Practical Biopharmaceutics RPHT-451List of subject/week Topics niroduction to biopharmaceuties Definitions and related terms Routes of drug administration and plasma level time curve Drug absorption, mechanism of drug absorption and drug transporters Partition coefficient: determination of partition coefficient of citric acid (drug) between water and chloroform Effect pH on partition coefficient of of citric acid (drug) between buffer solutions (Acetate buffer pH 4.5 and Phosphate buffer pH 7.2) and chloroform Reasoning questions on basic concept of absorption and related questions and answers Different dissolution apparatus and Dissolution Test for Aspirin Tablets Solubility phenomenon and attempt to increase the bioavailability of poorly water soluble drug by Dissolution Test; Biorelevant Dissolution Test 1g Plasma protein binding; Double reciprocal plot fo determine association constant and binding sites Scatchard method fo determine association constant and binding sites Basic concept of distribution and related questions and answers Diffusion Study of some market Gel and Creams Preparations Different dissolution apparatus and Dissolution Test for Aspirin TabletsLAB. 1 Definitions Pharmaceutics The area of science which deals with the formulation and performance of pharmaceutical dosage forms. In fact pharmaceutics fits into the overall scheme of pharmacy. Biopharmaceuti The study of the relationship between dosage formulation and the therapeutic response. Phat ‘okinetics The kinetic study of ADME (Absorption, Distribution, Metabolism and Elimination) of the drug. Drug Absorption The process of uptake of the compound from the site of administration into the systemic circulation ( the appearance of the drug in the blood ). A prerequisite for absorption is that the drug should be in aqueous solution. The only relatively rare exception is absorption by pinocytosis. Accumulation The increase of drug concentration in blood and tissue upon multiple dosing until steady state is reached. Metabolism The sum of all chemical reactions for biotransformation of endogenous and exogenous substances which take place in the living cell. Dosage Form Refers to the gross physical form in which a drug is administered to or used by a patient. Dosage Regimen Is the systematized dosage schedule 3Dosage Range Indicates the quantitative range or amounts of the drug that may be prescribed within the frame of usual medical practice. Drug Product A dosage form containing one or more active therapeutic ingredients along with other substances included during the manufacturing process. Loading Dose or Initial D The dose size used in initiating therapy so as to yield therapeutic concentration which will result in clinical effectiveness. Maintenance Dose The dose size required to maintain the clinical effectiveness or therapeutic concentration according to the dosage regimen. Per Indicates that the dosage form is swallowed and the drug is absorbed from the gastrointestinal tract (first pass effect possible). First Pass Effect The loss of drug during its first pass. It is the phenomenon that some drugs are already metabolized between the site of absorption and reaching systemic circulation. First pass effect may occur in the gut wall, in the mesenteric blood and /or in the liver. Area Under the Curve The integral of drug blood level over time from zero to infinity, and is a measure of the quantity of a drug absorbed in the body. Biliary Recy: z The phenomenon that drugs emptied via bile into the small intestine can be reabsorbed from the intestinal lumen into systemic circulation.Apparent Volume of Distribution Is an abstract volume or space which is circulated from the ratio of the amount of drug in the body to the concentration in plasma once partition is stabilized. Va = (Da) / (Co) Ck ate ec The volume of blood in ml which is completely cleared of the drug per unit time (minutes) by urinary excretion or metabolism. Total body clearance ( renal and hepatic ) = K . Va Renal Clearance The hypothetical plasma volume in ml ( volume of distribution ) of the un-metabolized drug which is cleared in one minute via the kidney. a ‘inine Cle: e The ratio of the excretion of creatinine in urine to the concentration of creatinine in plasma. The creatinine clearance decreases with renal impairment and with age. Bioavailability The bioavailability of a drug is defined as its rate and extent of absorption, Relative Bioavailability The bioavailability of a drug product as compared to a recognized standard. Absolute Bioavailability The bioavailability of a drug product as compared by IV administration. Bioequivalence Comparable bioavailability, indicates that two or more similar dosage forms reach the general circulation at the same relative rate and relative extent. Therapeutic Inequivalence Clinicaly important differences in bioavailability.Introduction Route of Drug Administration Drugs may be given by parenteral, enteral, inhalation, transdermal (percutaneous), or intranasal route for systemic absorption. Each route of drug administration has certain advantages and disadvantages. Some characteristics of the more common routes of drug administration are listed in . The systemic availability and onset of drug action are affected by blood flow to the administration site, the physicochemical characteristics of the drug and the drug product, and by any pathophysiologic condition at the absorption site. [Table 13.1 Common Routes of Drug Administration Route iavalbity ‘Aévantanes Disadventanes Parenteral Routes Intravenous bolus |Comelete (100%) ssteniecrug [prugisgven forimmedite eet ineased chance fr adverse reac, iw |absorton, [ate of bioavalabinyconsiered Possible anaphais. instantaneous. Irravenoue [complete 200%) systanic drug [plasma dug levalsmore preety [equescilin insrtonoiruion st fusion (Vnf)__|bsorton. controled. [Rate of drug bsorton controled [May meclage uid volumes. [sue damage at ste finecon (nitrabon, necro, | yintsion ae. ster abscess). Nav use drugs wth poor ine sci andjrintating drags Intremuscdar |Repidrom aquesus soliton, [Easertinectthan nzaveneusimtsingdruos maybe very pau, inetion 4) injection. [Sow absorton fom nonaquesus [Lager volumes may be used _[Dierentrats of absorption depending on muscle group (soos. comparedto subataneous injected and blood aw. sautor, ‘swbautzeous —[romptfrom aqueous suuon. [General usedforinsuin [Rate of dug absnption depends on lod fow and nection ($C) injection. nection von Siow absorption rom epostory formulations.Enteral Routes laxcalor (and absorron om nd sole [ao st pas ees. [ome drugs may be evalowed sublingual.) ergs (tte mst dug r uae wih igh does. oraiPo} Ibseotin may var. Ssfest and easiest rut of du [Some duge may hare erat: absorption be unstable inthe amination (stinesal toa, orbe metabozed by ver porto [yseme abecrpon (caer, slower sorption rate [hay useimediatesseare and Komparedta vbohs im |nodied release dup prods. inert. eda PR) —_[bserpton ay varyrom [usta when pant crmet—_(Absrpton maybe erat. ‘upper. swalon mediation (ore refsble bsortion rom [used forioal and systemic |suppestory may mirate to deen postion [nena saison}. eects. [Some patent dsanfot. (ther Rowes Transdermal [Sow absorption atemay vary. ransdamal vay system |Someitatonby patch or dua. lpsesea5/t0use Iinzeasedatsorpton wih [Used fortp-souble rugs wth Parmesity of sen varable wth contin anatome te, cde dressing ow dose and ow Mu age, and gerd. To of ream or ramen base alas dra release and absetin| Intaiton and Rap absorion. (nay be usedteriecaler system [Price Sze of rug deternnes anatomic paceretin| inanasal ef espkatry waa. Total doce absorbed svar a tilt cous ri, Some drug ay be swalowed Plasma Level-Time Curve The plasma level-time curve is generated by measuring the drug concentration in plasma taken at various time intervals after a drug product is administered. The concentration of drug in each plasma sample is plotted on rectangular coordinate graph against the corresponding time at which the plasma sample was removed. As the drug reaches the general (systemic) circulation, plasma drug concentrations will rise up to a maximum. The relationship of the drug level- pharmacological parameters for the drug is shown in the figure. MEC and MTC represent the minimum effective concentration and minimum toxic concentration of drug, respectively. ime curve and variousConcentration Onset tne TimeLAB. 3 — Drug Absorption Absorption is the process of movement of a drug from the site of application into the extracellular fluid of the body tissues. Systemic absorption of most drug products consists of a succession of rate processes viz. i) dissolution of the drug in an aqueous environment (requires water solubility); and ii) permeation across cell membran: circulation and, ultimately, to its site of action(requires lipid solubility). Absorption window Is the segment of the gastrointestinal tract from which the drug is well absorbed and beyond which the drug is either poorly absorbed or not absorbed at all. For drugs given orally, an anatomic absorption window may exist within the GI tract in which the drug is efficiently absorbed. Drugs contained in a nonbiodegradable controlled-release dosage form must be completely released into this absorption window to be absorbed before the movement of the dosage form into the large bowel. Anatomic and Physiologic Considerations Oral Cavity Saliva is the main secretion of the oral cavity, and it has a pH of about 7. Saliva contains ptyalin (salivary amylase), which digests starches. Mucin, a glycoprotein that lubricates food, is also secreted and may interact with drugs. About 1500 mL of saliva is secreted per day. Esophagus The esophagus connects the pharynx and the cardiac orifice of the stomach. The pH of the fluids in the esophagus is between 5 and 6. The lower part of the esophagus ends with the esophageal sphincter, whichprevents acid reflux from the stomach. Tablets or capsules may lodge in this area, causing local irritation. Very little drug dissolution occurs in the esophagus. Stomach The stomach is innervated by the vagus nerve. However, local nerve plexus, hormones, mechanoreceptors sensitive to the stretch of the GI wall, and chemoreceptors control the regulation of gastric secretions, including acid and stomach emptying. The fasting pH of the stomach is about 2 to 6. In the presence of food, the stomach pH is about 1.5 to 2, due to hydrochloric acid secreted by parietal cells. Stomach acid secretion is stimulated by gastrin and histamine. Gastrin is released from G cells, mainly in the antral mucosa and also in the duodenum. Gastrin release is regulated by stomach distention (swelling) and the presence of peptides and amino acids. A substance called intrinsic factor for vitamin B-12 absorption and various gastric enzymes, such as pepsin, which initiates protein digestion, are secreted into the gastric lumen to initiate digestion. Basic drugs are solubilized rapidly in the presence of stomach acid. Mixing is intense and pressurized in the antral part of the stomach, a process of breaking down large food particles described as antral milling. Food and liquid are emptied by opening the pyloric sphincter into the duodenum. Stomach emptying is influenced by the food content and osmolality. Fatty acids and mono- and diglycerides delay gastric emptying (). High-density foods generally are emptied from the stomach more slowly. The relation of gastric emptying time to drug absorption is discussed more fully in the next section. Duodenum A common duct from the pancreas and the gallbladder enters into the duodenum. The duodenal pH is about 6 to 6.5, because of the presence of bicarbonate that neutralizes