine (Merck) and phosphoric acid (Frutarom). Highpurified water was prepared by using a Millipore Milli-Q plus water purification system. - Equipment and tools: Equipment used are: balance (Ohous balance), viscometer (Brookfield), pH meter (Mettler Toledo MP225), dissolution apparatus (ERWEKA DT70), HPLC (HITACH), sonicater (BRANSON 8510), GastroPlus ™ software (version 9.0, Simulation Plus Inc, Lancaster, CA, USA). 22 - Media for Dissolution study:  Phosphate buffers (USP) for pH= 6.8 and pH= 4,5 was prepared by dissolving 47.6 g of potassium dihydrogen phosphate and 6.272 g of sodium hydroxide in 7 L of water, pH was adjusted to 6.8 using 0.2 N sodium hydroxide and to pH 4.5 using phosphoric acid.  0.1 N HCl (USP) pH 1.2 2.2 Preparation of the extemporaneous suspension In this study, an extemporaneous suspension containing (AML 5 mg/VAL 80 mg) , was prepared from commercial tablet Valzadepine® (5/80). The Detailed method of preparation are clarified in the following steps: 1. 100 tablets of Valzadepine® (AML 5/VAL 80) mg were crushed to a fine powder. 2. Then all the excipients in (Table 2) were weighed and mixed with the powder to achieve a final concentration of 0.2 mg/gm. 3. 16.02 gm were weighed and diluted with water in two steps up to 50 ml. Through which each 5 ml of suspension contains one crushed tablet with 5 mg AML and 80 mg VAL, and hence for the 50 ml bottle 10 crushed tablets are needed, given that each tablet weighs 0.2 g, then 2 g of crushed tablets are needed for each 50 ml bottle and 40 gm for 20 bottles. 23 Table 2: The composition of the AML/VAL 5/80 suspension formula. - * are from [67] - The average weight of Valzadepine® 5/80 mg tablets is 0.2 g. The resulting powder was divided into 20 amber glass bottles (16 gm powder in each 50 ml bottle), which were ready for reconstitution to form the 5/80 mg AML/VAL extemporaneous suspension (to be completed up to 50 ml water and to be shaken well before use). 2.3. pH measurements: The pH of the different media as well as the reconstituted suspension was determined using Mettler Toledo MP225 pH meter, each measurement was done in triplicate. 2.4 Viscosity measurements The rheological behaviour of the extemporaneous suspension was measured using Brookfield viscometer over a shear rate 90-100 s-1). The Material Function mg/g g/50ml bottle Gram Valzadepine® crushed tab 5/80 Active ingredients 0.20 2.00 40.00 Aspartame Sweetening agent* 0.01 0.10 2.00 Mannitol Flavoring agent * 1.36 13.60 272.00 Tri-sodium citrate pH modifier/Buffering agent * 0.016 0.16 3.20 Sodium hydroxide pH modifier /Buffering agent * 0.001 0.01 0.20 Guar gum Suspending agent * 0.015 0.15 3.00 Total weight 1.602 16.02 320.40 24 viscosity measurement was performed at 25°C in duplicate and the rheogram was obtained for the selected formula. 2.5 Stability study 2.5.1Chemical stability The stability study was conducted by storing 10 containers containing 50 ml of the extemporaneous suspension at room temperature. Another 10 bottles, containing the initial powder were kept for further analysis. The suspensions were analyzed using HPLC in duplicates in a weekly manner over a period of one month. The stability of the extemporaneous suspension was determined by calculating the percentage of the drugs remaining at the end of every week. 2.5.2 Physical stability The formulated suspension was tested for its physical properties such as: pH, viscosity, appearance, and its organoleptic properties. They were tested at the time of preparation and at the end of every week over one month at room temperature. 2.5.3 Microbiological stability 2.5.3.1 Preparation of culture media 28g of nutrient agar dehydrated powder was dissolved in 1L of distilled water. The prepared suspension was heated until boiling while being mixed roughly. The solution was placed in the autoclave at 125°C for 15 minutes 25 in order to get it sterilized. After sterilization, the solution was poured in already sterilized petri dishes. The petri dishes were placed in the refrigerator for 24 hrs. 2.5.3.2 Microbiological analysis After 24 hrs, 0.1 ml of each reconstituted suspension was placed on one of the petri dishes and they were placed in the incubator at 37 °C for 48 hrs. The analysis includes: total bacterial count and examine the presence of mold and yeast, Staphylococcusaureus, Pseudomonas aeroginosa and Candida albican. 2.6 Drug release study 2.6.1 Dissolution Dissolution rotating paddle apparatus II (Erweka dt70, Germany) was used to study the release of AML/VAL from the tablets as well as the extemporaneous suspension. 1000 ml medium was used for each vessel of the paddle apparatus that was rotating 75 rpm for 30 minutes, the temperature was set at (37 °C + 0.5 °C). 10 ml samples were withdrawn at predetermined time points; 5, 10, 15, 20, 30 minutes and replaced with fresh media, the samples were taken from the midway between the surface and the top of the rotating paddles not less than 1 cm from the vessel wall. Each sample was filtered through a 0.45- mcm microporous PTFE syringe filter, then they were introduced to HPLC analysis to figure AML/VAL concentration in the samples [68] 26 2.6.2 Statistical Analysis Similarity and difference factors (f2 and f1respectively) were used to assess the dissolution data as reported in equations 1 and 2 below. The f2 factor is a measure of the closeness of two profiles while f1 is a measure of the difference between two profiles: ………………………….. (1) …………………..……….. (2) where Rt and Tt are the percentages of drug dissolved at each time point for the reference and test products, respectively. When f1 value is greater than 15; this indicates no similarity, and when f2 value is greater than 50; then there is a significant similarity between the two products. 2.7 The HPLC analysis 2.7.1 Instruments, Solutions and Chromatographic Conditions The HPLC system consisted of Lachrom (Merck-Hitachi) equipped with model L-7100 pump, L-7200 autosampler, L-7300 column oven, DADL- 7450 photo diode array (PDA) detector, and D-7000 software HSM version 3.1 (Merck Hitachi, Kent, England). Weights were measured using Ohous balance, pH was identified using Toledo pH meter. 