**Drug Distribution and Drug Elimination**

Seng Kok-Yong and Lee Lawrence

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59929

**1. Introduction**

[3] Salvatore J. Turco, Robert E. King. (1994). Sterile Dosage Forms: Their Preparation and Clinical Application. (4th Revised Edition) Lippincott Williams & Wilkins. Phila‐

[4] Toutain PL, Bousquet-Mélou A. Volumes of distribution. J Vet Pharmacol Ther. 2004;

[5] Basic clinical pharmacokinetics, Page 32: Plasma protein binding By Michael E. Win‐ ter. (2003). Edition: 4, illustrated Published by Lippincott Williams & Wilkins.

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[7] Evans W E, Schentag J J, Jusko W J, Harrison H, eds (1992). In Evans W E, Schentag J J, eds. Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring, 3rd

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98 Basic Pharmacokinetic Concepts and Some Clinical Applications

ed. Oxford: Blackwell Scientific.

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Pharmacokinetics is a branch of pharmacology that examines how drug concentrations change with respect to time as a function of absorption, distribution, metabolism and excretion [1]. These are disparate but interrelated processes that occur between drug administration and its irreversible elimination from the body. Another way to consider pharmacokinetic processes is to group them into two components:

Once absorbed into the body, drug compounds are distributed reversibly to various tissues of the body including the eliminating organs, such as liver and kidney, which results in a decrease in blood or plasma drug concentration. The decrease in the blood concentration could be due to reversible loss of drug from the blood to the tissues, defined as distribution, or the irrever‐ sible loss of drug from blood, defined as elimination. Disposition is therefore a combination distribution and elimination.

### **2. Distribution**

Once in the systemic circulation, the blood or plasma concentrations of a drug will depend on how extensively it is distributed to extravascular sites [2]. Drug concentration in whole blood represents the total concentrations of drug in the circulatory system. Plasma concentration do not account for drug molecules that are sequestered into red or white blood cells. In general,

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the blood and the plasma concentrations are assumed to be equal unless the drug is preferen‐ tially sequestered by red blood cells. Drug distribution will be influenced by tissue/organ blood flow, whether the drug is able to passively diffuse across cell membranes or is a substrate for active uptake or efflux transporters, and its extent of binding to plasma protein and tissue sites.

#### **2.1. Tissue/organ blood flow**

The transfer of many drug compounds from the systemic circulation to various tissues/organs follows the perfusion-rate diffusion process. Here, we assume that cell membranes do not present any barrier to drug transfer. This typically applies to drug compounds that are lipid soluble. Under perfusion-rate diffusion, the rate of delivery from the systemic circulation to a specific tissue/organ is primarily dependent on the blood flow within an organ or tissue. Organs like the liver and the heart are highly perfused with blood. By contrast, the bone and the adipose tissues experience less blood perfusion. Therefore, drugs are likely to distribute more rapidly to tissues/organs that are more richly perfused with blood.

#### **2.2. Passive diffusion across cell membranes**

A major factor affecting drug distribution is the physicochemical properties of the drug [3] since these would influence the permeability of the drug to various tissues. A drug that is highly lipophilic, such as chloroquine, may readily cross the lipidic bilayer of endothelial cells and most cell membranes to reach into the intracellular space via passive transcellular diffusion. Lipid-soluble drugs, because of their high partition coefficient, can also accumulate in organs or sites with fat deposits. On the other hand, drugs that are more water soluble and polar, such as aminoglycosides, do not distribute well into most tissues/organs. For such drug molecules, entry into the tissue spaces may rely on either paracellular diffusion via gaps inbetween cells [4] or carrier-mediated uptake transport processes.

#### **2.3. Influx or efflux transporters**

Influx and efflux transporter are found in many tissues/organs and play a role in the distri‐ bution of drugs in the body [5]. The efflux transporter, P-glycoprotein (P-gp), which is expressed in the liver and the kidney, functions to keep drugs out of tissues [6]. By contrast, the influx transporter OATP1B1, an organic anion transporter expressed in the liver and the brain, acts on drug substrates to move them from the extracellular matrix into the tissue spaces. Since these transporters are subject to genetic polymorphisms, their underexpression or overexpression will result in differences in the extent of drug distribution between patients.

#### **2.4. Plasma protein and tissue binding**

Another factor influencing drug distribution is the preferential binding to plasma proteins and tissues [7]. It is the unbound or free portion of the drug that diffuses out of the plasma into the tissues/organs. Albumin and α1-acid glycoprotein are the two major proteins in plasma that are responsible for the binding of most drug compounds in the systemic circulation. The extent of plasma protein binding of a drug can be drug- or protein-concentration dependent, based on the affinity and capacity of the plasma protein. A drug's protein-binding characteristics also depend on its physicochemical properties, with lipophilic drugs more likely to bind to plasma proteins and consequently, less available to the intracellular spaces [8]. Table 1 lists the extent of plasma protein binding of selected drugs. Binding to tissues also affects drug concentrations in the blood/plasma and the tissues/organs. However, compared to plasma protein binding, much less is known about tissue binding or the sequestration of drugs, since reliable methods for estimating binding to tissue components *in vivo* are experimentally more challenging.

**Table 1.** Extent of plasma protein binding of selected drugs.

#### **3. Volume of distribution**

the blood and the plasma concentrations are assumed to be equal unless the drug is preferen‐ tially sequestered by red blood cells. Drug distribution will be influenced by tissue/organ blood flow, whether the drug is able to passively diffuse across cell membranes or is a substrate for active uptake or efflux transporters, and its extent of binding to plasma protein and tissue sites.

The transfer of many drug compounds from the systemic circulation to various tissues/organs follows the perfusion-rate diffusion process. Here, we assume that cell membranes do not present any barrier to drug transfer. This typically applies to drug compounds that are lipid soluble. Under perfusion-rate diffusion, the rate of delivery from the systemic circulation to a specific tissue/organ is primarily dependent on the blood flow within an organ or tissue. Organs like the liver and the heart are highly perfused with blood. By contrast, the bone and the adipose tissues experience less blood perfusion. Therefore, drugs are likely to distribute

A major factor affecting drug distribution is the physicochemical properties of the drug [3] since these would influence the permeability of the drug to various tissues. A drug that is highly lipophilic, such as chloroquine, may readily cross the lipidic bilayer of endothelial cells and most cell membranes to reach into the intracellular space via passive transcellular diffusion. Lipid-soluble drugs, because of their high partition coefficient, can also accumulate in organs or sites with fat deposits. On the other hand, drugs that are more water soluble and polar, such as aminoglycosides, do not distribute well into most tissues/organs. For such drug molecules, entry into the tissue spaces may rely on either paracellular diffusion via gaps in-

Influx and efflux transporter are found in many tissues/organs and play a role in the distri‐ bution of drugs in the body [5]. The efflux transporter, P-glycoprotein (P-gp), which is expressed in the liver and the kidney, functions to keep drugs out of tissues [6]. By contrast, the influx transporter OATP1B1, an organic anion transporter expressed in the liver and the brain, acts on drug substrates to move them from the extracellular matrix into the tissue spaces. Since these transporters are subject to genetic polymorphisms, their underexpression or overexpression will result in differences in the extent of drug distribution between patients.

Another factor influencing drug distribution is the preferential binding to plasma proteins and tissues [7]. It is the unbound or free portion of the drug that diffuses out of the plasma into the tissues/organs. Albumin and α1-acid glycoprotein are the two major proteins in plasma that are responsible for the binding of most drug compounds in the systemic circulation. The extent of plasma protein binding of a drug can be drug- or protein-concentration dependent, based

more rapidly to tissues/organs that are more richly perfused with blood.

between cells [4] or carrier-mediated uptake transport processes.

**2.2. Passive diffusion across cell membranes**

100 Basic Pharmacokinetic Concepts and Some Clinical Applications

**2.3. Influx or efflux transporters**

**2.4. Plasma protein and tissue binding**

**2.1. Tissue/organ blood flow**

A quantitative analysis of distribution is needed to understand pharmacokinetics of a drug. A drug can be characterised by the volume of fluids into which it distributes [9]. Since the volume of body fluids cannot be easily measured, it is assumed that the body simplifies into a tank of fluid into which the drug is placed. This volume is known as<s\$%&?>the volume of distribu‐ tion (*Vd*). The *Vd* of a drug is an important pharmacokinetic parameter and is defined as the ratio of the amount of drug in the body to the concentration in a biological matrix that is readily accessible, such as the plasma. The *Vd* has units of volume, such as litre.