the acidic chyme emptied from the stomach. The pH is optimum for enzymatic digestion of protein and peptide food. Pancreatic juice containing enzymes is secreted into the duodenum from the bile duct. Trypsin, chymotrypsin, and carboxypeptidase are involved in the hydrolysis of proteins into amino acids. Amylase is involved in the digestion of carbohydrates. Pancreatic lipase secretion hydrolyzes fats into fatty acid. The complex fluid medium in the duodenum helps to dissolve many drugs with limited aqueous solubility.The duodenum is a site where many ester prodrugs are hydrolyzed during absorption. The presence of proteolytic enzymes also makes many protein drugs unstable in the duodenum, preventing adequate absorption. Jejunum The jejunum is the middle portion of the small intestine, between the duodenum and the ileum. Digestion of protein and carbohydrates continues after addition of pancreatic juice and bile in the duodenum. This portion of the small intestine generally has fewer contractions than the duodenum and is preferred for in-vivo drug absorption studies. Tleum The ileum is the terminal part of the small intestine. This site has fewer contractions than the duodenum and may be blocked off by catheters with an inflatable balloon and perfused for drug absorption studies. The pH is about 7, with the distal part as high as 8. Due to the presence of bicarbonate secretion, acid drugs will dissolve. Bile secretion helps to dissolve fats and hydrophobic drugs. The ileocecal valve separates the small intestine from the colon. Colon The colon lacks villi and has limited drug absorption also, because of the more viscous and semisolid nature of the lumen contents. The colon is lined with mucin that functions as lubricant and protectant. The pH in this region is 5.5 to 7 (). A few drugs, such as theophylline and metoprolol, s region. Drugs that are absorbed well in this region are good candidates for an oral sustained-release dosage form. The colon contains both aerobic and anaerobic microorganisms that may metabolize some drugs. For example, L-dopa and lactulose are metabolized by enteric bacteria. Crohn's disease affects the colon and thickens the bowel wall. The microflora also become more anaerobic. Absorption of clindamycin and propranolol are increased, whereas other drugs have reduced absorption with this disease (). Rectum The rectum is about 15 cm long, ending at the anus. In the absence of fecal material, the rectum has a small amount of fluid (approximately 2 mL) with a pH about 7. The rectum is perfused by the superior, middle, and inferior hemorrhoidal veins. The inferior hemorrhoidal vein (closest to the anal sphincter) and the middle hemorrhoidal vein feed into the vena "cava and back to the heart. The superior hemorrhoidal vein joins the mesenteric circulation, which feeds into the hepatic portal vein and then to the liver. Drug absorption after rectal administration may be variable, depending on the placement of the suppository or drug solution within the rectum. A portion of the drug dose may be absorbed via the lower hemorrhoidal veins, from which the drug feeds directly into the systemic circulation; some drugs may be absorbed via the superior hemorthoidal vein, which feeds into the mesenteric veins to the hepatic portal vein to the liver, and be metabolized before systemic absorption. Effect of Disease States on Drug Absorption Drug absorption may be affected by any disease that causes changes in (1) intestinal blood flow, (2) gastrointestinal motility, (3) changes in stomach emptying time, (4) gastric pH that affects drug solubility, (5) intestinal pH that affects the extent of ionization, (6) the permeability of the gut wall, (7) bile secretion, (8) digestive enzyme secretion, or (9) alteration of normal GI flora. Some factors may dominate, while other sometimes cancel the effects of each other. Pharmacokinetic comparing subjects with and without the disease are generally ary to establish the effect of the disease on drug absorption. Patients in an advanced stage of Parkinson's disease may have difficulty swallowing and greatly diminished gastrointestinal motility. A case was reported in which the patient could not be controlled with regular oral levodopa medication because of poor absorption. Infusion of oral levodopa solution using a j-tube gave adequate control of his symptoms. The patient was subsequently placed on this mode of therapy. Achlorhydricpatients may not have adequate production of acids in the stomach; stomach HCI is essential for solubilizing insoluble free b: Many weak-base drugs that cannot form soluble salts will remain undissolved in the stomach when there is no hydrochloric acid present and are therefore unabsorbed. Salt forms of these drugs cannot be prepared because the free base readily precipitates out due to the weak basicity.Congestive heart failure (CHF) patients with persistent edema have reduced splanchnic blood flow and develop edema in the bowel wall. In addition, intestinal motility is slowed. The reduced blood flow to the intestine and reduced intestinal motility results in a decrease in drug absorption. For example, furosemide (Lasix), a commonly used loop diuretic, has erratic and reduced oral absorption in patients with CHF and a delay in the onset of action. Drugs that Affect Absorption of Other Drugs Anticholinergic drugs in general may reduce stomach acid secretion. Propantheline bromide is an anticholinergic drug that may slow stomach emptying and motility of the small intestine. Tricyclic antidepressants and phenothiazines also have anticholinergic side effects that may cause slower peristalsis in the GI tract. Slower stomach emptying may cause delay in drug absorption. Metoclopramide is a drug that stimulates stomach contraction, relaxes the pyloric sphincter, and, in general, increases intestinal