100 11 1                       n t t n t tt RTRf                   100)( 1 1log50 5.0 1 2 2 n t tt TR n f 27 The HPLC experimental conditions were optimized on a stainless steel column (250 cm ×4.6 mm) packed with octadecylsilyl silica gel for chromatography ( 5 µm). Mobile phase was prepared by mixing 2 solutions; solution A: solution B (1:1) in which solution A is: Methanol, Acetonitrile, and Buffer (175:75:250), and solution B is: Water, Acetonitrile, Glacial acetic acid (150:350:0.5), and the buffer was prepared by adding 7.0 ml of triethylamine into 1000 ml flask containing 900 ml of water, the pH of this buffer was adjusted to 3.0 + 1 with phosphoric acid, then diluted with water to the final volume of 1000 ml. The mobile phase was filtered through a 0.45-mcm microporous filter and degassed by sonication prior to use, the flow rate was 1.0 ml/minute with injection volume of 20 µL, and the UV-detector was set to 220 nm. The diluent was: Acetonitrile: Water (1:1) 2.7.2 Standard stock solution The standard solution of AML was prepared by dissolving 27.74 mg of AML besylate reference standard in diluent till reach 200 ml, the standard solution of VAL was prepared by dissolving 80 mg of VAL reference standard in 40 ml diluents then sonicated till dissolved and the volume completed to 50 ml with the diluent. Then the standard solution of the combination was prepared by taking 5 ml of each standard solution to 50 ml volumetric flask together and completed to 50 ml with the mobile phase. 28 2.7.3 Sample stock solution Sample stock solution was prepared by taking 5.5 gram of the suspension to 50 ml volumetric flask with 10 ml of water, 30 ml of diluent was added, stirred and sonicated then completed to the volume with the diluent, 5 ml of this sample stock solution was taken and diluted to 50 ml with the mobile phase, each sample was filtered through 0.45-µm syringe tip filter. The peak quantification was obtained by comparing sample & standard peak area ratios as a function of concentration. 2.8 Gastrointestinal simulation GastroPlus™ software (version 9.0, Simulations Plus Inc., Lancaster, CA, USA), which based on the Advanced Compartmental Absorption and Transit (ACAT) was used in this study. The approach used was to develop and verify absorption models for both AML and VAL from Valzadepine® tablet). The in silico models were initially constructed for immediate release (IR) tablet, and were afterwards implemented, to predict the in vivo profiles for both drugs from the extemporaneous suspension. Therefore, Two databases were established: one for AML and the other for VAL. Each database consists of two records; one for the tablet and the other for the suspension. GastroPlus™ as a single simulation mode was used to run the gastrointestinal simulation depending on the physicochemical, physiological, and the pharmacokinetics properties of AML and VAL, as 29 well as the in vitro dissolution data from both the tablet and the suspension. GastroPlus™ includes three modules: compound, physiology, and pharmacokinetics. For the compound and pharmacokinetics modules; the input data were collected from the literature. In the physiology module, the simulations were conducted using The Human Physiology Fasted mode. All the physiological parameters were fixed at default values. In the pharmacokinetic module: two compartment kinetics were followed for AML and for VAL as well, both exhibited zero order absorption and first order elimination[69]. The simulations were conducted using the Johnson model as a dissolution model. (IR tablet) mode, in GastroPlus™ was selected for simulations. The model for IR tablet was verified by comparing the simulated profiles to the observed in vivo pharmacokinetic profiles of (Valzadepine® tablet), which was obtained from Pharmacare Ltd (Table 3).The developed model for the “IR tablet” dosage form was then employed for predicting the in vivo performance of the suspension. The simulation of the suspension was performed using the “IR suspension” as the selected dosage form and by introducing the dissolution data for the formulated suspension. 30 Table 3: Plasma concentration-time profile of AML and VAL of Valzadepine® 5/80 tablets obtained from Pharmacare pharmaceutical company. Time (hr) AML (ng/ml) VAL (ng/ml) 0.0 0.00 0.00 0.5 - 153.75 1.0 0.6 458.45 1.5 - 611.95 2.0 - 689.80 2.5 1.8 747.30 3.0 - 689.35 3.5 - - 4.0 2.7 552.70 5/0 - 425.60 6/0 3.2 338.65 7/0 - 280.8 8.0 2.8 224.8 12.0 2.5 162.6 15.0 2.2 - 18.0 1.8 - 24.0 1.4 59.65 48.0 - 15.7 72.0 - 13.4 96.0 0.6 - 120.0 0.3 - 144.0 0.1 - The experimental in vitro dissolution profiles for both active ingredients from Valzadepine® tablet and suspension in the different pH media were 31 incorporated in the corresponding model. The summary of all input parameters for simulation is given in Table 4. Table 4: Simulation input data Parameter Value Amlodipine (as besylate) Valsartan Molecular weight (g/mole) 567.051 435.53 Partition/Distribution coefficient 2.66 (pH=7.4) a -0.34 (pH=7) b PKa1 8.7 c 3.9 d PKa2 - 4.73 d Solubility (mg/ml) 0.774 (pH 7.4) e 16.8 (pH=8) f Peff (Human jejunal permeability) (cm/sec) 0.0743 *10 -4 g (caco-2) 0.262*10 -4 h (rat) Dose (mg) 5 80 Dose volume (ml) 250 250 Mean precipitation time (sec) 900 i 900 i Diffusion coefficient (cm 2 /s) 4.2*10 -8 j 1.1*10 -8 k Drug particle density (g/ml) 1.2 i 1.2 i Blood plasma concentration ratio 1 i 1 i Body weight (kg) 70 70 Unbound percent in plasma (%) 2 l 5 m Clearance (l/hr) 28 n f n Volume of distribution, Vc(L/Kg) 17 n 0.23 n Elimination half-life (h) 27.03 o 5.58 o Simulation time (hr) 144 72 a From [45, 70] b From [52] c From [43, 71] d,f From[53] e From [72] g From[45] h From [73] i From Gastro Plus default values j,k From[74] l From [46] m From [75] n Gastro Plus calculated (using PBPKPlus™ Module) o Gastro Plus calculated (built-in calculation from PK parameters) 32 The percent of prediction error of the simulation (% PE) can be calculated by equation 3 below, this represents the percent of error between the predicted values and that of the in vivo observed data …………………….