The *Vd* of a drug given as an intravenous (i.v.) dose can be calculated by:

$$V\_d = \frac{A}{C\_p} \tag{1}$$

where *A* and *Cp* are the i.v. dose of the drug (units: mass) and the drug concentration (units, e.g.: g/L) in plasma at time zero, respectively. If direct measurement is impractical, this initial drug concentration is derived from the y-axis intercept of the extrapolated logarithmic concentration versus time line. In general, it is assumed that *Cp* is the total drug concentration of the drug (free and bound to plasma proteins).

The *Vd* of a drug is regarded as a hypothetical term as it has no direct correlation to anatomical spaces in the body. It denotes the apparent volume of space into which a drug can distribute after dosing, and is indicative of its relative storage in the plasma and in the tissue/organ spaces. For this reason, *Vd* has also been commonly called the apparent volume of distribution. The *Vd* provides an important guide when accessing the tissue penetration of a drug. Table 2 lists the *Vd* of selected drugs.

**Table 2.** The *Vd* of selected drugs.

The concept of *Vd* is illustrated in Figure 1.

**Figure 1.** A clarification of the concept of *Vd* of a drug dissolved in two containers containing water. The *Vd* is an appa‐ rent volume term that is determined from the amount of drug added and the resulting concentration. Given the same dose amount and the 100-fold reduction in drug concentration in Container 2, the *Vd* of the drug in Container 2 is 100 times larger than that of the same drug present in Container 1.

Let us imagine there are two containers of the same size filled with 1L of water: Container 1 and Container 2. Container 2 also contains a small quantity of sponge that adds no significant volume to the overall container volume. A one unit dose of the same drug is then added to each container. After complete dissolution of the drug in the volume of water within each container, the drug concentrations are measured. The concentrations in Container 1 and Container 2 are found to be 1unit/L and 0.01unit/L, respectively. Here, it is assumed that each container is a closed system after drug intake: no drug elimination occurs during the time for complete drug dissolution. By dividing the dose (one unit) by the measured concentration in each container (Eq. (1)), the volumes derived are 1L and 100L for Container 1 and Container 2, respectively. The explanation for this observation is that some of the drug in Container 2 is bound to the sponge, which renders it unavailable for measurement of the drug concentration in water. Consequently, the measured drug concentration in Container 2 is low and the calculated volume is high. However, each container still contains one unit of drug since none is eliminated. Put in another way, the *Vd* is an apparent or hypothetical quantity that relates the total drug amount in the system (dose) to its concentration in a matrix of measurement (water).

A physiology-driven formula has been proposed that accounts for the influence of blood or plasma volume, tissue volume (difference between total body water volume and plasma volume), drug binding to plasma proteins and drug binding to tissue sites on the *Vd* of a specific drug [10]. This formula is given by the following equation:

$$V\_d = V\_p + V\_t \times \frac{f\_{u,p}}{f\_{u,t}} \tag{2}$$

where *Vu,p*, *Vu,t*, *fu* and *ft* denote the plasma volume, tissue volume, fraction of the drug unbound in plasma, and fraction of the drug unbound in tissue, respectively. Drugs such as nortriptyline and chloroquine have a large distribution volume, which indicates significant uptake and binding to tissue binding sites. By contrast, large-sized drug compounds (e.g. heparin), drugs that preferentially bind to plasma proteins (e.g. warfarin) and monoclonal antibodies, have a small *Vd* indicating that these remain mainly in the vascular space.

#### **4. Elimination**

The *Vd* of a drug is regarded as a hypothetical term as it has no direct correlation to anatomical spaces in the body. It denotes the apparent volume of space into which a drug can distribute after dosing, and is indicative of its relative storage in the plasma and in the tissue/organ spaces. For this reason, *Vd* has also been commonly called the apparent volume of distribution. The *Vd* provides an important guide when accessing the tissue penetration of a drug. Table 2 lists the

Dose = one unit Dose = one unit

*Container 1 Container 2*

**Figure 1.** A clarification of the concept of *Vd* of a drug dissolved in two containers containing water. The *Vd* is an appa‐ rent volume term that is determined from the amount of drug added and the resulting concentration. Given the same dose amount and the 100-fold reduction in drug concentration in Container 2, the *Vd* of the drug in Container 2 is 100

Let us imagine there are two containers of the same size filled with 1L of water: Container 1 and Container 2. Container 2 also contains a small quantity of sponge that adds no significant volume to the overall container volume. A one unit dose of the same drug is then added to

Sponge

Concentration = 0.01unit/L

*Vd* of selected drugs.

**Table 2.** The *Vd* of selected drugs.

The concept of *Vd* is illustrated in Figure 1.

Concentration = 1unit/L

times larger than that of the same drug present in Container 1.

**Drug Vd (L)** Warfarin 8 Gentamicin 18 Digoxin 440 Diazepam 80 Nortriptyline 1700 Chloroquine > 15000

102 Basic Pharmacokinetic Concepts and Some Clinical Applications

Effective drug therapy involves achieving optimal efficacy without causing toxicity [11]. To this end, drug intake into and distribution within the body must be balanced with elimination so that appropriate concentrations at the receptor sites can be achieved. Elimination refers to the irreversible removal of a drug or its metabolite(s) from the body. For the majority of drugs, metabolism is the major pathway of elimination [12, 13]. The primary organ involved is the liver, although the gastrointestinal (GI) tract, kidney, lung and skin may also contain drug metabolising enzymes and may contribute to regional concentrations of the drug and the metabolites. Excretion of drugs and their metabolites mainly occur in the kidneys, but may also involve the GI tract and lung. This section describes the key aspects of liver metabolism and factors that may govern it, and the components of renal and biliary excretion.

#### **4.1. Liver metabolism**

In the liver, a wide array of enzymes exists to biotransform drugs, producing less active (or in some cases more active) metabolites [14]. Drug metabolism is defined as the biotransformation of lipid-soluble chemicals into water-soluble forms, so that these can be excreted in the urine. Metabolism is divided into two phases (Figure 2). Drugs may undergo one phase only, or be metabolised through both phases sequentially.

**Figure 2.** The phases of drug metabolism. Phase I reaction functionalises the drug with a 'reactive' group. Phase II me‐ tabolism typically results from the conjugation of an endogenous molecule to the 'reactive' group.

#### *4.1.1. Phase I metabolism*

Phase I reactions involve the introduction into or unveiling of a polar functional group (e.g. – OH, –SH) on the drug molecule, rendering it a suitable substrate for conjugation with another molecule during phase II metabolism. Such reactions typically involve oxidation, reduction or hydrolysis processes. Often, the by-product of phase I metabolism, called a derivative, is pharmacologically inactive but more chemically reactive than the parent drug, and may be toxic or even carcinogenic.

The major liver enzyme system involved in phase I metabolism (oxidation) is the cytochrome P450 (CYP) enzyme system [15]. Thus far, 18 CYP families have been identified in mammals, although only CYP1, CYP2, CYP3 and CYP4 are involved in drug metabolism, with CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 being responsible for the biotransforma‐ tion of greater than 90% of drugs undergoing phase I metabolism (Table 3).

**Table 3.** Selected CYP enzyme-substrate drugs and their respective inducers and inhibitors.

In addition to genetic polymorphism, liver CYP enzymes are subject to induction and inhibi‐ tion by certain drugs. As a consequence, elimination of such CYP enzyme–substrate drugs administered concomitantly may increase or decrease. Additionally, where metabolic path‐ ways involve the production of pharmacologically active or toxic metabolites, induction or inhibition of CYP enzymes could result in unanticipated changes in plasma drug concentra‐ tions, with potential clinical relevance to its therapeutic or toxicity profile. Some examples of inducers or inhibitors of CYP enzymes are provided in Table 3.

#### *4.1.2. Phase II metabolism*

**4.1. Liver metabolism**

*4.1.1. Phase I metabolism*

toxic or even carcinogenic.

CYP1A2 Acetaminophen, caffeine, theophylline

metabolised through both phases sequentially.

104 Basic Pharmacokinetic Concepts and Some Clinical Applications

**Drug**

tabolism typically results from the conjugation of an endogenous molecule to the 'reactive' group.

tion of greater than 90% of drugs undergoing phase I metabolism (Table 3).

