Drug distribution & clearance

1. DRUG DISTRIBUTION &CLEARANCE GUIDED BY: Dr.Satyabrata bhanja M. Pharm., Ph. D Presentation by: G.Shekhar (256213886011) Department of Pharmaceutics (1st year -2 nd sem ) MALLA REDDY COLLEGE OF PHARMACY

2. DRUG DISTRIBUTION Contents: * Introduction * Volume of distribution * Factors effecting on drug distribution * Protein & tissue binding * Kinetics * Determination of rate constant & different plots (direct , scatchard ,&reciprocal )

3. Introduction *DRUG DISTRIBUTION refers to the reversible transfer of drug from one locatio n to another within the body (or) which involves reversible transfer of a drug between compartments. * Definitive information on the distribution of a drug requires its measurement in various tissues. Such data has been obtained in animals, but is essentially lacking in humans. * Much useful information on the rate and extent of distribution in humans can be derived from blood or plasma data.

4. * Distribution is a Passive Process , for which the driving force is the Conc. Gradient between the blood and Extravascular Tissues. * The Process occurs by the Diffusion of Free Drug until equilibrium is established. * As the Pharmacological action of a drug depends upon its concentration at the site of action Distribution plays a significant role in the Onset, Intensity, and Duration of Action. * Distribution of a drug is not Uniform throughout the body because different tissues receive the drug from plasma at different rates and to different extents.

5. * The Volume of distribution (V D ), also known as Apparent volume of distribution , is used to quantify the distribution of a drug between plasma and the rest of the body after oral or parenteral dosing. * It is called as Apparent Volume because all parts of the body equilibrated with the drug do not have equal concentration. * It is defined as the volume in which the amount of drug would be uniformly distributed to produce the observed blood concentration. Volume of Distribution

6. Redistribution Highly lipid soluble drugs when given by i.v . or by inhalation initially get distributed to organs with high blood flow, e.g. brain, heart, kidney etc. Later, less vascular but more bulky tissues ( muscles,fat ) take up the drug and plasma concentration falls and drug is withdrawn from these sites. If the site of action of the drug was in one of the highly perfused organs, redistribution results in termination of the drug action. Greater the lipid solubility of the drug, faster is its redistribution.

7. The real volume of distribution has physiological meaning and is related to the Body Water.

8. The volume of each of these compartments can be determined by use of specific markers or tracers. Physiological Fluid Compartments the Markers Used Approximate volume (liters) Plasma Evans Blue, Indocyanine Green 4 Extracellular fluid Inulin , Raffinose , Mannitol 14 Total Body Water D 2 O, Antipyrine 42 The intracellular fluid volume can be determined as the difference between total body water and extracellular fluid.

9. Drugs which bind selectively to Plasma proteins e.g. Warfarin have Apparent volume of distribution smaller than their Real volume of distribution. The Vd of such drugs lies between blood volume and total body water i.e. b/w 6 to 42 liters. Drugs which bind selectively to Extravascular Tissues e.g. Chloroquine have Apparent volume of distribution larger than their Real volume of distribution. The Vd of such drugs is always greater than 42 liters.

10. Several factors influence drug distribution to various tissues of the body. They are listed below . 1) Physicochemical properties of the drug * molecular size * oil\water partition coefficient ( Ko \w) * degree of ionization that depends on pKa 2) Physiological factors * organ or tissue size * blood flow rate * physiological barriers to the diffusion of drugs - blood capillary membrane - cell membrane - specialized barriers - blood brain barrier - blood cerebrospinal fluid barrier Factors effecting on drug distribution

11. _placental barrier -blood testis barrier 3) Drug binding in the blood 4) Drug binding to the tissue and other macromolecules 5) Miscellaneous factors a) age b) Pregnancy c) Obesity d) Diet e) Disease states f) Drug interactions

12. Physicochemical properties of the drug Drugs having molecular wt. less than 400 daltons easily cross the Capillary Membrane to diffuse into the Extracellular Interstitial Fluids. Now, the penetration of drug from the Extracellular fluid (ECF) is a function of :- Molecular Size : Small ions of size < 50 daltons enter the cell through Aq. filled channels where as larger size ions are restricted unless a specialized transport system exists for them. Ionisation : A drug that remains unionized at pH values of blood and ECF can permeate the cells more rapidly. Blood and ECF pH normally remains constant at 7.4, unless altered in conditions like Systemic alkalosis/acidosis.