………………(3) %100% observed observed    PK PKPK PE predicted 33 Chapter Three Results 3.1 The formulation The AML/VAL suspension was successfully prepared and well suspended upon brief shaking with acceptable appearance, smell and palatable taste. Its pH value was 5.5 3.2 Viscosity Determination The viscosity of the extemporaneous suspension was examined at different shear rates. The behavior is shown to be dilatant, i,e, the viscosity increases with the increase in the shear rate. The data is shown in Table 5 and Figure 4. Table 5: The rheological behavior of the extemporaneous suspension over different shear rates. Shear rate(rpm) 0 5 10 12 20 30 50 60 100 Viscosity (Cp) 0 0 25.6 160 377 410 422 425 470 34 Figure 4: The rheological behavior of the extemporaneous preparation over different shear rates 3.3. Drug release study The in vitro release of AML from both the IR tablet and the suspension was investigated in media with different pH (1.2, 4.5 and 6.8). The dissolution profiles for AML from both formulations are shown in Figure 5. As can be seen, AML exhibited very rapid dissolution in phosphate buffers (4.5 and 6.8) with more than 85% was dissolved within 15 minutes, and has a rapid dissolution in 0.1 N HCL with more than 85% was dissolved within 30 minutes and an f2value of 51.74. 35 Whereas for VAL; media pH has shown to have a marked effect on its release from both dosage forms (Figure 6). At pH=6.8, the percentage of VAL released was more than 85% within 15 minutes, however, in pH 4.5 and 1.2 media, the dissolution was much slower. At pH=4.5 less than 70% of the drug released within 30 minutes. Whereas, at pH= 1.2, the apparent amount of VAL released was not more than 26% within 30 minutes from both dosage forms. This decrease in the dissolution rates with the reduction in the media pH reflects the pH-dependent solubility of VAL. f1 and f2 values were calculated for each drug from each dosage form. Where the IR tablet was the reference and the extemporaneous suspension was the test. Figure 5: Release profiles of AML from the tablet and the suspension at different pH values. 36 Figure 6: Release profiles of VAL from the tablet and the suspension at different pH values. Table 6: Dissolution of AML and VAL from Valzadepine® tablets % Dissolved of Amlodipine + (SD) Tablet Suspension Medium 15 min 30 min 15 min 30 min pH 1.2 84.4 + (2.14) 90.3 + (1.81) (f2 =51.74) 85.6 + (1.32) 93.3 + (1.59) pH 4.5 95.6 + (1.67) 98.7 + (1.73) 90.4 + (1.68) 100.1 + (1.97) pH 6.8 87.8 + (1.97) 94.3 + (0.78) 87.1 + (0.87) 89.9 + (1.05) % Dissolved of Valsartan + (SD) pH 1.2 17.7 + (2.07) 25.8 + (1.91) (f2 =51.80) 16.1 + (1.96) 23.5 + (1.45) pH 4.5 49.6 + (2.18) 68.1 + (1.89) (f2 =51.63) 44.4 + (1.84) 67.9 + (2.76) pH 6.8 104.4 +(1.83) 103.4 + (1.22) 99.8 + (0.56) 100.7 +(1.74) 37 The results of similarity were more than 50 for each dissolution showed latency in 85% within 15 minutes indicating the similarity in the release from both formulations. They are shown in Table 6 and Figures 5 and 6. As pH 6.8 is the recommended media by FDA and USP [68]. For AML, it was very rapidly dissolving with average of 87.3% and 88.1% was dissolved within 10 minutes from the tablet and the suspension respectively. The same in case of VAL; it was very rapidly dissolving with 104.8% and 98.7% dissolved within 10 minutes for the tablet and the suspension respectively, the data are shown in Table 7. Table 7: The percentage of AML and VAL released from the tablet and suspension formulations at pH 6.8 as recommended by FDA and USP. % Dissolved of Amlodipine Time (min) Tablet SD Suspension SD 5.0 75.6 1.4 80.3 2.0 10.0 87.3 1.2 88.1 1.9 15.0 87.8 0.9 87.1 1.9 20.0 89.4 1.8 86.6 1.5 30.0 94.3 1.1 89.9 0.8 % Dissolved of Valsartan Time (min) Tablet SD Suspension SD 5.0 102.9 1.0 98.9 1.9 10.0 104.8 0.3 98.7 1.8 15.0 104.3 0.6 99.8 1.8 20.0 103.1 1.9 98.9 1.8 30.0 103.4 1.7 100.7 1.2 38 3.4. Stability study 3.4.1 Physical stability: There were no changes observed in the appearance, odour, colour and pH. 3.4.2 Chemical stability The suspension was chemically stable throughout the four weeks period. The mean percentages of the remaining active ingredients were over 90% within the four weeks period (Table 8).The mean concentrations of AML and VAL on the thirty day were 97.3% and 101.1% respectively at room temperature. 39 Table 8: The mean percentage of the active ingredient in AML/VAL suspension throughout 4 weeks period at room temperature. Week initial Week 1 Week 2 Week 3 Week 4 AML VAL AML VAL AML VAL AML VAL AML VAL % remained 102.1 106.2 101.8 105.3 99.1 102.2 98.3 101.9 97.3 101.1 3.4.3 Microbial Stability The formulated AML/VAL suspension passed the microbial testing study through the four weeks period. No microbial contamination was observed in the suspension during the study period. The results are described in Table 9. Table 9: Microbial study results. Microrganism Total microbial count Mold and yeast < 10 cfu/ml.g S. aureus Negative P. aeroginosa Negative C. albicans Negative 40 3.5 HPLC analysis AML eluted first at about 4 minutes, and VAL was next at about 11 minutes, standard peaks are shown in Figure 6. Figure 7: standard peaks of AML and VAL as eluted in HPLC analysis. 