**CYP enzyme Examples of substrate drugs Induced by Inhibited by**

CYP2C9 Warfarin, phenytoin Phenytoin, carbamazepine Fluoxetine CYP2C19 Omeprazole, phenytoin Phenytoin, carbamazepine Fluvoxamine CYP2D6 Codeine, risperidone Glutethimide Fluoxetine CYP2E1 Acetaminophen, ethanol Ethanol Disulfram

**Table 3.** Selected CYP enzyme-substrate drugs and their respective inducers and inhibitors.

CYP3A4 Midazolam, simvastatin Rifampicin Ritonavir, ketoconazole

In the liver, a wide array of enzymes exists to biotransform drugs, producing less active (or in some cases more active) metabolites [14]. Drug metabolism is defined as the biotransformation of lipid-soluble chemicals into water-soluble forms, so that these can be excreted in the urine. Metabolism is divided into two phases (Figure 2). Drugs may undergo one phase only, or be

**Phase I metabolism**

**Phase II metabolism**

**Figure 2.** The phases of drug metabolism. Phase I reaction functionalises the drug with a 'reactive' group. Phase II me‐

Phase I reactions involve the introduction into or unveiling of a polar functional group (e.g. – OH, –SH) on the drug molecule, rendering it a suitable substrate for conjugation with another molecule during phase II metabolism. Such reactions typically involve oxidation, reduction or hydrolysis processes. Often, the by-product of phase I metabolism, called a derivative, is pharmacologically inactive but more chemically reactive than the parent drug, and may be

The major liver enzyme system involved in phase I metabolism (oxidation) is the cytochrome P450 (CYP) enzyme system [15]. Thus far, 18 CYP families have been identified in mammals, although only CYP1, CYP2, CYP3 and CYP4 are involved in drug metabolism, with CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 being responsible for the biotransforma‐

Smoking Ciprofloxacin, fluvoxamine

The derivative from phase I metabolism may be excreted via the urine immediately if high aqueous solubility is achieved. If not, the derivative undergoes a phase II reaction that brings about the conjugation of its functional group(s) to various hydrophilic endogenous com‐ pounds [16]. Examples of phase II reactions include sulfation, glucuronidation and glutathione conjugation. Sufficient water solubility is normally achieved in conjugates, which facilitate renal excretion. In addition, the insertion of a large polar substrate to the parent drug or derivative would make it more amenable for active secretion into the bile for subsequent excretion into the GI tract.

#### *4.1.3. Prodrugs*

After a phase I metabolism reaction, a drug may become "activated" or pharmacologically active. This biotransformation process is the basis for the development and usage of prodrugs [17]. The prodrug is typically a structural derivative of the active drug and synthesised by adding or changing a functional group(s) on the active drug structure. The ester is a common prodrug form of drug with hydroxyl or carboxylic groups. Esters can be synthesised with desired degrees of lipophilicity or hydrophilicity, and with controlled rates of the activating hydrolytic reaction. Once the prodrug gets inside the body, enzymes work to metabolically cleave the prodrug in order to form the active drug. Examples of prodrugs include levodopa, which is an amino acid derivative form of dopamine, and codeine, which is metabolised in the body to form morphine for analgesic effect.

There are many reasons to administer a prodrug in lieu of the active drug. The active drug may be too polar or hydrophilic for sufficient absorption and oral bioavailability to be attained, or for transfer across into the lipidic cell membranes to reach receptor sites, such as in neurons. Under such conditions, a functional group, such as carboxylic or hydroxyl group, may be attached to the active drug in order to enhance membrane transport. After absorption and distribution to the site of action, the functional group is cleaved via metabolism to release the active drug. In this regard, esterases found in almost all tissues make conversion of prodrugs into active drugs relatively straightforward. Other reasons for synthesising prodrugs are poor stability or poor patient acceptability (odour, pain on injection, gastric irritation) of the active drug, or a need to prolong the stay of the drug in the body.

#### **4.2. Drug excretion**

Excretion is the principal mode of termination of drug and metabolite effects. Drugs and their metabolite(s) are most commonly removed from the body via two main routes: renal and biliary excretion.

#### *4.2.1. Renal excretion*

About 25% of cardiac output goes to the kidney at which a significant portion of foreign compounds are filtered out. Renal excretion incorporates the processes of glomerular filtration, reabsorption from the renal tubular lumen, and tubular secretion as the drug passes through the nephron, the functional excretory unit of the kidney [18].

As blood passes through the glomerulus, entities within it are filtered to form the renal filtrate in the tubular lumen. The process of filtration is passive in nature and is driven by a combi‐ nation of the large hydrostatic and concentration gradients present across the glomerulus-Bowman's capsule junction. Nevertheless, large-sized components cannot be filtered through the glomerular membrane, which implies that large drugs (e.g. heparin), plasma proteins and plasma protein-bound drugs (e.g. warfarin) cannot cross into the tubular filtrate.

Water is reabsorbed along the nephron tubule so that only 1% of the original filtrate is passed out of the body as urine. Approximately 99% of substances filtered at the glomerulus are reabsorbed along the renal tubules. The majority of filtered, unmetabolised drug molecules are also reabsorbed, especially if these are lipophilic. This is because such drugs are more likely to cross the membranes of the cells lining the tubules. By contrast, polar drugs e.g. gentamicin and digoxin, are unable to do this. Such drugs will therefore be excreted unchanged in the urine because they do not need to undergo biotransformation to increase their water solubility.

Active secretion into the renal tubules occurs for some drugs that are not readily filtered in the glomerulus. This pathway occurs via a carrier mechanism and is sufficiently efficient as to not depend on binding between plasma proteins and drugs, ensuring almost complete clearance of drugs such as penicillin [19]. Other drugs excreted by this process include anti-inflammatory drugs and methotrexate.

There may be competition for the active transport sites amongst drug compounds [20]. This may be exploited for therapeutic care, such as probenecid inhibiting the active secretion of penicillin from the kidney, increasing the latter's elimination half-life and prolonging its effect on the body. Conversely, competition for transport sites may lead to increased morbidity, e.g. aspirin can inhibit the secretion of uric acid, leading to gout aggravation.

Renal clearance of a drug can be measured with timed collection of urine and analysis of the drug concentration in the urine using the following equation [21]:

$$\text{CL}\_{\mathbb{R}} = \frac{\mathbb{C}\_{\boldsymbol{u}} \times \mathbb{Q}\_{\boldsymbol{u}}}{\mathbb{C}\_{p}} \tag{3}$$

where *CLR* is renal clearance, *Cu* is the concentration of the drug in urine, *Qu* is the volume of urine formation per unit time, and *Cp* is the concentration of the drug in plasma. Here, *Cu*× *Qu* represents the excretion rate of drug in urine.

#### *4.2.2. Biliary excretion*

**4.2. Drug excretion**

106 Basic Pharmacokinetic Concepts and Some Clinical Applications

biliary excretion.

*4.2.1. Renal excretion*

drugs and methotrexate.

Excretion is the principal mode of termination of drug and metabolite effects. Drugs and their metabolite(s) are most commonly removed from the body via two main routes: renal and

About 25% of cardiac output goes to the kidney at which a significant portion of foreign compounds are filtered out. Renal excretion incorporates the processes of glomerular filtration, reabsorption from the renal tubular lumen, and tubular secretion as the drug passes through

As blood passes through the glomerulus, entities within it are filtered to form the renal filtrate in the tubular lumen. The process of filtration is passive in nature and is driven by a combi‐ nation of the large hydrostatic and concentration gradients present across the glomerulus-Bowman's capsule junction. Nevertheless, large-sized components cannot be filtered through the glomerular membrane, which implies that large drugs (e.g. heparin), plasma proteins and

Water is reabsorbed along the nephron tubule so that only 1% of the original filtrate is passed out of the body as urine. Approximately 99% of substances filtered at the glomerulus are reabsorbed along the renal tubules. The majority of filtered, unmetabolised drug molecules are also reabsorbed, especially if these are lipophilic. This is because such drugs are more likely to cross the membranes of the cells lining the tubules. By contrast, polar drugs e.g. gentamicin and digoxin, are unable to do this. Such drugs will therefore be excreted unchanged in the urine because they do not need to undergo biotransformation to increase their water solubility.