peristalsis, which may reduce the effective time for the absorption of some drugs and thereby reduce the peak drug concentration and the time to reach peak drug concentration. For example, digoxin absorption from a tablet is reduced by metoclopramide but increased by an anticholinergic drug, such as propantheline bromide. Allowing more time in the stomach for the tablet to dissolve generally helps with the dissolution and absorption of a poorly soluble drug, but would not be helpful for a drug that is not soluble in stomach acid. Cholestyramine is a nonabsorbable ion-exchange resin for the treatment of hyperlipemia. Cholestyramine adsorbs warfarin, thyroxine, and loperamide, similar to activated charcoal, thereby reducing absorption of these dru; Absorption of calcium in the duodenum is an active process facilitated by vitamin D, with calcium absorption as much as four times more than that in vitamin D deficiency states. It is believed that a calcium-binding protein, which increases after vitamin D administration, binds calcium in the intestinal cell and transfers it out of the base of the cell to the blood circulation. 13Mechanisms of Drug Absorption 1- Simple nonionic diffusion or passive diffusion 2- Carrier — Mediated Diffusion a) Active Transport b) Facilitated Diffusion 3- Vesicular Transport 4- Ion-Pair Absorption Drug transporters Several transport proteins are expressed in the intestinal epithelial cells. Although some transporters facilitate absorption, other transporters, such as P-glycoprotein may effectively inhibit drug absorption. 1- Efflux transporter Several members of the ATP binding cassette (ABC) transporter protein by ability to efflux xenobiotics from the cytoplasm and across the cellular membrane in an energy-dependent manner e.g; p-gp. P-gp, an energy-dependent, membrane-bound protein, is an_ efflux transporter that mediates the secretion of compounds from inside the cell back out into the intestinal lumen, thereby limiting overall absorption. Thus, drug absorption may be reduced or increased by the presence or absence of efflux proteins. Many drugs and chemotherapeutic agents, such as cyclosporin A, verapamil, terfenadine, fexofenadine, are substrates of P-gp. In addition, individual genetic differences in intestinal absorption may be the result of genetic differences in P-gp and other transporters.Apical Membrane (lumen) (pooia) auesquiayy jexrejosea 2- Influx transporter Several different uptake transporters are localized on the apical (brush border) membrane of enterocytes that facilitate the entry of non- membrane-permeable molecules into the blood circulation. Examples: 1- peptide transporter 1 (PEPT!) is responsible for the uptake of di- and tripeptides formed during the digestion of proteins (80%). 152- many B-lactam antibiotics (e.g., oral cephalosporin and penicillin drugs) and some angiotensin-converting enzyme inhibitors (e.g., captopril and enalapril) are known substrates of PepT1. 3- anumber of amino acid or dipeptide-conjugated prodrugs (e.g., valacyclovir, valyl ester prodrug of the potent antiviral agent acyclovir)undergo PepT 1-mediated transport. Prodrugs of this type are commonly referred to as PepT1 targeted prodrugs. Clinical Example Multidrug resistance (MDR) to cancer cells has been linked to efflux transporter proteins such as P-gp that either exclude or extrude chemotherapeutic agents from the cells. Paclitaxel (Taxol) is an example of coordinated metabolism, and efflux to induce efflux protein. P-gp is responsible for 85% of paclitaxel excretion back into the GI tract. Which Absorption Path Dominates Drug Absorption? Although different mechanisms of oral drug absorption have been shown in small intestinal regions, under physiological conditions, several routes may contribute to drug absorption at the same time. Usually, the fastest route dominates the absorption of a particular compound. In general, passive diffusion is the main mechanism for absorption of many lipophilic compounds, while the carrier-mediated process governs the absorption of transporter substrates. In some cases, paracellular junction is the route for the absorption of some small hydrophilic compounds with molecular weight le:LAB. 4 Partition coefficient The partition coefficient of a drug between a fat-like solvent, such as chloroform, and water or aqueous buffer approximating the pH of the absorption site, is an important factor in drug absorption. A direct correlation was found between partition coefficient and absorption for drugs having nearly the same pKa, e.g. barbiturates. Determination One of the most common ways of measuring partition coefficients is to use the shake flask method. This relies on the equilibrium distribution of a drug between an oil and an aqueous phase. Prior to the experiment the aqueous phase should be saturated with the oil phase and vice versa. The experiment should be carried out at constant temperature. The drug should be added to the aqueous phase and the oil phase which, in the case of octanol, as it is less dense than water, will sit on top of the water. The system is mixed and then left to reach equilibrium (usually at least 24 hours). The two phases are separated and the concentration of drug is measured in each phase and a partition coefficient calculated. Add analyte @ Measure analyte in () mix both phases \\ di) separate | p= Sone inowg.. _ cone. inaqu. Organic log P= 0.954 Buffer solventPartition Coefficient of Citric Acid (drug) between Water aqueous phase) and chloroform (non aqueous phase). AIM Determination of the Partition Coefficient of Citric Acid (drug) between Water (aqueous phase) and chloroform (non aqueous phase). CHEMICALS. in the aqueous phi Apparatus 100 ml separating funnel, titration apparatus, 5 ml pipette Chloroform, 0.2 M Citric acid (drug already dissolved —water), 0.1 M NaOH, phenolphthalein indicator PROCEDURE 1. Record the room temperature. 