3.6 Drug absorption simulation 3.6.1 Gastrointestinal simulation In silico simulation was used to build models describing the in vivo absorption of both AML and VAL from IR tablet based upon the physicochemical, physiological and the in vitro dissolution data. The simulated plasma profiles for AML and VAL together with the in vivo observed curves following the intake of Valzadepine® IR tablet are presented in Figures 8 and 10. The simulated profiles for both drugs from the solid dosage form were in good agreement with the in vivo observed curves. The simulated and the in vivo pharmacokinetic parameters (Cmax and AUC0-∞) for both drugs are presented in Tables 10 and 11. The percent 41 prediction errors obtained were less than 10% for all pharmacokinetic parameters, indicating good predictability. The developed models for the IR tablet dosage form were implemented to predict the in vivo performance of the extemporaneous suspension using the in vitro dissolution data of the suspension. Figures 9 and 11 for AML and VAL respectively compare the predicted absorption profiles for the suspension and the in vivo plasma profile observed for IR tablet. Then in silico pharmacokinetic parameters for suspension were compared with that observed in vivo for IR tablet. The extemporaneous preparation is predicted to be similar with the IR tablet dosage form, since the 90% confidence intervals for Cmax and AUC0-∞for both active ingredients fall within the limits of 80–125% for IR release tablet. Figure 8: The simulated plasma profile of AML from Valzadepine® tablet ( __: in silico, : in vivo data) 42 The pharmacokinetics parameters that are predicted by the in silico method for AML suspension indicates good predictability with the percent of prediction error maintained less than 10%, the data and predicted profiles of AML suspension are shown in Table 10and Figure 9. Table 10: AML observed and predicted pharmacokinetic parameters with percentage of prediction error. Figure 9: The simulated plasma profile of AML suspension ( ___ : in silico predicted using in vitro data, : in vivo data of the tablet). Parameter Calculated for the tab Observed Calculated for the susp Cmax (ng/ml) 3.0302 PE= 5.306% 3.20 3.027 PE= 5.406% AUC0-∞ (ng.hr/ml) 169,81 PE= 5.76% 160.65 169.78 PE= 5.74% 43 Table 11: VAL observed and predicted pharmacokinetic parameters with percentage of prediction error. Figure 10: The simulated plasma profile of VAL from Valzadepine® tablet ( ___: in silico, : in vivo data). Parameter Calculated for the tab Observed Calculated for the susp Cmax (ng/ml) 704,55 PE= 5.72% 747.30 707,22 PE= 5,36% AUC (ng.hr/ml) 8517.70 PE= 4,826% 8949.70 8517.60 PE= 4,82% 44 Figure 11: The simulated plasma profile of VAL suspension ( ___: in silico predicted using in vitro data, : in vivo data of the tablet). 45 Chapter Four Discussion For paediatric or geriatric patients with swallowing difficulties, the liquid preparations are the most convenient ones. A wide variety of medications in the pharmaceutical market are lacking the liquid oral dosage forms. that’s why many researchers tend to prepare extemporaneous suspensions to cover up the shortage in the pharmaceutical market especially for paediatric medications [1]. Considering a research conducted by Sharon Conroy et al about 65% of medications that are used in an intensive care unit of children’s hospital are off-lable or un-licensed [9].paediatric patients are considered therapeutic orphans especially with the large decrease in medications bearing labels for paediatric administration combined with the insufficient safety data making their prescription and use are limited as off-lable medications [7, 76, 77]. Such medications are not registered or approved by FDA. Moreover, no bioequivalence studies are conducted in such situations, which makes these suspensions questionable in terms of efficacy and safety. The combination of AML and VAL as anti -hypertensive medications is an example of such medications with no liquid oral formulation available. In the current study, an extemporaneous suspension of a combination of AML and VAL was prepared from available commercial tablets Valzadepine® 5/80 mg as a source of the active ingredients. This 46 AML/VAL extemporaneous preparation proved its stability in all aspects; physically, microbiologically and therapeutically. A sugar free 5/80 mg AML/VAL per 5 ml suspension in a 50 ml bottle was adopted upon patient usual dose as well as stability period after reconstitution of the suspension, which is convenient for patients who have a co-existing diabetes as well. The usual daily dose of AML/VAL combination ( 5/80 mg) can be obtained in 5 ml of this extemporaneous suspension, the 50 ml bottle will be sufficient for 10 days period through which the suspension still stable and effective. Provided that the liquid preparations like this suspension provide flexible dosing capacity with the ability of administration of parts of the 5ml dose, the 50 ml volume of the was chosen as a final volume of this suspension in consideration of paediatric hypertensive patients for which the amount will be saved for longer period when parts of the 5 ml dose will be given notifying them to discard the remaining amount at the fourth week after reconstitution. The stability, efficacy and bioequivalence of these extemporaneous preparations are lacking.In vitro dissolution is considered as a potential surrogate marker of bioequivalence. In vitro dissolution analysis of extemporaneously prepared suspensions coupled with in silico modelling can help in predicting the bioequivalence of these preparations. To investigate if the extemporaneous preparation is bioequivalent to the tablet dosage form, GastroPlus was used to build an in silico model for both VAL and AML using their respective in 47 vitro dissolution profiles as input. In this study, the in vitro dissolution of the extemporaneous suspension was conducted against the tablets, where the IR tablets was the reference and the extemporaneous suspension was the test, for the two formulations to be bioequivalent they must have similar dissolution behaviour; either having a very rapid dissolution with >85% dissolved within 15 minutes, or >85% dissolved within 30 minutes with similar dissolution profile confirmed with similarity factor f2 > 50 And difference factor f1<15 Both of AML and VAL release was investigated from both the IR tablet and the suspension in media with different pH (1.2, 4.5 and 6.8). Since AML is a BCS class 1 with high solubility and high permeability, AML exhibited very rapid dissolution in phosphate buffers (pH 4.5 and 6.8) with more than 85% was dissolved within 15 minutes, and has a rapid dissolution in 0.1 N HCL with more than 85% being dissolved within 30 minutes and f2value of 51.74 and f1 value of 2.14 confirming the similarity of AML release from the IR tablets and from the extemporaneous suspension. Whereas for VAL; media pH has shown to have a marked effect on its release from both dosage forms. At pH=6.8, the percentage of VAL