Active secretion into the renal tubules occurs for some drugs that are not readily filtered in the glomerulus. This pathway occurs via a carrier mechanism and is sufficiently efficient as to not depend on binding between plasma proteins and drugs, ensuring almost complete clearance of drugs such as penicillin [19]. Other drugs excreted by this process include anti-inflammatory

There may be competition for the active transport sites amongst drug compounds [20]. This may be exploited for therapeutic care, such as probenecid inhibiting the active secretion of penicillin from the kidney, increasing the latter's elimination half-life and prolonging its effect on the body. Conversely, competition for transport sites may lead to increased morbidity, e.g.

Renal clearance of a drug can be measured with timed collection of urine and analysis of the

*u u*

´ <sup>=</sup> (3)

*p*

aspirin can inhibit the secretion of uric acid, leading to gout aggravation.

*R*

*C Q CL C*

drug concentration in the urine using the following equation [21]:

plasma protein-bound drugs (e.g. warfarin) cannot cross into the tubular filtrate.

the nephron, the functional excretory unit of the kidney [18].

While in the liver, drugs or metabolites can also be secreted into the bile in much the same manner as the kidney secretes drugs into the nephron tubular filtrate [22, 23]. Biliary excretion is facilitated by active transport systems located in the canalicular membrane of the hepatocyte, and can be an important hepatic elimination pathway for many compounds. Since bile is an aqueous solution, it is suitable for dissolving hydrophilic drugs. In addition, bile acids allow solubilisation of lipid-soluble drugs. Thus, all types of species (anionic, cationic and un-ionised drugs), polar and lipophilic, can be secreted into the bile. These include drug metabolites that have undergone conjugation with glucuronate during phase II metabolism. The main criterion for significant biliary excretion seems to be molecular weight > 500.

Once bile and its constituents enter into the intestines, many organic biliary constituents, including bile salts and cholesterol, are reabsorbed from intestines back into the blood with high efficiency. These components then return to the liver via the hepatic portal vein. Drugs or metabolites excreted in the bile may recirculate in the same manner. If the drug has favourable physicochemical properties, it can be partially reabsorbed from the intestines back into the blood stream just like an orally ingested drug. Metabolites with glucuronate or sulfate groups may be removed by enzymes produced by the resident bacteria of the lower small intestine and colon, and the now-active drug is able to be reabsorbed. One example is myco‐ phenolic acid. This immunosuppressant drug undergoes conjugation to glucuronate in the liver. The glucuronide metabolite of mycophenolic acid is secreted into the bile, cleaved in the small intestines, and reabsorbed back into the systemic circulation as the parent drug com‐ pound. Thus, a reservoir of the drug is established in the enterohepatic circulation, with an ongoing cycle of absorption, metabolism, secretion into the bile and reabsorption. Enterohe‐ patic circulation hence increases the persistence of drugs in the body, and reduces overall clearance in the bile.

The clearance of a drug from various eliminating tissues occurs in parallel so the total body clearance of the drug (*CLT*) is equal to the sum of the clearances of the individual tissues:

$$\text{CL}\_{T} = \text{CL}\_{R} + \text{CL}\_{H} + \text{CL}\_{L} + \text{CL}\_{\text{other}} \tag{4}$$

where *CLH* is hepatic clearance of the drug, *CLL* is clearance of the drug from the lung, and *CLother* denotes the respective clearance values from the other eliminating tissues.

Clearance is a constant that describes the relationship between drug concentration (*C*(*t*)) in the body and the rate of elimination of the drug from the body and has units of volume per time.

Figure 3 shows a way of schematically visualising *CL*.

**Figure 3.** Clearance may be viewed as the volume of plasma from which the drug is totally removed over a specified time period.

Here, it is assumed that a container is present that contains 1L of water with a drug concen‐ tration of 1ng/mL immediately after drug intake (Figure 3, left container). The container is used to denote the human body (the system) and water is used to denote the plasma. After one hour, the drug concentration is measured in the 1L of water and found to decrease to 0.5ng/mL due to drug elimination (Figure 3, middle container). Another way of viewing this is that, after one hour, half of the water volume contains the original drug concentration (1ng/mL), whereas the other half of the water volume is completely voided of the drug (Figure 3, right container). This means that the clearance of the drug is 500mL/h.

Clearance can also be understood as the product of the perfusion of the eliminating organ (Q) and the intrinsic ability of the organ to eliminate the drug termed extraction (E):

$$\text{CL} = \mathbb{Q} \times \text{E} \tag{5}$$

wher*e E* is calculated by *Cin* − *Cout Cin* , and *Cin* and *Cout* are the drug concentrations in the blood entering and leaving the organ, respectively. Since *E* is unitless, *CL* has the same units as perfusion (volume per time). The *CL* of the drug is always constant and is considered a primary parameter. This is a physiologically appealing definition of *CL* in the sense that alterations in perfusion and extraction can be shown to change *CL* in a predictable manner.

#### **5. Introduction to compartmental modelling**

The body can be regarded as compartmental systems to describe the many processes involved in the absorption, distribution, metabolism and excretion of drugs. Three different approaches can be used to describe the pharmacokinetics of drugs in the body:

One hour later

This means that the clearance of the drug is 500mL/h.

*Cin* − *Cout*

**5. Introduction to compartmental modelling**

can be used to describe the pharmacokinetics of drugs in the body:

Volume = 1L

Concentration = 0.5ng/mL

**Figure 3.** Clearance may be viewed as the volume of plasma from which the drug is totally removed over a specified

Here, it is assumed that a container is present that contains 1L of water with a drug concen‐ tration of 1ng/mL immediately after drug intake (Figure 3, left container). The container is used to denote the human body (the system) and water is used to denote the plasma. After one hour, the drug concentration is measured in the 1L of water and found to decrease to 0.5ng/mL due to drug elimination (Figure 3, middle container). Another way of viewing this is that, after one hour, half of the water volume contains the original drug concentration (1ng/mL), whereas the other half of the water volume is completely voided of the drug (Figure 3, right container).

Clearance can also be understood as the product of the perfusion of the eliminating organ (Q)

entering and leaving the organ, respectively. Since *E* is unitless, *CL* has the same units as perfusion (volume per time). The *CL* of the drug is always constant and is considered a primary parameter. This is a physiologically appealing definition of *CL* in the sense that alterations in

The body can be regarded as compartmental systems to describe the many processes involved in the absorption, distribution, metabolism and excretion of drugs. Three different approaches

and the intrinsic ability of the organ to eliminate the drug termed extraction (E):

perfusion and extraction can be shown to change *CL* in a predictable manner.

Volume = 0.5L Concentration = 0ng/mL

Volume = 0.5L Concentration = 1ng/mL

=

*CL Q E* = ´ (5)

*Cin* , and *Cin* and *Cout* are the drug concentrations in the blood

Volume = 1L Concentration = 1ng/mL

108 Basic Pharmacokinetic Concepts and Some Clinical Applications

time period.

wher*e E* is calculated by

**3.** Physiologically based pharmacokinetic (PBPK) model

The NCA approach (using statistical moment analysis) does not require the assumption of any compartments for the purpose of data analysis [24]. This method is based on the area under the drug concentration versus time curve (AUC) and the mean residence time (MRT). Although the NCA approach can be applied to most pharmacokinetic data, it lacks the ability to predict pharmacokinetic profiles when there are changes to the dosing regimen since it cannot estimate the concentration value at a specific time point.

The compartmental approach divides the body into a series of pharmacokinetically distinct compartments, each of which denotes a collection of tissues and organs that have similar rate of change of the drug concentration [25]. Drugs may exhibit single- or multi-compartment plasma concentration versus time profiles, with the number of compartments referring to the total number of disposition compartments. The compartment model assumes that each of the compartments is a well-stirred, kinetically homogenous unit. In addition, it is often assumed when constructing a compartmental model that the rate of elimination of the drug from the compartment and the transfer of the drug between the compartments (for a multi-compart‐ ment model) follows first-order (linear) kinetics. Under first-order kinetics, the rate of change of the drug amount or concentration is directly proportional to the remaining drug amount or concentration within the compartment.

**Figure 4.** A physiologically based pharmacokinetic (PBPK) model for the description of drug pharmacokinetics in ana‐ tomically relevant tissues and organs. Here, the drug is administered as a bolus dose into the venous blood compart‐ ment.

Unlike the aforementioned compartment model, a PBPK model (Figure 4) comprises com‐ partments that are defined based on anatomy, e.g. a compartment each for the liver and the brain. In addition, the PBPK model is also based on actual physiological and biochemical factors important in the input and disposition of the drug [26]. Such factors include cardiac output, organ blood flow rates, blood-to-plasma drug concentration ratio, partition coefficients in each organ, and transporter activities.