2. Using suitable apparatus pour 25 ml of the 0.2 M Citric acid solution and 25 ml of 2-methylpropan-l-ol into a 100 cm3 separating funnel. Stopper the funnel and shake vigorously for 6 minutes. (Release pressure in the funnel by occasionally opening the tap.) 3. Separate approximately 15 ml of EACH layer and collect them in TWO clean beakers. (Discard the fraction near the junction of the two 4.Pipette 5 ml of the aqueous layer into a titration flask and titrate it with 0.1M sodium hydroxide solution using 2 drops of phenolphthalein indicator. 5. Using another pipette, deliver 5 ml of the non-aqueous layer into a titration flask and titrate it with 0.1 M sodium hydroxide solution using 2 drops of phenolphthalein indicator 7. For each experiment, calculate the ratio of the concentration of citric acid in the non-aqueous layer in relation to the aqueous layer Partition coefficient / distribution coefficient, K (constant) = (Concentration of solute in solvent A / Concentration of solute in solvent B Aqueous layer sno. |Initialreading [Final reading [difference [Mean Organic layer Sno. |Initialreading [Final reading _| difference [MeanFor separating bottle Norg. = burette reading x 0.1 5 Naq = burette reading x 0.1 5 Distribution coefficient k1 = Report — The partition coefficient of citric acid between chloroform and water is when k is greater than | it means drug is more soluble in organic phase when k is lesser than | it means drug is more soluble in aqueous phase when k is | it means solubility of drug in organic phase is equal to that in aqueous phaseLAB. 5 drugs Aim: Determination of the effect of pH on Partition Coefficient of Citric Acid (drug) between (aqueous phase) and chloroform (non aqueous phase). Introduction For a drug to cross a membrane barrier it must normally be soluble in the lipid material of the membrane to get into membrane it has to be soluble in the aqueous phase as well to get out of the membrane. Many drugs have polar and non-polar characteristics or are weak acids or bases. For drugs which are weak acids or bases the pH of the GI tract fluid and the pH of the blood stream will control the solubility of the drug and thereby the rate of absorption through the membranes lining the GI tract. Brodie et al. stated that when a drug is ionized it will not be able to get through the lipid membrane, but only when it is non-ionized and therefore igher lipid solubility, Chemicals--- Organic solvent, 0.2 M Citric acid (drug already dissolved in the aqueous phase — Buffer solutions with different pH ), 0.1 M NaOH, phenolphthalein indicator Apparatus 100 ml separating funnel, titration apparatus, 5 ml pipette Procedure 1. Record the room temperature. 2. Using suitable apparatus pour 25 ml of the 0.2 M Citric acid solution and 25ml of organic solvent into a separating funnel. Stopper the funnel and shake vigorously for 6 minutes. (Release pressure in the funnel by occasionally opening the tap). 3. Separate approximately 15 ml of each layer and collect them in two clean beakers. (Discard the fraction near the junction of the two layers.) 4. Pipette 5 ml of the aqueous layer into a titration flask and titrate it with 0.1 M sodium hydroxide solution using two drops of phenolphthalein indicator. 5. Using another pipette, deliver 5 ml of the non-aqueous layer into a titration flask and titrate it with 0.1 M sodium hydroxide solution using 2 drops of phenolphthalein indicator. 206. Repeat steps (2) to (5) with another separating funnel using either one of the following as aqueous solutions a. Acetate buffer pH 4.5 b. Phosphate buffer Ph 7.2 7. For each experiment, calculate the ratio of the concentration of citric acid in the non-aqueous layer in relation to the aqueous layer 8.Take burette reading individually for aqueous solvent, Acetate buffer pH 4.5 and Phosphate buffer Ph 7.2 and Calculate K value and write the results pH 4. Aqueous layer sno. [Initial reading [Final reading | difference [Mean Organic layer sno. [Initial reading [Final reading | difference [Mean Norg = burette reading x 0.1 5 Naq = burette reading x 0.1 5 Distribution coefficient k = atFor pk 17.2 Aqueot yer Initial reading Final reading difference Mean Organi ic layer S.nO. Initial reading Final reading difference Mean Norg = burette reading x 0.1 5 Naq = burette reading x 0.1 Distribution coefficient k = Results--- 5 For aqueous solvent, Acetate buffer pH buffer pH 7.2 for solvent is 22 4.5 and PhosphateLAB.6 Reasoning questions 1. Why are some drugs absorbed better with food and others are retarded by food? 2. If a drug is administered orally as a solution, does it mean that all of the drug will be systemically absorbed? 3. What is the biggest biological factor that contributes to delay in drug absorption? 4. A recent bioavailability study in adult human volunteers demonstrated that after the administration of a single enteric-coated aspirin granule product given with a meal, the plasma drug levels resembled the kinetics ofas stained-release drug product. In contrast, when the product was given to fasted subjects, the plasma drug levels resembled the kinetics of an immediate-release drug product. Give a plausible explanation for this observation. 5. What are biopharmaceutics, absorption and an" absorption window? 6. Which region of the gastrointestinal tract is most populated by bacteria? What types of drugs might affect the gastrointestinal flora? 7. Allowing drug more time in the stomach may be helpful oruseless. Explain 8. Efflux transporter. Explain and give examples. 9. PepT1 targeted prodrugs. 