released was more than 85% within 15 minutes, however, in pH 4.5 and 1.2media,the dissolution was much slower, with less than 70% of the drug released within 30 minutes in pH 4.5 (f2 and f1 are 51.63and 3.33 respectively).Whereas, at pH=1.2, the apparent amount of VAL released was not more than 26% within 30 minutes from both dosage forms (f2 and 48 f1 are 51.80 and 8.68 respectively. This decrease in the dissolution rates with the reduction in the media pH reflects the pH- dependent solubility of VAL. These findings are in agreement with previous studies which reported a reduction in VAL solubility at lower pH values. [78-80]. f1 and f2 value were calculated for each drug from each dosage form.. The results of similarity were more than 50 for each dissolution in which the release showed latency in 85% within 15 minutes indicating the similarity in the release from both formulations. According to USP and FDA, dissolution studies of AML/VAL IR tablets have to be conducted in phosphate buffer pH 6.8, the percentage released of both AML and VAL exceeded 85% within 15 minutes in which bioequivalence of the extemporaneous suspension with the IR tablets is guaranteed. According to BCS, AML which is highly soluble and highly permeable as a BCS class 1 member, then a biowaiver is granted [81]. Whereas in case of VAL, there is a conflict about its BCS classification . Some literature considered VAL as BCS class 2 in which it must have a high permeability and low solubility due to the shortage of VAL solubility at low pH levels [82, 83], others considered VAL as a special case with pH dependent solubility taking into consideration that VAL solubility increase 1000 folds when pH increase from 4 to 6 [84], keeping in mind that VAL site of absorption is the upper gastrointestinal tract where it remains ionized[85] and hence barely absorbed with fraction of dose absorbed and 49 systemically available after oral administration about 0.23[56]. Then it is more likely to be BCS class 3[55]with high solubility and low permeability [84]. To be more precise, VAL can be identified as intermediate class 2/3 rather than class 2 or class 3 as it is suggested by Chi-Yuan and Wu and Leslie Z. Benet for ciprofloxacin and erythromycin [55] . Similar situation was identified by Arthur Okumu and others for assigning etoricoxib as intermediate class 1/2 [86]. Accordingly, VAL is eligible for biowaiver if the release of VAL exceeds 85% within 15 minutes as it is suggested by BCS [87]. Nevertheless, the extent of VAL release from this extemporaneous suspension is in agreement with AN Zaid et al study conducted on VAL extemporaneous suspension prepared from commercial tablets in which more than 85% of VAL was released within 10 minutes [65]. The in vitro dissolution profiles were used in adjacent to in silico modelling that was applied to predict the bioavailability of this suspension in order to confirm the bioequivalence of the suspensions with the IR tablets. Recently, In silico modelling developed a new insight in the prediction of bioavailability depending on in vitro dissolution testing.[33], in which in silico method beside in vitro dissolution could be a valuable and reliable tool in predicting the bioavailability of new dosage forms and in this work for our extemporaneous compounded suspension. 50 GastroPlus TM simulation was used for developing a model for each of AML and VAL in order to predict the absorption of them from the IR tablet and from the extemporaneous suspension. The simulations were carried out using the in vitro dissolution profiles of the IR tablet and the suspension. The predicted absorption profiles correspond well with in vivo observed data of the IR tablet, for the suspension the simulated profiles were compared with the in vivo data of the IR tablets, because there is no available in vivo data for the suspension, considering the IR tablets is the reference and the extemporaneous suspension is the test product in a way to test the bioequivalence. Both the IR tablet and the suspension matched well due to the closeness of dissolution profiles with prediction error values for simulated data which indicates good predictability while maintained below 10%. Arthur okumu and others suggested that similar in vitro dissolution profiles could justify a biowaiver when they are in compliance with in silico predictive profiles [86]. Considering FDA regulations, two products are said to be bioequivalent if the 90% CI of the relative mean Cmax and AUC0-∞ of the test product to reference product is within 80%-125% range[88]. In this study The 90% CIs of the geometric mean ratios (test: reference) for bioequivalent analysis obtained from pharmacokinetic parameters (Cmax and AUC0-∞) of AML/VAL 5/80 mg extemporaneous suspension was predicted by GastroPlus TM and compared to that observed for the tablet considering the suspension as the test where the tablet is the reference in order to investigate BE (Table 12). 51 Table 12: Confidence interval of pharmacokinetic parameters of AML/VAL suspension. parameter Test vs Reference Ratio 90% confidence interval Cmax AUC0-∞ Cmax AUC0-∞ AML 0.94593 1.05683 95% 106% VAL 0.946366 0.951719 95% 95% Depending on the simulation data and the in vitro dissolution data combined with BE terms that are achieved, then the compounded suspension of AML/VAL appears to be bioequivalent to the commercial tablets Valzadepine® as both are giving similar profiles that gives efficient insight into in vivo behaviour of this extemporaneous suspension. For the compounded anti-hypertensive extemporaneous suspension it must preserve its efficacy and safety over an eligible period of time, for any formulation to be considered stable it must retain > 90% of the initial concentration of the drug, AML/VAL suspension preserves 97.3% and 101.1% respectively of its initial concentration over the 30 days period of time. Moreover, no changes in the appearance, pH, colour or odour was observed, no microbial growth was detected as well throughout the 4 weeks. 