#### **5.1. One-compartment model**

Although more complex pharmacokinetic models may be necessary, a one-compartment model with first-order input provides a reasonable description of the time course for many drugs given at therapeutic doses (Figure 5), for example [27-29].

**Figure 5.** A one-compartment model with first-order input for describing the drug plasma concentration versus time profile. The arrows indicate the movement of the drug with respect to the body. Further, the diagram depicts that part of the drug may be excreted unchanged and some part may be metabolised by enzymes in the body.

In the one-compartment model, all the tissues of the body are lumped together as a single kinetically homogeneous compartment, commonly referred to as the central compartment. The one-compartment model is typically applied to drugs that distribute to only richlyperfused tissues and organs, such as liver, kidney and brain, in addition to the systemic circulation. The strongest indication that the body behaves as a single pharmacokinetically homogenous compartment for a drug is given by the presence of a mono-exponential decline in concentration values with respect to time when the concentrations are plotted on a loga‐ rithmic scale (Figure 6). For such a drug, it is assumed that all tissues and organs have a similar rate of change of the drug concentration as that of the systemic circulation, i.e. the source compartment.

Unlike the aforementioned compartment model, a PBPK model (Figure 4) comprises com‐ partments that are defined based on anatomy, e.g. a compartment each for the liver and the brain. In addition, the PBPK model is also based on actual physiological and biochemical factors important in the input and disposition of the drug [26]. Such factors include cardiac output, organ blood flow rates, blood-to-plasma drug concentration ratio, partition coefficients

Although more complex pharmacokinetic models may be necessary, a one-compartment model with first-order input provides a reasonable description of the time course for many

Excreted drug

Metabolised drug

Excreted metabolite

Body (Central compartment)

**Figure 5.** A one-compartment model with first-order input for describing the drug plasma concentration versus time profile. The arrows indicate the movement of the drug with respect to the body. Further, the diagram depicts that part

In the one-compartment model, all the tissues of the body are lumped together as a single kinetically homogeneous compartment, commonly referred to as the central compartment. The one-compartment model is typically applied to drugs that distribute to only richlyperfused tissues and organs, such as liver, kidney and brain, in addition to the systemic circulation. The strongest indication that the body behaves as a single pharmacokinetically homogenous compartment for a drug is given by the presence of a mono-exponential decline in concentration values with respect to time when the concentrations are plotted on a loga‐ rithmic scale (Figure 6). For such a drug, it is assumed that all tissues and organs have a similar rate of change of the drug concentration as that of the systemic circulation, i.e. the source

of the drug may be excreted unchanged and some part may be metabolised by enzymes in the body.

*Intake Disposition*

in each organ, and transporter activities.

110 Basic Pharmacokinetic Concepts and Some Clinical Applications

drugs given at therapeutic doses (Figure 5), for example [27-29].

**5.1. One-compartment model**

Drug

compartment.

**Figure 6**: Plasma concentration versus time profiles following i.v. bolus administration on a semi‐logarithmic graph for a drug exhibiting a one‐compartment model (upper panel) and a two‐compartment model (lower panel). For the two‐compartment model, the slopes and denote the rates of combined distribution plus elimination, and elimination, respectively, of the drug from the central compartment. **Figure 6.** Plasma concentration versus time profiles following i.v. bolus administration on a semi-logarithmic graph for a drug exhibiting a one-compartment model (upper panel) and a two-compartment model (lower panel). For the twocompartment model, the slopes *α* and *β* denote the rates of combined distribution plus elimination, and elimination, respectively, of the drug from the central compartment.

time *t* may be represented as: After intravenous (i.v.) bolus administration, the blood or plasma drug concentrations (*C*(*t*)) at time *t* may be represented as:

After intravenous (i.v.) bolus administration, the blood or plasma drug concentrations (*C*(*t*)) at

$$\mathbf{C}\left(t\right) = \frac{\text{Dose}\_{lv}}{V\_d} \times e^{-k\_s \times t} \tag{6}$$

where *Doseiv*, *Vd* and *ke* are the administered i.v. dose (units: mass), volume of distribution (units: volume), and first-order elimination rate constant (units: reciprocal time), respectively. This equation may be converted to the natural logarithm to yield:

$$\ln \mathbf{C} \left( t \right) = \ln \left( \frac{Dose\_{iv}}{V\_d} \right) - k\_\epsilon \times t \tag{7}$$

The total body clearance (*CLT*) and elimination half-life, *t*1/2 (units: time), can be calculated by:

$$\mathbb{CL}\_T = k\_c \times V\_d \tag{8}$$

$$t\_{1/2} = \frac{0.693}{k\_e} \tag{9}$$

For an i.v. administered drug displaying one-compartmental pharmacokinetic behaviour, drug concentrations of tissues will decay in parallel with plasma concentrations. However, it does not imply that the concentration in the plasma is equal to the concentration in these body tissues.

When a drug is administered extravascularly, such as oral ingestion, it has to be absorbed through biological barriers prior to reaching the central compartment (blood or plasma). Only when the drug enters the blood or plasma will it be regarded as systemically available. The process of absorption is complex and is governed by myriad factors including the adminis‐ tration route, formulation type, dose amount, and the physicochemical properties of the drug. Following extravascular administration, compartment models become more complicated because now the drug's absorption rate constant (*ka*) needs to be considered in the model. Despite the complexity involved, drug absorption is generally regarded as a first-order input process. For this first-order input, one-compartment model, the drug concentration at any time *t* is given by:

$$C\left(t\right) = \frac{F \times k\_a \times Dose\_{ce}}{V\_d \times \left(k\_a - k\_c\right)} \times \left(e^{-k\_s \times t} - e^{-k\_s \times t}\right) \tag{10}$$

where *F* and *Doseex* are the bioavailability (no units) and extravascular dose (units: mass), respectively. *F* denotes the proportion of extravascularly-administered drug that reaches the system circulation after drug administration. The maximum concentration (*Cmax*) after extrava‐ scular administration and the time at which *Cmax* is attained (*tmax*) are calculated by:

$$\mathcal{C}\_{\text{max}} = \frac{F \times Dose\_{cx}}{V\_d} \times e^{-k\_s \times t\_{\text{max}}} \tag{11}$$

$$t\_{\text{max}} = \frac{\ln\left(k\_a / k\_e\right)}{k\_a - k\_e} \tag{12}$$

#### **5.2. Two-compartment model**

Sometimes, drugs may display two or more phases during the declining portion of the concentration versus time profile [30]. This phenomenon may be encountered when multiple blood samples are collected during the drug's distribution and elimination phases. When the plasma drug concentration exhibits a bi-exponential decay (Figure 6, lower panel) on a semilogarithmic scale following an i.v. bolus injection, a two-compartment model is necessary to describe the underlying pharmacokinetics. The two-compartment model divides the body into the central compartment as per the one-compartment model, as well as a peripheral compart‐ ment that lumps together slowly-perfused tissues, such as fat and muscle. It is generally assumed that drug is eliminated still from the central compartment that comprises the liver and the kidney, since most compounds are metabolised by the liver and/or undergo renal excretion.

As portrayed by Figure 6 (lower panel), the concentration versus time profile after a single i.v. bolus dose on the semi- logarithmic scale has a rapidly declining phase followed by a shallower declining phase. The initial rapid decline in concentration is a consequence of simultaneous elimination (to the external environment) and distribution of the drug from the plasma to tissues lumped under the peripheral compartment. After the distribution process is completed and equilibrium is established between drug concentrations in the central compartment and the peripheral compartment, the drug concentration in the central compartment decreases at a rate dependent on the drug elimination rate. In general, the drug elimination rate is lower than the initial rate of decline in concentration due to the simultaneous distribution and elimination of the drug from the central compartment.

Without specifically describing the micro rate constants, the drug concentration at any time *t* after a single i.v. bolus administration of a two-compartment system is represented by:

$$\mathbf{C}\begin{pmatrix} t \\ \end{pmatrix} = A \times e^{-\kappa \times t} + B \times e^{-\rho \times t} \tag{13}$$

where *A*, *B*, *α* and *β* are derived from the intercepts and slopes of the respective distribution plus elimination, and elimination phases of the drug concentration versus time profile by curve fitting, such as nonlinear regression analysis.