10. Relationship between MDR and transporters 23LAB. 7 Revision 24LAB. 8 First midterm exam 25LAB. 9 Solubility phenomenon So dispersion Solid dispersion technology is the science of dispersing one or more active ingredient in an inert matrix in the solid state in order to achieve increased dissolution rate, sustained release of drugs and thus improve solubility and stability. The bioavailability of many poorly water soluble drugs is limited by their dissolution rates, which are in-turn controlled by surface area that they present for dissolution. Aschematic representation of the bioavailability enhancement of a poorly water-soluble drug by solid dispersion compared with conventional tablet or capsule s POORLY WATER- SOLUBLE DRUG — - m Solid dispersion/ Table capsule —____ Dosage form —_— Soluson Disintegration Disini€gration Large solid particle Colloidal particlesi (usually 5-100 Drug in GI tract ily globules microns) (usually <1 microns) —~ ABSORPTION Lower disohtion INTO BODY Higher dissohtion Solid dispersion improves the bioavailability of poorly water soluble drugs by combining drug with a polymer. Suitable carrier polymer that are useful in formation of solid drug dispersion include polyvinyl pyrrolidone (PVP), high molecular weight polyethylene glycol (PEG), urea, mannitol, citric acid, copolymer, acrylic polymers, sugars, 26etc. The carrier of choice is most dispersions is polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG). METHODS OF PREPARATIONS Physical Mixture: This method involves mixing, an accurately weighted quantities of drug and carrier in suitable/ required proportion in a mortar and sieved through mesh No. 100. Fusion Method: Accurately weighed quantity of carrier was melted in a porcelain dish and to this calculated quantity of drug was added with thorough mixing. The molten mass was rapidly cooled with constant stirring using glass rod. The resulting solid dispersion was crushed pulverized and passed through 44 mesh sieve and stored in a dessicator as shown in Solvent Evaporation Method: Accurate amount of drug and carrier were weighted. The carrier was dissolved in organic solvent and drug was added in parts with continuous stirring until dissolved. The solvent was evaporated at 40°C under vacuum until solid dispersion was achieved a7LAB. 10 Assessment of biopharmaceutical properties The key biopharmaceutical properties that can be quantified and therefore give an insight into the absorption of a drug are its: + Release from its dosage form into solution at the absorption site The solubility of a drug across the gastrointestinal pH range will be one of the first indicators as to whether dissolution is liable to be rate limiting in the absorption process. A knowledge of the solubility across the gastrointestinal pH range can be determined by measuring the equilibrium solubility in suitable buffers or by using an acid or a base titration method. An in vitro/in vivo correlation may only be possible for those drugs where dissolution is the rate-limiting step in the absorption process. Determining full dissolution profiles of such drugs in a number of different physiologically representative media will aid the understanding of the factors affecting the rate and extent of dissolution. Aims of Dissolution Testing 1- to develop and evaluate the performance of new formulations by examining drug release from dosage forms, evaluating the stability of these formulations, monitoring and assessing the formulation consistency and changes 2- to obtain an in vitro—in vivo correlation (IVIVC; predictive mathematical model describing the relationship between an in vitroproperty of a dosage form and a relevant in vivo response.) and other biorelevant information that will guide bioavailability and/or bioequivalence assessment of drug products. 3- an important QC tool which is used to verify manufacturing and product consistency. It is employed to evaluate the quality of the product during its shelf life, as well as to assess postapproval changes. 28Biorelevant Dissolution Testing The biorelevant dissolution method should be able to simulate the in vivo environment where the majority of the drug is released from the formulation. In principle, the design of such a system should, at minimum, account for the following factors to reflect the physiological conditions in the GI tract: 1. pH Conditions 2. Key aspects of the composition of the GI contents (e.g., osmolarity, ionic strength, surface tension, bile salts, and phospholipids) 3. Volume of the GI contents. 4, Transit times 5. Motility pattern 6. Dosing conditions (e.g., administered with food) Table 18.1 Dissolution medium to simulate gastric | conditions in the fasted state (proposed by Dressman et al 1998) Component Concentration/amount Hydrochloric acid 0.01-0.05 M | Sodium iaury! sulphate 259 Sodium chloride 29 Distilled water qs to 1000 mL 29Table 18.2 Dissolution medium to simulate intestinal | conditions in the fasted state (proposed by Dressman et al 1998) Component Concentration/amount Potassium dinydrogen phosphate 0.029 M Sodium hydroxide gs to pH 6.8 Sodium taurocholate (bile salt) 5mM Lecithin 1.5mM | Potassium chloride 0.22M Distilled water gs to 1000 mL pH = 6.8, osmolarity = 280-310 mOSm. | Buffer capacity = 10 + 2 mEqiL/pH. Table 18.3 Dissolution medium to simulate intestinal conditions in the fed state (proposed by Dressman et al 1998) Component Concentration/amount | Acetic acid 0.144 M Sodium hydroxide gs to pH5 | Sodium taurocholate (bile salt) 15 mM Lecithin 4mM Potassium chloride 0.19M | Distilled water gs to 1000 mL pH =5, osmolarity = 485-535 mOSm. Butfer capacity = 76 + 2 mEq/L/pH 30In-vitro dissolution testing The most commonly used dissolution apparatus for solid oral dosage forms are i an {) Rotating basket ‘method Advantages: 1-Suited for QC dissolution testing 2-Easy to operate 3-Standardized 4- Robust + Disadvantages + Fixed (limited) volume of medium makes it unsuitable for testing of poorly soluble drugs + Formulation may clog the basket mesh @ Small disintegrated particles may fall out 312- the paddle