13. Lipophilicity : Only unionized drugs that are lipophilic rapidly crosses the cell membrane. e.g. Thiopental, a lipophilic drug, largely unionized at Blood and ECF pH readily diffuses the brain where as Penicillins which are polar and ionized at plasma pH do not cross BBB. Effective Partition Coefficient for a drug is given by: Effective K o/w = Fraction unionized at pH 7.4 X K o/w of unionized drug

14. Perfusion Rate is defined as the volume of blood that flows per unit time per unit volume of the tissue. Greater the blood flow, faster the distribution. Highly perfused tissues such as lungs, kidneys, liver, heart and brain are rapidly equilibrated with lipid soluble drugs. The extent to which a drug is distributed in a particular tissue or organ depends upon the size of the tissue i.e. tissue volume. Organ / Tissue Size and Perfusion Rate

15. A stealth of endothelial cells lining the capillaries. It has tight junctions and lack large intra cellular pores. Further, neural tissue covers the capillaries. Together , they constitute the BLOOD BRAIN BARRIER. Astrocytes : Special cells / elements of supporting tissue are found at the base of endothelial membrane. * The blood-brain barrier (BBB) is a separation of circulating blood and cerebrospinal fluid (CSF) maintained by the choroid plexus in the central nervous system (CNS). PENETRATION OF DRUGS THROUGH BLOOD BRAIN BARRIER

16. Since BBB is a lipoidal barrier * It allows only the drugs having high o/w partition coefficient to diffuse passively where as moderately lipid soluble and partially ionized molecules penetrate at a slow rate. * Endothelial cells restrict the diffusion of microscopic objects (e.g. bacteria ) and large or hydrophillic molecules into the CSF, while allowing the diffusion of small hydrophobic molecules (O 2 , CO 2, hormones). * Cells of the barrier actively transport metabolic products such as glucose across the barrier with specific proteins.

17. Various approaches to promote crossing BBB: Use of Permeation enhancers such as Dimethyl Sulfoxide . Osmotic disruption of the BBB by infusing internal carotid artery with Mannitol . Use of Dihydropyridine Redox system as drug carriers to the brain ( the lipid soluble dihydropyridine is linked as a carrier to the polar drug to form a prodrug that rapidly crosses the BBB )

18. PENETRATION OF DRUGS THROUGH PLACENTAL BARRIER *Placenta is the membrane separating Fetal blood from the Maternal blood. *It is made up of Fetal Trophoblast Basement Membrane and the Endothelium. *Mean thickness in early pregnancy is (25 µ) which reduces to (2 µ) at full term.

19. * Many drugs having mol. wt. < 1000 Daltons and moderate to high lipid solubility e.g. ethanol,sulfonamides,barbiturates,steroids , anticonvulsants and some antibiotics cross the barrier by simple diffusion quite rapidly . * Nutrients essential for fetal growth are transported by carrier mediated processes.

20. Blood – Cerebrospinal Fluid Barrier : The Cerebrospinal Fluid (CSF) is formed mainly by the Choroid Plexus of lateral, third and fourth ventricles. The choroidal cells are joined to each other by tight junctions forming the Blood – CSF barrier which has permeability characteristics similar to that of BBB. Only high lipid soluble drugs can cross the Blood – CSF barrier.

21. Blood – Testis Barrier: It has tight junctions between the neighbouring cells of sertoli which restricts the passage of drugs to spermatocytes and spermatids .