#### **6. Conclusion**

The total body clearance (*CLT*) and elimination half-life, *t*1/2 (units: time), can be calculated by:

0.693 *e*

For an i.v. administered drug displaying one-compartmental pharmacokinetic behaviour, drug concentrations of tissues will decay in parallel with plasma concentrations. However, it does not imply that the concentration in the plasma is equal to the concentration in these body

When a drug is administered extravascularly, such as oral ingestion, it has to be absorbed through biological barriers prior to reaching the central compartment (blood or plasma). Only when the drug enters the blood or plasma will it be regarded as systemically available. The process of absorption is complex and is governed by myriad factors including the adminis‐ tration route, formulation type, dose amount, and the physicochemical properties of the drug. Following extravascular administration, compartment models become more complicated because now the drug's absorption rate constant (*ka*) needs to be considered in the model. Despite the complexity involved, drug absorption is generally regarded as a first-order input process. For this first-order input, one-compartment model, the drug concentration at any time

( ) ( ) ( ) *e a <sup>a</sup> ex kt kt*

where *F* and *Doseex* are the bioavailability (no units) and extravascular dose (units: mass), respectively. *F* denotes the proportion of extravascularly-administered drug that reaches the system circulation after drug administration. The maximum concentration (*Cmax*) after extrava‐

*e max ex k t*

*d ae F k Dose C t e e V kk*

scular administration and the time at which *Cmax* is attained (*tmax*) are calculated by:

*d F Dose C e V*

ln / ( ) *a e*

*a e k k*

Sometimes, drugs may display two or more phases during the declining portion of the concentration versus time profile [30]. This phenomenon may be encountered when multiple

*max*

*max*

*t*

1/2

*t*

112 Basic Pharmacokinetic Concepts and Some Clinical Applications

tissues.

*t* is given by:

**5.2. Two-compartment model**

*CL k V Ted* = ´ (8)

´ ´ -´ -´ = ´- ´ - (10)

´ - ´ = ´ (11)

*k k* <sup>=</sup> - (12)

*<sup>k</sup>* <sup>=</sup> (9)

Drug disposition refers to the combination of distribution and elimination. Distribution is a reversible process of movement of drugs from and to the site of measurement, typically the plasma or blood. Elimination comprises metabolism and excretion, and represents the total irreversible loss of the drug from the body. Drug disposition and elimination contribute to overall efficacy or toxicity and hence, up-to-date understanding of these pharmacokinetic processes is essential to safe, professional practice around medications and drug treatment. Compartmental modelling is a useful method to understand distribution and excretion processes [27, 31].

#### **Author details**

Seng Kok-Yong 1 and Lee Lawrence2\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Defence Medical & Environmental Research Institute, DSO National Laboratories, Singa‐ pore, Singapore

2 Department of Medicine, National University Health System, Singapore, Singapore

#### **References**

[12] Wienkers LC, Heath TG. Predicting in vivo drug interactions from in vitro drug dis‐ covery data. Nature Reviews Drug Discovery. 2005;4(10):825–833. doi: 10.1038/ nrd1851

**Author details**

Seng Kok-Yong 1

pore, Singapore

**References**

and Lee Lawrence2\*

114 Basic Pharmacokinetic Concepts and Some Clinical Applications

\*Address all correspondence to<EMAIL_ADDRESS>
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## **Biopharmaceutics and Pharmacokinetics**

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K. Ruckmani and R. Thirumurugan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61160

**1. Introduction**

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[28] Cooper JM, Duffull SB, Saiao AS, Isbister GK. The pharmacokinetics of sertraline in overdose and the effect of activated charcoal. British Journal of Clinical Pharmacolo‐

[29] Wiczling P, Liem RI, Panepinto JA, Garg U, Abdel-Rahman SM, Kearns GL, et al. Population pharmacokinetics of hydroxyurea for children and adolescents with sick‐ le cell disease. Journal of Clinical Pharmacology. 2014;54(9):1016–1022. doi: 10.1002/

[30] Jusko WJ, Gibaldi M. Effects of change in elimination on varous parameters of the two-compartment open model. Journal of Pharmaceutical Sciences. 1972;61(8):1270–

[31] Seng KY, Fan L, Lee HS, Yong WP, Goh BC, Lee LS. Population pharmacokinetics of modafinil and its acid and sulfone metabolites in Chinese males. Therapeutic Drug

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116 Basic Pharmacokinetic Concepts and Some Clinical Applications

jcph.303

1273.

Drug research is a specific process toward the development of new therapeutic agents in this era to meet the current medical needs. Drug discovery and development are the two major stages in the development of new therapeutic drug substance. Drug discovery involves identification and characterization of new targets (enzymes or receptors), synthesis of new lead molecules, screening of new lead molecules for its in vitro and/or in vivo biological activities, and physicochemical characterization of leads. The drug discovery and develop‐ ment process requires close interaction among the different scientific discipline members for as many as 10–12 years. It is estimated that only 1 out of 5000 screened compounds is approved as a new drug. On an average, every new drug molecule requires 12±15 years to reach the patient and costs a staggering amount of US \$ 400±650 million [1, 2].

**Active pharmaceutical ingredient (API):** Any substance or mixture of substances intended to be used in the manufacture of a pharmaceutical dosage form and that, when used so, becomes an active ingredient of that pharmaceutical dosage form [3].

*Steps involved in developing a new drug are:*

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Human body composition**

Human body is composed of a series of membrane barriers divided by aqueous-filled com‐ partments. These membrane barriers are principally composed of the phospholipid bilayers resulting from the orientation of the lipids (phospholipids, glycolipids, and cholesterol) in the aqueous medium, which surround the cells and also form intracellular barriers around the organelles present in cells (mitochondria, nucleus, etc.). The phospholipids are amphipathic in nature and have aligned polar head groups and lipid "tails," so the polar head groups of phospholipid orientate toward the aqueous phases and the lipid tails form a highly hydro‐ phobic inner core. Hence, the drug substance releases its hydration element and becomes hydrophobic. The drug disposition across the membrane depends on its lipophilicity and partition coefficient. Here, the protein binding plays an important role [4, 5].

The polar molecules will be dissociated in an aqueous environment; thereby, the hydrophilicity arises and vice versa in the case of nonpolar molecules in a lipophilic environment. Every component of an organic compound has a defined lipophilicity. Absorption and bile elimina‐ tion rate are molecular weight dependent. Lower-molecular-weight compounds have better absorption and less bile excretion when compared to the higher-molecular-weight com‐ pounds. Drugs with higher lipophilicity can be better absorbed from the intestine [5, 6].

#### **3. Biopharmaceutics**

Biopharmaceutics is a major branch in pharmaceutical sciences which relates between the physicochemical properties of a drug in dosage form and the pharmacology, toxicology, or clinical response observed after its administration [7]. Drug efficacy and safety are dependent on the dosing regimen. The optimal dosage and dosing intervals can be quite different for different drugs. Moreover, for a single drug, the optimal dosage can be different widely between patients [8].

It is not sufficient to know what the drug does to the body; it is also crucial to know what the body does to the drug. The knowledge of the pharmacodynamic and pharmacokinetic properties of the drug and its metabolites in humans and animals is crucial to understand its different effects among species and for adjusting drug dosing [9, 10].

The plasma concentration of the drug is the basic concept of pharmacokinetics. Based on protein binding of the drug, the concentration of free drug available in the circulation influ‐ ences greatly the dose calculations. The concentration of drug in the plasma is in equilibrium with some tissues in the body [11].

#### **4. Bioanalytical method**

Blood is the transporter of many vital substances and nutrients for the entire body and thus contains many endogenous and exogenous compounds in different concentrations. Biological samples (tissue extracts, plasma, serum, or urine) are extremely complex matrices comprised of many components that can interfere in estimation/quantification; hence, biological samples cannot normally be injected directly into the analyzing system for the determination of active principle. Sample pretreatment is required for achieving sufficient sensitivity and selectivity to determine the active principle. Chemical assays of high quality which include adequate sensitivity, selectivity and reproducibility are essential for obtaining valuable data. Bioanalysis is a subdiscipline of analytical chemistry covering the quantitative measurement drugs and their metabolites in biological systems. Bioanalysis technique can provide a quantitative measure of the active drug and/or its metabolite(s) for the purpose of pharmacokinetics. Various analytical instrument methods such as high-performance liquid chromatography (HPLC) or gas chromatography (GC) or ultra performance liquid chromatography (UPLC) with variety of detectors such as UV, fluorescent, diode array, flame ionization, electron capture and mass spectrometry, and capillary electrophoresis–mass spectrometry may be used. For macromolecule, ELISA or RIA method can be used for quantification [1, 12].