method (USP Apparatus Il), ve Advantages: 1-Suited for QC dissolution testing 2-Easy to operate 3-Standardized 4- Robust: Stability in physiological fluids + Disadvantages Fixed (limited) volume of medium makes it unsuitable for testing of poorly soluble drugs Floating dosage forms (e.g. capsules) require sinkers 3the reciprocating cylinder (USP Apparatus Il) 32Advantages: Media change fully automated Ease of sampling Suitable for MR products Small media volumes suitable for predictive dissolution Hydrodynamics more similar to those in the gastrointestinal tract + Disadvantages Not suitable for dosage forms that disintegrate into small particles The use of surfactants is discouraged as they can cause foaming Small-medium volume unsuitable for QC dissolution testing of poorly soluble drugs Media evaporation for tests of long duration 4-the flow-through cell system (USP Apparatus IV). Collection Heating Syringe Media coil pump reservoir Advantages: Unlimited fluid supply makes it ideal for testing poorly soluble drugs Allows for rapid media change Continuous sampling 33+ Disadvantages Limited experience Complex design makes it inappropriate for QC dissolution testing Results very dependent on the type of pump used Q Stability in physiological fluids The stability of drugs in physiological fluids depends on two factors: stability of the drug across the gastrointestinal Ph range, ice. the drug's pH-stability profile between pHI and pH 8, 2- susceptibility to enzymatic breakdown by the gastrointestinal fluids. The stability of a drug in gastrointestinal fluids can be assessed by simulated gastri inal media. In general the drug is incubated with either real or simulated fluid at 37°C for a period of 3 hours and the drug content analysed. A loss of more than 5% of drug indicates 1- the chemi and inte: potential instability. : Permeability It depends mainly on logP (partition coefficient). : Susceptibility to presystemic clearance. Pres reaches the sys stemic metabolism is the metabolism that occurs before the drug such as brush ‘mic circulation. Intestinal cell fractions, s border membrane preparations which contain an abundance of hydrolytic enzymes, and homogenized preparations of segments of rat intestine, can also be used to determine intestinal presystemic metabolism. Drugs are incubated with either brush border membrane preparations or gut wall homogenate at 37°C and the drug content analysed. 34solution Test for Aspirin Tablets Conditions: o Apparatus : I (basket) © Medium : 500 ml of 0.05 M acetate buffer pH 4.5 o Temp. : 37+ 0.5 oC 0 Speed : 50 rpm o Time : 45 min. Procedure: 1. Place one tablet in the basket, immerse in the vessel, and then start the apparatus at the above conditions. 2. At specified time intervals (5, 10, 15, 20, 25, 30 and 45 min) withdraw 1 ml sample from the dissolution medium, through a Millipore filtration unit (polyethylene tube with a cotton), and place the sample in a test tube. 3. Replace the withdrawn sample with 1 ml fresh acetate buffer kept at 37 + 0.50 C. 4, Dilute 1 ml of the collected sample to 5, 10, 20 or 25 ml (dilution factor =1:5, 1:10, 1:20 or 1:25) with fresh acetate buffer (in a volumetric flask),mix well. (Dilution is made if necessary.) 5. Read the absorbance for the diluted samples at 265 nm against a blank of acetate buffer. 7. Plot the dissolution curve of aspirin (% released vs. time). 8. From the dissolution curve, determine the time required for 80% of the labeled amount of the drug to be released (go into solution), i.e., t 80%. 35Results of the dissolution of aspirin tablets Time (min) Abs. at 265 nm Dilution factor = (total vol / vol taken from the sample) Conc. = _abs x dil factor x 5 (mg/S00ml) % released = cone._x 100 ‘strength 20 25 30 45 36LAB. 11 Drug Distribution Plasma Protein Binding Drugs may bind to various macromolecular components in the blood including albumin, alpha acid glycoprotein, _ lipoproteins, immunoglobulin, and erythrocytes (RBC). Factors affecting drug-protein binding: a) The drug Physicochemical properties of the drug. Total concentration of the drug in the body. b) The protein Quantity of protein available for drug-protein binding. Quality or physicochemical nature of the protein synthesized. c) The affinity between the drug and the protein, including the magnitude of the association constant. d) Drug interaction Competition for the drug by other substances at a protein-binding site. Alteration of the protein by a substance that modifies the affinity of the drug for the protein; e.g. aspirin acetylates lysine residues of albumin. e) The patho-physiological condition of the patient; e.g. drug-protein binding may be reduced in uremic patients and in patients with hepatic disease. Kinetics of Protein Binding The kinetics of reversible drug-protein binding can be described by the law of mass action, as follows: Protein + drug —] drug-protein complex or 37() + D) @ @D) From the equation and the law of mass action, an association constant (Ka ) can be expressed as the ratio of the molar concentration of the products and the molar concentration of the reactants, Ka = [(PD)/(P)(D)] The extent of drug-protein complex formed is dependent on the association binding constant (Ka). The magnitude of (Ka) yields information on the degree of drug-protein binding. Drugs strongly bound to protein have a very large (Ka) and exist mostly as the drug-protein complex. With such drugs a large dose may be needed to obtain a reasonable therapeutic concentration of free drug. Most kinetic studies in-vitro use purified albumin as a standard protein source, because this protein is responsible for the major portion of plasma drug-protein binding. Experimentally, both the free drug (D) and the protein-bound drug (PD) as well as the total protein concentration (P) + (PD), may be determined. To study the binding behaviour