22. Miscellaneous Factors Diet: A Diet high in fats will increase the free fatty acid levels in circulation thereby affecting binding of acidic drugs such as NSAIDS to Albumin. Obesity: In Obese persons, high adipose tissue content can take up a large fraction of lipophilic drugs. Pregnancy: During pregnancy the growth of the uterus, placenta and fetus increases the volume available for distribution of drugs. Disease States: Altered albumin or drug – binding protein conc. Altered or Reduced perfusion to organs /tissues Altered Tissue pH

23. Factor affecting drug-Protein binding , Significant , Kinetics of drug-protein binding

24. Protein Molecular Weight ( Da ) concentration (g/L) Drug that bind Albumin 65,000 3.5–5.0 Large variety of drug α1 - a cid glycoprotein   44,000 0.04 – 0.1 Basic drug - propranolol , imipramine , and lidocaine . Globulins, corticosteroids . Lipoproteins 200,000–3,400,000 .003-.007 Basic lipophilic drug Eg - chlorpromazine α 1 globulin α 2 globulin 59000 13400 .015-.06 Steroid , thyroxine Cynocobalamine Vit . –A,D,E,K BIND TO BLOOD PROTEIN PLASMA PROTEIN- DRUG BINDING

25. α 1 globulin bind to a number of steroidal drug cortisone , prednisolone $ thyroxine , cynocobalamine α2 globulin ( ceruloplasmin ) bind to Vit . A D E K β1-globulin ( transferrin ) bind to ferrous ion β 2-globulin bind to carotinoid γ- globulin bind to antigen Binding of drug to globulin

26. majority of drug bind to extravascular tissue- the order of binding -: liver > kidney > lung > muscle liver – epoxide of number of halogenated hydrocorban , paracetamol lung – basic drug imipramine , chlorpramazine , antihistaminis , kidney – metallothionin bind to heavy metal , lead, Hg , Cd , skin – chloroquine $ phenothiazine eye - chloroquine $ phenothiazine Hairs - arsenicals , chloroquine , $ PTZ bind to hair shaft . Bone – tetracycline Fats – thiopental , pesticide- DDT Tissue binding of drug

27. Factor affecting drug protein binding 1. factor relating to the drug Physicochemical characteristic of drug Concentration of drug in the body Affinity of drug for a particular component 2. factor relating to the protein and other binding component Physicochemical characteristic of the protein or binding component Concentration of protein or binding component Num. Of binding site on the binding site 3. drug interaction 4. patient related factor

28. Physicochemical characteristics of drug Protein binding is directly related to lipophilicity lipophilicity = the extent of binding e.g. The slow absorption of cloxacilin in compression to ampicillin after i.m . Injection is attributes to its higher lipophilicity it binding 95% letter binding 20% to protein Highly lipophilic thiopental tend to localized in adipose tissue . Anionic or acidic drug like . Penicillin , sulfonamide bind more to HSA Cationic or basic drug like . Imepramine alprenolol bind to AAG Drug related factor

29. Physicochemical property of protein / binding component – lipoprotein or adipose tissue tend to bind lipophilic drug by dissolving them to lipid core . The physiological pH determine the presence of anionic or cationic group on the albumin molecule to bind a variety of drug Concentration of protein / binding component Mostly all drug bind to albumin b/c it present a higher concentration than other protein number of binding sites on the protein Albumin has a large number of binding site as compare to other protein and is a high capacity binding component Protein or tissue related factor

30. *Tissue binding , apparent volume of distribution and drug storage A drug that bind to blood component remains confined to blood have small volume of distribution. Drug that show extra-vascular tissue binding have large volume of distribution . the relationship b/w tissue drug binding and apparent volume of distribution- Vd = amount of drug in the body = X plasma drug concentration C the amount of drug in the body X = Vd . C SIMILAR , amount of drug in plasma = Vp . S Amount of drug in extravascular tissue = Vt .Ct

31. The total amount of drug in the body Vd . C = Vp.C+Vt . Ct where , Vp is volume of plasma Vt is volume of extravascular tissue Ct is tissue drug concentration Vd = Vp + Vt Ct/C ………………….(1) Dividing both side by C in above equation The fraction of unbound drug in plasma (fu) The fraction unbound drug in tissue ( fut ) fut = Cut Ct