#### **5. Pharmacodynamics**

**2. Human body composition**

118 Basic Pharmacokinetic Concepts and Some Clinical Applications

**3. Biopharmaceutics**

between patients [8].

with some tissues in the body [11].

**4. Bioanalytical method**

Human body is composed of a series of membrane barriers divided by aqueous-filled com‐ partments. These membrane barriers are principally composed of the phospholipid bilayers resulting from the orientation of the lipids (phospholipids, glycolipids, and cholesterol) in the aqueous medium, which surround the cells and also form intracellular barriers around the organelles present in cells (mitochondria, nucleus, etc.). The phospholipids are amphipathic in nature and have aligned polar head groups and lipid "tails," so the polar head groups of phospholipid orientate toward the aqueous phases and the lipid tails form a highly hydro‐ phobic inner core. Hence, the drug substance releases its hydration element and becomes hydrophobic. The drug disposition across the membrane depends on its lipophilicity and

The polar molecules will be dissociated in an aqueous environment; thereby, the hydrophilicity arises and vice versa in the case of nonpolar molecules in a lipophilic environment. Every component of an organic compound has a defined lipophilicity. Absorption and bile elimina‐ tion rate are molecular weight dependent. Lower-molecular-weight compounds have better absorption and less bile excretion when compared to the higher-molecular-weight com‐ pounds. Drugs with higher lipophilicity can be better absorbed from the intestine [5, 6].

Biopharmaceutics is a major branch in pharmaceutical sciences which relates between the physicochemical properties of a drug in dosage form and the pharmacology, toxicology, or clinical response observed after its administration [7]. Drug efficacy and safety are dependent on the dosing regimen. The optimal dosage and dosing intervals can be quite different for different drugs. Moreover, for a single drug, the optimal dosage can be different widely

It is not sufficient to know what the drug does to the body; it is also crucial to know what the body does to the drug. The knowledge of the pharmacodynamic and pharmacokinetic properties of the drug and its metabolites in humans and animals is crucial to understand its

The plasma concentration of the drug is the basic concept of pharmacokinetics. Based on protein binding of the drug, the concentration of free drug available in the circulation influ‐ ences greatly the dose calculations. The concentration of drug in the plasma is in equilibrium

Blood is the transporter of many vital substances and nutrients for the entire body and thus contains many endogenous and exogenous compounds in different concentrations. Biological

different effects among species and for adjusting drug dosing [9, 10].

partition coefficient. Here, the protein binding plays an important role [4, 5].

Pharmacodynamics refers to the relationship between drug concentration at the site of action and the resulting effect, including the time course and intensity of therapeutic and its adverse effects. Studies are designed to investigate all primary and secondary effects related to the desired therapeutic effects, extensions of the therapeutic effect that might produce toxicity at higher doses, and effects related to interactions with other drugs.

#### **6. Pharmacokinetics**

Pharmacokinetics refers to the study of the time course of a drug within the body (extent and duration of systemic exposure to the drug) and also incorporates the process about the drug's *absorption*, *distribution*, *metabolism*, and *excretion* (ADME) pattern. In general, pharmacokinetic parameters are derived from the measurement of drug concentrations in blood or plasma [1].

#### **7. Absorption**

Absorption studies generally involve serial determinations of drug concentration in blood and urine after dosing to indicate the rate and extent of absorption.

Drug absorption refers to the passage of drug molecules from the site of administration into the circulation. Drug absorption requires that drugs cross one or more layers of cells and cell membranes.

Solubility is manipulated mainly by the structure of the drug. In general, solubility is inversely proportional to the number and type of lipophilic functions within the molecule and tightness of the crystal packing of the molecule. Solubility decreases when there is increase in crystal packing or lipophilicity.

The concentration of drug in solution is the driving force of the membrane transfer of drug into the body, and low aqueous solubility often continues to present itself as a problem even after formulation improvements.

Factors that influence drug absorption through oral route are:

#### **8. Drug absorption**

Drugs may be either weak acids or bases that exist in both ionized and non-ionized forms in the body. Drug in the non-ionized form is sufficiently soluble in membrane lipids and can cross cell membranes. The rate of absorption depends upon the ratio of the two forms at a particular site and is also a factor in distribution and elimination. The protonated form of a weak acid is non-ionized, whereas the protonated form of a weak base is ionized. The pKa is the negative log of the ionization constant, particular for each acidic or basic drug. Protonated form predominates when the pH is less than the pKa, whereas nonprotonated form predominates when pH is greater than the pKa. In the stomach, with a pH of 1, weak acids and bases are highly protonated. At this site, the non-ionized form of weak acids (pKa = 4 ± 1) and the ionized form of weak bases (pKa = 9 ± 1) will prevail upon. Weak acids are absorbed without dissoci‐ ation than weak base from the stomach and exactly opposite in the intestine where weak bases are absorbed readily than weakly acidic drugs. In intestine, weakly acidic drugs are also found to be absorbed even though they are ionized due to the large surface area [15].

Absorption takes place across the biological membrane by two methods. Lipid drugs are absorbed by transcellular mechanism where the drug distributes into the lipid core of the membrane which diffuses into the other side of the membrane. The solute may also diffuse across the cell membrane and enter into the circulation. Another mechanism is the paracellular absorption. The aqueous-filled pores in between the cells aid absorption of the drugs. Watersoluble drugs are readily absorbed, but the molecule size of the particle plays an important role [5, 12].

Drug absorption through transcellular and paracellular pathways is shown in Figure 1.

**Figure 1.** Drug absorption through transcellular and paracellular pathways

#### **9. Transport across cell membranes**

#### **9.1. Passive diffusion**

of the crystal packing of the molecule. Solubility decreases when there is increase in crystal

The concentration of drug in solution is the driving force of the membrane transfer of drug into the body, and low aqueous solubility often continues to present itself as a problem even

**i.** Biological factors: Permeation of the drug across the membrane, GI transit, site

**ii.** Pharmaceutical factors: Excipients, type of dosage forms, process of preparation,

**iii.** Other factors: Solubility of the drug; partitioning properties; dissociation character‐

Drugs may be either weak acids or bases that exist in both ionized and non-ionized forms in the body. Drug in the non-ionized form is sufficiently soluble in membrane lipids and can cross cell membranes. The rate of absorption depends upon the ratio of the two forms at a particular site and is also a factor in distribution and elimination. The protonated form of a weak acid is non-ionized, whereas the protonated form of a weak base is ionized. The pKa is the negative log of the ionization constant, particular for each acidic or basic drug. Protonated form predominates when the pH is less than the pKa, whereas nonprotonated form predominates when pH is greater than the pKa. In the stomach, with a pH of 1, weak acids and bases are highly protonated. At this site, the non-ionized form of weak acids (pKa = 4 ± 1) and the ionized form of weak bases (pKa = 9 ± 1) will prevail upon. Weak acids are absorbed without dissoci‐ ation than weak base from the stomach and exactly opposite in the intestine where weak bases are absorbed readily than weakly acidic drugs. In intestine, weakly acidic drugs are also found

to be absorbed even though they are ionized due to the large surface area [15].

Absorption takes place across the biological membrane by two methods. Lipid drugs are absorbed by transcellular mechanism where the drug distributes into the lipid core of the membrane which diffuses into the other side of the membrane. The solute may also diffuse across the cell membrane and enter into the circulation. Another mechanism is the paracellular absorption. The aqueous-filled pores in between the cells aid absorption of the drugs. Watersoluble drugs are readily absorbed, but the molecule size of the particle plays an important

Drug absorption through transcellular and paracellular pathways is shown in Figure 1.

polymorphism; prodrugs; and stereotype and its formation [8, 13, 14]

specificity, first-pass metabolism, metabolism in the liver, excretion as bile, excretion

istics; salt formation; particle size, shape, volume, and its distribution; crystallinity;

packing or lipophilicity.

**8. Drug absorption**

role [5, 12].

after formulation improvements.