52 Conclusion The extemporaneous suspension could be successfully prepared using available commercial tablets as a source of the active ingredients even for the combinations medications. Such suspensions should be carefully evaluated in different aspects; volume, organoleptics, stability and bioavailability which is lacking in such circumstances. AML/VAL extemporaneous suspension can preserve its stability over four weeks period when stored in room temperature, in silico modelling could be applied adjacent to in vitro testing to predict PKs and prove similarity of an extemporaneous suspension with the tablets. Pharmaceutical companies should include a section in their leaflets regarding the compounding and stability of those suspensions when the alternative liquid dosage form in not available in the market which could be a life-saving for a patient in need. 53 References 1.Abdallah, Y.M.M., Attitudes, Knowledge and Practices of Health- Care Practitioners Toward Splitting or Crushing Oral Solid Dosage Forms in Palestine: Safety and Therapeutic Implications. 2015, Faculty of Graduate Studies, An-Najah National University. 2.Green, G., et al. Pharmacopeial standards for the subdivision characteristics of scored tablets. in Pharmacopeial Forum. 2009. 3.Kawachi, I., Economic factors in the initiation of antihypertensive therapy. Pharmacoeconomics, 1992. 2(4): p. 324-334. 4.Atkin, P.A., et al., Functional ability of patients to manage medication packaging: a survey of geriatric inpatients. Age and ageing, 1994. 23(2): p. 113-116. 5.Tahaineh, L.M. and S.F. Gharaibeh, Tablet splitting and weight uniformity of half-tablets of 4 medications in pharmacy practice. Journal of pharmacy practice, 2012. 25(4): p. 471-476. 6.Dawson, L.M. and M.C. Nahata, Guidelines for compounding oral medications for pediatric patients. Journal of Pharmacy Technology, 1991. 7(5): p. 168-175. 7.Nahata, M.C., Lack of pediatric drug formulations. Pediatrics, 1999. 104(Supplement 3): p. 607-609. 54 8.Eileen Kairuz, T., et al., Quality, safety and efficacy in the ‘off- label’use of medicines. Current Drug Safety, 2007. 2(1): p. 89-95. 9.Conroy, S., J. McIntyre, and I. Choonara, Unlicensed and off label drug use in neonates. Archives of Disease in Childhood-Fetal and Neonatal Edition, 1999. 80(2): p. F142-F145. 10.Woods, D.J., Extemporaneous Formulations -Problems and Solutions. Paediatric and Perinatal Drug Therapy, 1997. 1(1): p. 25-29. 11.Allen, L.V. and M.A. Erickson, Stability of labetalol hydrochloride, metoprolol tartrate, verapamil hydrochloride, and spironolactone with hydrochlorothiazide in extemporaneously compounded oral liquids. American journal of health-system pharmacy, 1996. 53(19): p. 2304-2309. 12.Thompson, K.C., et al., Characterization of an extemporaneous liquid formulation of lisinopril. American journal of health-system pharmacy, 2003. 60(1). 13.Mohamed‐Ahmed, A.H., et al., Quality and stability of extemporaneous pyridoxal phosphate preparations used in the treatment of paediatric epilepsy. Journal of Pharmacy and Pharmacology, 2017. 69(4): p. 480-488. 14.Lyszkiewicz, D.A., et al., Bioavailability of a pediatric amlodipine suspension. Pediatric Nephrology, 2003. 18(7): p. 675-678. 55 15.Talamonti, W., R.F. Wagner, and H. Wen, Pharmaceutical Formulation of Valsartan. 2008, Google Patents. 16.Zaid, A.N., et al., Preparation and stability evaluation of extemporaneous oral suspension of valsartan using commercially available tablets. International journal of pharmaceutical compounding, 2013. 18(2): p. 169-174. 17.Young, D., Student’s death sparks concerns about compounded preparations. 2005, ASHP. 18.Turner, S., et al., Adverse drug reactions to unlicensed and off‐label drugs on paediatric wards: a prospective study. Acta Paediatrica, 1999. 88(9): p. 965-968. 19.Glass, B.D. and A. Haywood, Stability considerations in liquid dosage forms extemporaneously prepared from commercially available products. 2015. 20.Provenza Bernal, N., et al., Development, physical-chemical stability and release studies of four alcohol-free spironolactone suspensions for use in pediatrics. Dissolution Technologies, 2014, vol. 21, num. 1, p. 19- 30, 2014. 21.Abdel‐Rahman, S.M., et al., The bioequivalence of nizatidine (Axid®) in two extemporaneously and one commercially prepared oral liquid formulations compared with capsule. The Journal of Clinical Pharmacology, 2003. 43(2): p. 148-153. 56 22.McGinity, J.W., Drug Stability: Principles and Practices. Journal of Pharmaceutical Sciences, 1991. 80(1): p. 98. 23.Guideline, I.H.T., Stability testing of new drug substances and products. Q1A (R2), current step, 2003. 4. 24.Kommanaboyina, B. and C. Rhodes, Trends in stability testing, with emphasis on stability during distribution and storage. Drug development and industrial pharmacy, 1999. 25(7): p. 857-868. 25.Lawrence, X.Y., et al., Biopharmaceutics classification system: the scientific basis for biowaiver extensions. Pharmaceutical research, 2002. 19(7): p. 921-925. 26.Amidon, G.L., et al., A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharmaceutical research, 1995. 12(3): p. 413-420. 27.Dokoumetzidis, A. and P. Macheras, A century of dissolution research: from Noyes and Whitney to the biopharmaceutics classification system. International journal of pharmaceutics, 2006. 321(1): p. 1-11. 28.Marroum, P.J., History and evolution of the dissolution test. Dissolution Technol, 2014. 21(3): p. 11-16. 29.Anand, O., et al., Dissolution testing for generic drugs: an FDA perspective. The AAPS journal, 2011. 13(3): p. 328. 57 30.Dressman, J.B., et al., Dissolution testing as a prognostic tool for oral drug absorption: immediate release dosage forms. Pharmaceutical research, 1998. 15(1): p. 11-22. 31.Sirisuth, N. and N.D. Eddington, In vitro-in vivo correlation, definitions and regulatory guidance. International Journal of Generic Drugs, 2002. 2: p. 1-11. 32.Van De Waterbeemd, H. and E. Gifford, ADMET in silico modelling: towards prediction paradise? Nature reviews. Drug discovery, 2003. 2(3): p. 192. 33.da Silva Honório, T., et al., In vitro–in vivo correlation of efavirenz tablets using GastroPlus®. AAPS PharmSciTech, 2013. 14(3): p. 1244- 1254. 