32. Assuming that equilibrium unbound or free drug conc. In plasma and tissue is equal C t = fu C fut mean Cu = Cut then , Vd = Vp + Vt . fu fut substituting the above value in equa . 1 It is clear that greater the unbound or free concentration of drug in plasma larger its Vd

33. The kinetics of reversible drug–protein binding for a protein with one simple binding site can be described by the law of mass action , as follows: or ………………1 The law of mass action, an association constant, K a , can be expressed as the ratio of the molar concentration of the products and the molar concentration of the reactants. This equation assumes only one-binding site per protein molecule ……………………….…2 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 behavior of drugs, a determinable ratio (r )is defined, as follows Kinetics of protein drug binding

34. moles of drug bound is [ PD ] and the total moles of protein is [ P ] + [ PD ], this equation becomes Substituting the value of PD from equa . This equation describes the simplest situation, in which 1 mole of drug binds to 1 mole of protein in a 1:1 complex. This case assumes only one independent binding site for each molecule of drug. If there are n identical independent binding sites per protein molecule, then the following is used:

35. * Protein molecules are quite large compared to drug molecules and may contain more than one type of binding site for the drug. If there is more than one type of binding site and the drug binds independently on each binding site with its own association constant, then Equation 6 expands to The values for the association constants and the number of binding sites are obtained by various graphic methods.

36. 1. Direct plot It is made by plotting r vresus (D) 2. Double reciprocal plot The reciprocal of Equation 6 gives the following equation

37. A graph of 1/ r versus 1/[ D ] is called a double reciprocal plot . The y intercept is 1/ n and the slope is 1/ nKa . 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 for n is known. 3. Scatchard plot The Scatchard plot spreads the data to give a better line for the estimation of the binding constants and binding sites r = n Kas – r Kas [DF]

38. *Drug clearance is a pharmacokinetic term for describing drug elimination from the body without identifying the mechanism of the process. *instead of describing the drug elimination rate in terms of amount of drug removed per unit time ( eg,mg /min). *Drug clearance is defined as the fixed volume of fluid (containing the drug) cleared of drug per unit time *the units for the clearance are volume /time (ml/ min,l /hr). *for example ,if the clt of penicillin is 15mL/min in a patient and penicillin has a VD of 12 L, then from the clearance definition.15 ml of the 12 L will be cleared of drug per minute. * Alternatively,CLT may be defined as the rate of drug elimination divided by the plasma drug concentration. CLEARANCE

39. CLT= elimination rate plasma concentration(CP) CLT= dDE / dt = mL /min CP Elimination rate= dDE = CpClT dt Just as the elimination rate constant (k) represents the sum total of all the rate constants for drug elimination, including excretion and biotransformation,CLT is the sum total of all the clearance processes in the body,including clearance through the kidney (renal clearance), lung,and liver (hepatic clearance).

40. Rather than describing in terms of amount of drug removed per unit time, Clearance is described as volume of plasma cleared of drug per unit time (volume/time) 10 Litres L/hr 100 mg/L 1000 mg Drug Simplest case- a beaker…

41. DRUG URINE ke Metabolites km KIDNEY Bile k bile LIVER BODY IV Vd But the body’s not a beaker- multiple systems involved…..