of drugs, a determinable quantity (r) is defined as follows: r = (moles of drug bound) / (total moles of protein) Because moles of drug bound is (PD) and the total moles of protein is (P) + (PD), this equation becomes: r= {(PD)/[(PD) + (P)]} According to the equation of the law of mass action, (PD) = Ka(P)(D) 1 = [Ka (P) (D)]/ [Ka (P) (D) + (P)] = [Ka (D)]/ [1 + Ka(D)] This equation describes the simplest situation, in which | mole of the drug binds to 1 mole of protein in a 1:1 complex. This case assumes only one independent binding site for each mole of drug. If there are (n) identical independent binding sites per protein molecules, there the following is used: 38r= [n Ka (D)]/[1 + Ka(D)] Protein molecules are quite large compared to drug molecules and may contain more than one type of binding site for the drug. Determination of binding constants and binding sites by graphical methods In vitro methods (known protein concentration) The values for the association constants and the number of binding sites are obtained by various graphical methods. The reciprocal of the last equation gives the following equation: Vr=[1 + Ks (D)]/[n Ks(D)] = [(1) /(n Ka (D)] + (1/n) a) Double reciprocal plot: A graph of 1/r versus 1/(D) . The y intercept is I/n and the slope is I/n Ka. From this graph, the number of binding sites may be determined from the y intercept, and the association constant may be determined from the slope if the value of n is known, If the graph of 1/r versus 1/(D) does not yield a straight line, then the drug-protein binding process is probably more complex. The original equation assumes one type of binding site and no interaction among the binding sites. Frequently, the last equation is of binding sites and binding constants, using computerized iteration methods. used to estimate the number 391/ [8] Hypothetical binding of drug to protein. The line was obtained with the double reciprocal equation Problem Determine the number of binding sites (n) and the association constant (K ) from the following data using double reciprocal equation. tr D 0.15 1.7 0.26 1.48 0.4 1.29 0.6 1.1 0.75 0.95 40Rearrangement of the original equation. The Scatchard plot spreads the data to give a better line for the estimation of the binding constants and binding sites. From the original equation we obtain. [n Ka (D)]/ [1 + Ka(D)] r+rKa(D)=nKa(D) r=nKa(D)—r Ks(D) 1/(D) =n Ka —r Ka A graph constructed by plotting r/(D) versus r yields a straight line with the intercepts and slope shown in the figure. Hypothetical binding of drug to protein. The line was obtained with Scatchard equation a1Problem Determine the number of binding sites (n) and the association constant (K a) from the following data using the Scatchard equation. r (D x 10 4 M) 0.40 0.33 0.80 0.89 1.20 2.00 1.60 5.33 42LAB. 13 Reasoning questions 1. Do all drugs that bind proteins lead to clinically significant interactions? 2. How does drug protein binding affect drug elimination? 3. How does a physical property, such as partition coefficient, affect drug distribution? 4. What are the factors to consider when adjusting the drug dose for a patient whose plasma protein concentration decreases to half that of normal? 5. When a body organ is equilibrated with drug from the plasma, the drug concentration in that organ should be the same as that of the plasma. True or false? 6. Can a protein-bound drug be metabolized? 7. Why is the zone of inhibition in an antibiotic disc assay larger for the same drug concentration in water than in serum? 8. Discuss the clinical significance of drug protein binding on the following: a. Drug elimination b. Drug—drug interactions c. "Percent of drug-bound" data d. Liver disease 439. Explain the effects of plasma drug-protein binding and tissue drug— protein binding on the apparent volume of distribution and drug elimination. 10. Naproxen is a nonsteroidal anti-inflammatory drug (NSAID) that is highly bound to plasma proteins, >99%. Explain why the plasma concentration of free (unbound) naproxen increases in patients with chronic alcoholic liver disease and probably other forms of cirrhosis, whereas the total plasma drug concentration decreases. 11. It is often assumed that linear binding occurs at therapeutic dose. What are the potential risks of this assumption? 12. When a drug is 99% bound, it means that there is a potential risk of saturation. True or false? 44LAB. 14 Diffusion Process Aim-To perform the diffusion study of given sample Apparatus and chemicals---Franz cell apparatus, OR tube assembled glass diffusion apparatus, sample buffer solutions, | UV spectrophotometer, diclofenac sodium and ibuprofen topical preparations Diffusion is a process where molecules or particles move from a high concentrated area to a low concentrated area Donor Compound — Dener Chamber Flat Ground « rou Joint Membrane —p —— Sampling Port teeter = , Circulator - 3 nN. = ” ‘Chamber Water Jacket —>| Proceedure---The diffusion studies were performed using a diffusion cell. The cell was locally fabricated and had a 25 ml receptor compartment. The dialysis membrane was mounted between the donor and receptor compartments. The cream formulation was applied uniformly on the dialysis membrane and the compartments were clamped together. 45The receptor compartment was filled with the phosphate buffer (pH 7.4) and the hydrodynamics in the receptor compartment were maintained by stirring with a magnetic bead. The study was carried out for 24 hrs with the interval of 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hrs. Iml of samples was withdrawn from the receptor compartment at pre-determined time intervals and an equal volume of buffer was replaced. The samples were analyzed after appropriate dilution for drug content spectrophotometrically Observation and result~ 46LAB. 15 Revision 47LAB. 16 Second midterm exam 48