34.Agoram, B., W.S. Woltosz, and M.B. Bolger, Predicting the impact of physiological and biochemical processes on oral drug bioavailability. Advanced drug delivery reviews, 2001. 50: p. S41-S67. 35.Parrott, N. and T. Lavé, Prediction of intestinal absorption: comparative assessment of GASTROPLUS™ and IDEA™. European journal of pharmaceutical sciences, 2002. 17(1): p. 51-61. 36.Ilić, M., I. Kovačević, and J. Parojčić, Deciphering nifedipine in vivo delivery from modified release dosage forms: Identification of food effect. Acta pharmaceutica, 2015. 65(4): p. 427-441. 58 37.Kuentz, M., et al., A strategy for preclinical formulation development using GastroPlus™ as pharmacokinetic simulation tool and a statistical screening design applied to a dog study. European journal of pharmaceutical sciences, 2006. 27(1): p. 91-99. 38.Saxena, A., et al., Prediction of pH dependent absorption using in vitro, in silico, and in vivo rat models: Early liability assessment during lead optimization. European Journal of Pharmaceutical Sciences, 2015. 76: p. 173-180. 39.Organization, W.H., http://www. who. int/mediacentre/factsheets/fs104/en. Geneva: WHO. Estimated incidence, prevalence and TB mortality [homepage on the internet].[one screen], 2004. 40.Sookram, C., et al., WHO’s supported interventions on salt intake reduction in the sub-Saharan Africa region. Cardiovascular diagnosis and therapy, 2015. 5(3): p. 186-190. 41.Murdoch, D. and R.C. Heel, Amlodipine. Drugs, 1991. 41(3): p. 478- 505. 42.Bisognano, J., et al., Incremental effectiveness of amlodipine besylate in the treatment of hypertension with single and multiple medication regimens. American journal of hypertension, 2004. 17(8): p. 676-683. http://www/ 59 43.Van Zwieten, P., Amlodipine: an overview of its pharmacodynamic and pharmacokinetic properties. Clinical cardiology, 1994. 17(9 Suppl 3): p. III3-6. 44.Olusola, A.M., et al., Equivalence of two generic brands of amlodipine besylate under biowaiver conditions. Int. J. Pharm. Pharm. Sci, 2012. 4(2): p. 265-268. 45.Patel, H.J., et al., Permeability studies of anti hypertensive drug amlodipine besilate for transdermal delivery. Asian J Pharm Clin Res, 2010. 3(1): p. 31-34. 46.Meredith, P.A. and H.L. Elliott, Clinical pharmacokinetics of amlodipine. Clinical pharmacokinetics, 1992. 22(1): p. 22-31. 47.Faulkner, J., et al., The pharmacokinetics of amlodipine in healthy volunteers after single intravenous and oral doses and after 14 repeated oral doses given once daily. British journal of clinical pharmacology, 1986. 22(1): p. 21-25. 48.Stopher, D., et al., The metabolism and pharmacokinetics of amlodipine in humans and animals. Journal of cardiovascular pharmacology, 1988. 12: p. S55&hyhen. 49.Abernethy, D.R., Pharmacokinetics and pharmacodynamics of amlodipine. Cardiology, 1992. 80(Suppl. 1): p. 31-36. 60 50.Abernethy, D.R., The pharmacokinetic profile of amlodipine. American heart journal, 1989. 118(5): p. 1100-1103. 51.Burnier, M. and H. Brunner, Angiotensin II receptor antagonists. The Lancet, 2000. 355(9204): p. 637-645. 52.Yamashiro, W., et al., Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans. Drug metabolism and disposition, 2006. 34(7): p. 1247-1254. 53.Criscione, L., et al., Valsartan: preclinical and clinical profile of an antihypertensive angiotensin‐II antagonist. Cardiovascular Therapeutics, 1995. 13(3): p. 230-250. 54.Varma, M.V., et al., pH-dependent solubility and permeability criteria for provisional biopharmaceutics classification (BCS and BDDCS) in early drug discovery. Molecular pharmaceutics, 2012. 9(5): p. 1199-1212. 55.Wu, C.-Y. and L.Z. Benet, Predicting drug disposition via application of BCS: transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system. Pharmaceutical research, 2005. 22(1): p. 11-23. 61 56.Flesch, G., P. Müller, and P. Lloyd, Absolute bioavailability and pharmacokinetics of valsartan, an angiotensin II receptor antagonist, in man. European journal of clinical pharmacology, 1997. 52(2): p. 115-120. 57.Waldmeier, F., et al., Pharmacokinetics, disposition and biotransformation of [14C]-radiolabelled valsartan in healthy male volunteers after a single oral dose. Xenobiotica, 1997. 27(1): p. 59-71. 58.Fogari, R., et al., Effect of valsartan addition to amlodipine on ankle oedema and subcutaneous tissue pressure in hypertensive patients. Journal of human hypertension, 2007. 21(3): p. 220. 59.Brachmann, J., et al., Effective and safe reduction of blood pressure with the combination of amlodipine 5 mg and valsartan 160 mg in hypertensive patients not controlled by calcium channel blocker monotherapy. Advances in therapy, 2008. 25(5): p. 399-411. 60.Flack, J., et al., Efficacy and safety of initial combination therapy with amlodipine/valsartan compared with amlodipine monotherapy in black patients with stage 2 hypertension: the EX-STAND study. Journal of human hypertension, 2009. 23(7): p. 479-489. 62 61.Philipp, T., et al., Two multicenter, 8-week, randomized, double- blind, placebo-controlled, parallel-group studies evaluating the efficacy and tolerability of amlodipine and valsartan in combination and as monotherapy in adult patients with mild to moderate essential hypertension. Clinical therapeutics, 2007. 29(4): p. 563-580. 62.Flynn, J.T., W.E. Smoyer, and T.E. Bunchman, Treatment of hypertensive children with amlodipine. American journal of hypertension, 2000. 13(10): p. 1061-1066. 63.Wells, T., et al., Effectiveness and safety of valsartan in children aged 6 to 16 years with hypertension. The Journal of Clinical Hypertension, 2011. 13(5): p. 357-365. 64.Flynn, J.T., et al., Efficacy and safety of the angiotensin receptor blocker valsartan in children with hypertension aged 1 to 5 years. Hypertension, 2008. 52(2): p. 222-228. 65.Zaid, A.N., et al., Preparation and stability evaluation of extemporaneous oral suspension of valsartan using commercially available tablets. International journal of pharmaceutical compounding, 2014. 18(2): p. 169-174. 66.Talamonti, W., R.F. Wagner, and H. Wen, Pharmaceutical Formulation of Valsartan. 2010, Google Patents. 63 67.Kulshreshtha, A.K., O.N. Singh, and G.M. Wall, Pharmaceutical suspensions. From Formulation Development to Manufacturing. Springer, New York, 2010. 