42. The calculation of clearance from k and VD assumes (sometimes incorrectly) a defined model, whereas clearance estimated directly from the plasma drug concentration time curve does not assume any model. Physiologic/Organ Clearance : Clearance may be calculated for any oxygen involved in the irreversible removal of drug from the body. Many organs in the body have the capacity for drug elimination, including drug excretion and biotransformation. Rate of elimination by an organ = Rate of presentation organ input – rate of extraction organ output = Q.C in-Q.C out CLEARANCE MODELS

43. Clearance= Q(ER) If the drug concentration in the blood (Ca) entering is greater than the drug concentration of blood ( Cv ) leaving the organ, then some of the drug has been extracted by the organ. The ER is Ca- Cv divided by the entering drug concentration (Ca). ER= Ca- Cv Ca Hepatic clearance: CLH= = rate of eliminated by liver plasma concentration(C) CLH =CLT-CLR

44. Clearance is commonly used to describe first-order drug elimination from compartment models such as the one-compartment model. Model-independent methods are non compartment model approaches used to calculate certain pharmacokinetic parameters such as clearance and bioavailability (F).The major advantage of model-independent methods is that no assumption for a specific compartment model is required to analyze the data. Moreover, the volume of distribution and the elimination rate constant need not be determined MODEL –INDEPENDENT METHODS

45. * Compartment model: static volume and first-order elimination is assumed. Plasma flow is not considered. Clt =k Vd . Physiologic model: clearance is the product of the plasma flow (Q) and the extraction ratio (ER).Thus CLt =Q ER Model independent: volume and elimination rate constant not defined.

46. Renal clearance, Clr , is defined as the volume of plasma that is cleared of drug per unit of time through the kidney. Similarly, renal clearance may be defined as a constant fraction of the Vd in which the drug is contained that is excreted by the kidney per unit of time. More simply, renal clearance is defined as the urinary drug excretion rate ( dDu / dt ) divided by the plasma drug concentration (Cp). CLr = rate of eliminated by kidney plasma concentration(c) = dDu / dt Cp RENAL CLEARANCE

47. * Rate of drug passing through kidney = rate of drug excreted Clr Cp= Qu Cu mL /min mg/ mL = mL /min mg/ mL

48. Comparison of drug excretion methods renal clearance may be measured without regard to the physiologic mechanisms involved in this process. From a physiologic viewpoint, however, renal clearance may be considered as the ratio of the sum of the glomerular filtration and active secretion rates less the reabsorption rate divided by the plasma drug concentration: clr =filtration rate+ secretion rate – reabsorptionrate cp * the actual renal clearance of a drug is not generally obtained by direct measurement .

49. Filtration only If glomerular filtration is the sole process for drug excreation and no drug is reabsorbed. Then tha amount of drug filtered at any time (t) will always be cp*GFR likewise if the clr of the drug is by glomerular filtration only as in the case of inulin then clr =GFR otherwise clr represents all the processes by which the drug is cleared through the kidney, including any combination of filtration, reabsorption , and active secretion. dDu = ke VD Cp (compartment) (6.24) dt dDu = Clr Cp (physiologic) (6.25) dt From above eqns : Ke VD CP =CLR CP KE= CLR VD (6.26)

50. FILTRATION AND REABSORPTION: for a drug with a reabsorption fraction of fr , the drug excretion rate is reduced and equation6.25 is restarted as equation 6.27: dDu = clr (1-fr)cp (6.27) dt equating the right sides of equations 6.27 and 6.24 indicates that the first-order rate constant ( ke ) in the compartment model is equivalent to clr (1-fr)/ vd.in this case, the excretion. Rate constant is affected by the reabsorption fraction ( fr ) and the GFR because these two parameters generally remain constant the general adoption of a first-order elimination process to describe renal drug excretion is a reasonable approach.

51. Filtration and active secretion for a drug that is primarily filtered and secreted, with negligible reabsorption , the overall excretion rate will exceed GFR at low drug plasma concentrations, active secretion is not saturated and the drug is excreted by filtration and active secretion at high concentrations the percentage of drug excreted by active secretion decreases due to saturation. Clearance decreases because excretion rate decreases . Clearance decreases because the total excretion rate of the drug increases to the point where it is approximately equal to the filtration rate.

52. Model-Independent Methods * Clearance rates may also be estimated by a single ( nongraphical ) calculation from knowledge of the (AUG) 0 to infinite, the total amount of drug absorbed,FD0, and the total amount of drug excreted in the urine, D u to infinite. For example, if a single IV bolus drug injection is given to a patient and the (AUG) 0 to infinite is obtained from the plasma drug level-time curve, then total body clearance is estimated by Clt = Do [AUG] 0 to infinite * If the total amount of drug excreted in the urine D u to infinite, has been obtained, then renal clearance is calculated by Clt = D 0 to infinite [AUG] 0 to infinite

53. * Clearance can also be calculated from fitted parameters. If the volume of distribution and elimination constants are known, body clearance ( Clt ), renal clearance ( Clr ), and hepatic clearance ( Clh ) can be calculated according to the following expressions: Clt = kVd (6.35) Clr = keVd (6.36) Clh = kmVd 6.37) * Total body clearance ( Clt ) is equal to the sum of renal clearance and hepatic clearance and Is based on the concept that the entire body acts as a drug-eliminating system. Clt = Clr+Clh (6.38)

54. By substitution of equations 6.35 and 6.36 into equation 6.38, kVd = keVd+kmVd Dividing by Vd on both sides of equation 6.39, k = ke+km Total body clearance : it is estimated of dividing the rate of elimination by each organ with concentration of drug present. CL T = CL H + CL R +CL BILE +...

55. Protein-bound drugs *Protein-bound drugs are not eliminated by glomerular filtration. The bound drugs are usually excreted by active secretion, following capacity-limited kinetics. The determination of clearance that separates the two component results in a hybrid clearance. *There is no simple way to overcome this problem. *Clearance values for a Protein-bound drug is therefore calculated with the following equation: Clr = rate of unbound drug excretion conc of unbound drug in the plasma *plasma protein binding has very little effect on the renal clearance of actively secreted drugs such as penicillin.

56. *drugs and their metabolites may also be excreted by routes other than the renal route ,called as the extra renal or non renal routes of drug excretion . * Biliary excretion * pulmonary excretion * salivary excretion * Mammary excretion * Skin/dermal excretion * Gastrointestinal excretion * Genital excretion Non renal routes of drug excretion

57. * Biliary excretion: The ability of liver to excrete the drug in the bile is expressed by biliary clearance. biliary clearance = biliary clearance rate plasma drug concentration = bile flow* biliary drug clearance plasma drug concentration *pulmonary excretion: Gaseous & volatile substances such as the general anaesthetics (e.g. halothane) are absorbed through the lungs by simple diffusion. *salivary excretion: Excretion of drugs in saliva is also a passive diffusion process & therefore predictable on the basis of pH-partition hypothesis.

58. *Mammary excretion: *Excretion of a drug in milk is a important since it can gain entry into the breast – feeding infant. * Excretion of drugs in milk is a passive process & is dependent upon pH-partition behaviour , molecular weight, lipid solubility & degree of ionisation . *Skin/dermal excretion : *Drugs excreted through the skin via sweat also follow pH-partition hypothesis. * compounds such as benzoic acid , salicylic acid , alcohol & antipyrine & heavy metals like lead , mercury & arsenic are excreted in sweat.

59. *Gastrointestinal excretion: * Excretion of drugs into the GIT usually occurs after parenteral administration when the concentration gradient for passive diffusion is favourable . * The process is reverse of GI absorption of drugs. *Genital excretion : * reproductive tract & genital secretions may contain the excreted drugs. Some drugs have been detected in semen. * Drugs can also get excreted via the lachrymal fluid.

60. Relationship of clearance to elimination half life & volume of distribution CL T = KVd & Therefore,by substitution, CL T = 0.693 * Vd / t½ t½ = 0.693 * Vd / CL T t½ inversely related to CL T t½ also dependent on volume of distribution. K and t½ are dependent on both CL T &V K = 0.693/ t½

61. REFERENCES * Leon shargel , Susanna WU-Pong , Andrew B.C. YU , Applied Biopharmaceutics & Pharmacokinetics * V Venkateswarulu Biopharmaceutics & Pharmacokinetics Second Edition * Brahmankar , D.M., Jaiswal , S.B., Biopharmaceutics & Pharmacokinetics

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