68.Pharmacopeia US and national formulary, U.-N., United States Pharmacopeia and National Formulary (USP 40- NF 35) Rockville, MD: United States Pharmacopeial Convention, 2017. 2(2763). 69.Heo, Y.A., et al., Quantitative model for the blood pressure‐lowering interaction of valsartan and amlodipine. British journal of clinical pharmacology, 2016. 82(6): p. 1557-1567. 70.Caron, G., et al., Ionization, lipophilicity, and molecular modeling to investigate permeability and other biological properties of amlodipine. Bioorganic & medicinal chemistry, 2004. 12(23): p. 6107-6118. 71.Burges, R. and D. Moisey, Unique pharmacologic properties of amlodipine. The American journal of cardiology, 1994. 73(3): p. A2-A9. 72.McDaid, D. and P. Deasy, Formulation development of a transdermal drug delivery system for amlodipine base. International journal of pharmaceutics, 1996. 133(1-2): p. 71-83. 73.Lozoya‐Agullo, I., et al., In Situ Perfusion Model in Rat Colon for Drug Absorption Studies: Comparison with Small Intestine and Caco‐2 Cell Model. Journal of pharmaceutical sciences, 2015. 104(9): p. 3136-3145. 64 74.Erden, P.E., et al., Simultaneous determination of valsartan and amlodipine besylate in human serum and pharmaceutical dosage forms by voltammetry. Int J Electrochem Sci, 2014. 9: p. 2208-2220. 75.Thürmann, P.A., Valsartan: a novel angiotensin type 1 receptor antagonist. Expert opinion on pharmacotherapy, 2000. 1(2): p. 337-350. 76.Ali, A.A., N.A. Charoo, and D.B. Abdallah, Pediatric drug development: formulation considerations. Drug development and industrial pharmacy, 2014. 40(10): p. 1283-1299. 77.Rakhmanina, N.Y. and J.N. van den Anker, Pharmacological research in pediatrics: from neonates to adolescents. Advanced drug delivery reviews, 2006. 58(1): p. 4-14. 78.Chella, N., N. Shastri, and R.R. Tadikonda, Use of the liquisolid compact technique for improvement of the dissolution rate of valsartan. Acta Pharmaceutica Sinica B, 2012. 2(5): p. 502-508. 79.Dixit, A.R., S.J. Rajput, and S.G. Patel, Preparation and bioavailability assessment of SMEDDS containing valsartan. AAPS pharmscitech, 2010. 11(1): p. 314-321. 80.Biswas, N. and K. Kuotsu, Chronotherapeutically Modulated Pulsatile System of Valsartan Nanocrystals—an In Vitro and In Vivo Evaluation. AAPS PharmSciTech, 2017. 18(2): p. 349-357. 65 81.Food and D. Administration, Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutics classification system. Food and Drug Administration, Rockville, MD, 2000. 82.Cappello, B., et al., Improvement of solubility and stability of valsartan by hydroxypropyl-\boldbeta-cyclodextrin. Journal of inclusion phenomena and macrocyclic chemistry, 2006. 54(3-4): p. 289. 83.Pradhan, R., et al., Preparation and characterization of spray-dried valsartan-loaded Eudragit® E PO solid dispersion microparticles. asian journal of pharmaceutical sciences, 2016. 11(6): p. 744-750. 84.Saydam, M. and S. Takka, Bioavailability file: valsartan. FABAD J Pharm Sci, 2007. 32: p. 185-196. 85.Siddiqui, N., et al., Pharmacological and Pharmaceutical Profile of Valsartan: A Review. 2011. 86.Okumu, A., M. DiMaso, and R. Löbenberg, Computer simulations using GastroPlus™ to justify a biowaiver for etoricoxib solid oral drug products. European Journal of Pharmaceutics and Biopharmaceutics, 2009. 72(1): p. 91-98. 87.Cheng, C.-L., et al., Biowaiver extension potential to BCS Class III high solubility-low permeability drugs: bridging evidence for metformin immediate-release tablet. European Journal of Pharmaceutical Sciences, 2004. 22(4): p. 297-304. 66 88.Food and D. Administration, Guidance for industry: bioavailability and bioequivalence studies for orally administered drug products— general considerations. Food and Drug Administration, Washington, DC, 2003. 67 الوطنية النجاح جامعة العميا الدراسات كمية ركيب معمق فوري من االممودبين والفالسارتان باستخدام النمذجة ت الفسيولوجية إعداد وفاء جاسم محمود عابد إشراف أسماء رضوان. د الصيدالنية، العموم في الماجستير درجة عمى الحصول لمتطمبات استكماالا األطروحة هذه قدمت .فمسطين -نابمس الوطنية، النجاح جامعة في العميا، الدراسات بكمية 8102 ب النمذجة الفسيولوجية باستخدامالفالسارتان االممودبين و ركيب معمق فوري منت عدادإ وفاء جاسم محمود عابد إشراف أسماء رضوان. د الممخص كبسولةال إذابة القرص او سحق فإن سائمة، جرعات شكلتوفر الدواء عمى عدم حالة في: الخمفية ذلك، ومع. البمع عسر من يعانون الذين المرضى في األدوية ىذه الستخداماألنسب الخيار سيكون األمر ،ىذه الحاالتمثل في األدويةصالحية ثباتأو الحيوي لمتكافؤ دراسات أي إجراء يتم ال التنبؤ عمى البرمجيات االلكترونية أثبتت .اوسالمتيا بفعاليتي يتعمق فيما االرتباك إلى يؤدي الذي وعمى سموكو وذائبيتو في الكيميائية الفيزيائية خصائصو عمى اعتماًدا الحي الجسم في األدوية بأداء المستحضرات سوقفي ممودبين والفالسارتانألمزيج ا من سائمة تركيبة توجد ال. المختبر .الدم ضغط بارتفاع المصابين األطفال لتناسب الصيدالنية من فالسارتانالو ممودبيناأل فوري من مزيج معمق إعداد ىو الدراسة ىذه من اليدف كان: اليدف .المركب عمقلمم الذائبية و الثبات خصائص وتقييم المتاحة، التجارية األقراص في السوق المحمي المتاحة التجارية األقراص من فالسارتان/ أمموديبين المعمق إعداد تم: الطريقة Valzadepine® تقييم التغييرات الفيزيائية إلى إضافة دراسة سموك و ذائبية المعمق في درجات الحموضة المختمفةو الغرفة لضمان الثبات وتحديد فترة والكيميائية والميكروبية عمى مدى شير واحد في درجة حرارة GastroPlus رونيتبرنامج التنبؤ االلك صالحية المعمق، ثم تم استخدام TMامتصاص نماذج ناءلب األدويةممفات سموك مقارنة وتمت. المختبر في الذوبان بيانات أساس عمى األدوية من لكل خاصة .في المختبر مع ممفات التنبؤ لمتأكد من التكافؤ الحيوي بين المختبر والجسم الحي ج من ناحية المون مقبولة خصائص مع بنجاح فالسارتان/ األمموديبين تعميق إعداد تم: النتائج الفيزيائية بخصائصو االحتفاظ مع أسابيع أربعة لمدة مستقراً معمقال كان. والرائحة والطعم خصائص الذائبية واالمتصاص لممعمق كانت مماثمة لألقراص التي ىي مصدر المواد والكيميائية، الفعالة ومكافئة لما تنبأ بو البرنامج االلكتروني من تكافؤ حيوي بين السموك في المختبر والجسم .الحي . حةالمتا التجارية قراصاأل من فالسارتانمعمق فوري من مزيج االممودبين وال إعداد يمكن :خاتمة GastroPlus االلكترونيبرنامج التنبؤ تطبيق يمكن ذلك، عمى وعالوة TM مع جنب إلى جنبا عند تركيب معمق فوري من مطحون الحيوي التكافؤ تأكيد أجل من المختبر في الذوبان خصائص .المتوفرة األقراص