120 Basic Pharmacokinetic Concepts and Some Clinical Applications

Factors that influence drug absorption through oral route are:

through bladder, and protein binding of drugs

stability testing, and storage directions

The concentration gradient provides energy for the transportation of the drug across the membrane, and also partitioning of the drug in favor of the lipid membrane decides the quantity of the drug absorbed. The unionized drug is absorbed markedly higher than the ionized form. Passive diffusion could be explained with Fick's first law which relates the diffusive flux to the concentration under the assumption of steady state. It postulates that the flux goes from regions of high concentration to regions of low concentration, with a magnitude that is proportional to the concentration gradient, or in simplistic terms, the concept that a solute will move from a region of high concentration to a region of low concentration across a concentration gradient.

#### **9.2. Active transport**

Active transport is the movement of molecules across the lipid cell membrane against con‐ centration gradient, i.e., moving from an area of lower concentration in the GIT to an area of higher concentration in the plasma. The absorption sites are at a specific place in the GIT. Active transport is usually associated with accumulating high concentrations of molecules that the cell needs, such as ions, glucose, and amino acids. This active transport process uses chemical energy, such as from adenosine triphosphate (ATP). These energy molecules are site specific – the drugs are transported at a particular site in the GIT, they are limited in number, and they act like a ferry service: it picks a molecule from the GIT, ferries across, leaves in the cytoplasm, and comes back to pick another molecule. The concentration of the drug in the plasma is maintained constant because of this "ferry" service, and the energy/carrier molecules are nothing but ATP-dependent proteins

#### **9.3. Endocytosis**

Endocytosis is an energy-using process by which cells absorb molecules (such as proteins) by engulfing them. It is used by large polar molecules that cannot pass through the hydrophobic plasma or cell membrane. The opposite process is exocytosis. Phagocytosis is a specific form of endocytosis involving the vascular internalization of solids such as bacteria by an organism and is therefore distinct from other forms of endocytosis such as the vesicular internalization of various liquids (pinocytosis). Phagocytosis is involved in the acquisition of nutrients for some cells. Pinocytosis, otherwise known as cell drinking, fluid endocytosis, and bulk-phase pinocytosis, is a mode of endocytosis in which small particles are brought into the cell, forming an invagination and then suspended within small vesicles [14, 16-21]. Various types of endocytosis are shown in Figure 2.

**Figure 2.** Various pathways of endocytosis

#### **10. Models for drug absorption**

Various in vitro, in situ, and in vivo tools and techniques are used to characterize the absorption of drug substance to determine the rate and extent of absorption.

Various models from low-throughput (in situ rat model) to high-throughput (in silico) models are used. Screening models for absorption such as human colon adenocarcinoma cell lines Caco-2 and HT-29 are widely used; recently, MDCK cell line is used as an alternative one.

Other in vitro methods are:

**1.** *Cell culture models*

In vitro cell culture models have been utilized to assess the permeability and metabolism of drugs, to elucidate molecular mechanism of drug transport to provide information on pathways of drug degradation, and to explore the influence of structure in the absorption of new chemical entities.

Several human colon carcinoma cells lines, such as the Caco-2, HT-29, SW116, LS174T, and SW480, are investigated for absorption. The cultured epithelial cells undergo enterocyte-like differentiation in culture and spontaneously differentiate into polarized columnar cells that are representative of the small intestine, with developed microvilli and polarized distribution of brush border enzymes. When grown on plastic membrane, epithelial cells result in a confluent monolayer and therefore serve as a model to study drug absorption.

#### **2.** *Isolated mucosal cells*

plasma or cell membrane. The opposite process is exocytosis. Phagocytosis is a specific form of endocytosis involving the vascular internalization of solids such as bacteria by an organism and is therefore distinct from other forms of endocytosis such as the vesicular internalization of various liquids (pinocytosis). Phagocytosis is involved in the acquisition of nutrients for some cells. Pinocytosis, otherwise known as cell drinking, fluid endocytosis, and bulk-phase pinocytosis, is a mode of endocytosis in which small particles are brought into the cell, forming an invagination and then suspended within small vesicles [14, 16-21]. Various types of

Various in vitro, in situ, and in vivo tools and techniques are used to characterize the absorption

Various models from low-throughput (in situ rat model) to high-throughput (in silico) models are used. Screening models for absorption such as human colon adenocarcinoma cell lines Caco-2 and HT-29 are widely used; recently, MDCK cell line is used as an alternative one.

In vitro cell culture models have been utilized to assess the permeability and metabolism of drugs, to elucidate molecular mechanism of drug transport to provide information on pathways of drug degradation, and to explore the influence of structure in the absorption of

Several human colon carcinoma cells lines, such as the Caco-2, HT-29, SW116, LS174T, and SW480, are investigated for absorption. The cultured epithelial cells undergo enterocyte-like differentiation in culture and spontaneously differentiate into polarized columnar cells that are representative of the small intestine, with developed microvilli and polarized distribution of brush border enzymes. When grown on plastic membrane, epithelial cells result in a

confluent monolayer and therefore serve as a model to study drug absorption.

of drug substance to determine the rate and extent of absorption.

endocytosis are shown in Figure 2.

122 Basic Pharmacokinetic Concepts and Some Clinical Applications

**Figure 2.** Various pathways of endocytosis

Other in vitro methods are:

**1.** *Cell culture models*

new chemical entities.

**10. Models for drug absorption**

Isolated mucosal cell suspensions have been used to study enzyme activity, drug transport, and cellular metabolism. The use of mucosal cells in drug absorption and transport studies is limited due to rapid autolysis.

#### **3.** *Brush border membrane vesicles*

Isolation of brush border membrane vesicles has been used extensively to study mucosal uptake process especially to investigate factors that influence mucosal uptake without interference of intracellular metabolism.

	- **a.** Everted sac technique: To prepare everted sac, a small length of the intestine is excised, turned inside out, filled, and ligated at both ends. The sac is immersed in an oxygenated solution that contains a drug. The fluid inside the sac is assayed for the drug, and the rate of drug transfer across the membrane provides an estimate of drug permeability.
	- **b.** Intestinal rings: Prepared by excising a portion of the intestine, everting it over a glass road, and cutting it into rings approximately 30–50 mg. The rings are then incubated in an oxygenated culture media that contain a drug. At the end of the incubation, the tissues are extracted and the unchanged drug is measured. Intestinal ring preparation can be used to measure the rate of uptake and accumulation of a drug from the intestines.

#### **5.** *Isolated tissue technique*

In this technique, the epithelium is mounted as a flat sheet between two chambers. The solution on each side of the chamber is oxygenated and maintained at physiological temperature. The test drug and markers for volume fluctuation or tissue viability are placed in the chambers. The chambers can be stirred using a gas lift of O/Co2 (95 %/5 %) at a flow rate of 15–2 ml/min. Samples can be obtained from the serosal and mucosal chambers to study diffusion and permeability.

#### *In situ methods*

#### **11. Distribution**

Distribution provides information on the extent and time course of tissue accumulation and the elimination of drug and/or its metabolites.

The disposition of drug into the organs and tissues via circulation depends upon the nature of the drug. The more lipophilic the drug is, the better will be the distribution into the organs and tissues. Hydrophilic drugs are normally concentrated in cells and they are referred to as *ion trapping*.

When a drug is introduced into the body, the rate of distribution is dependent upon the following:

#### **Reasons for the variation in concentration of drug distribution are:**

#### **11.1. Volume of distribution**

The volume of distribution (Vd), also known as apparent volume of distribution, is a pharma‐ cological, theoretical volume that the total amount of administered drug would have to provide the same concentration as it is in blood plasma.

If the amount of drug (*X*) and the resulting concentration (*C*) are known, then the volume of distribution (*Vd*) can be calculated using the simplified equations:

*X =VdC*, *where X* = amount of drug in body, *Vd* = volume of distribution, and *C* = concentration in the plasma.

Lipid-insoluble drugs are mainly confined to the plasma and interstitial fluid; most do not enter the brain following acute dosing. Lipid soluble drugs reach all compartments and may accumulate in fat. For drugs that accumulate outside the plasma compartment, Vd may exceed the total body volume.

*Factors involved in drug distribution and diffusion across blood tissue barrier are:*

**1.** Blood flow

and tissues. Hydrophilic drugs are normally concentrated in cells and they are referred to as

When a drug is introduced into the body, the rate of distribution is dependent upon the

**1.** Tissues with the highest blood flow receive the drug: The rate at which a drug is distrib‐ uted to various organs after a drug dose is administered depends largely on the proportion

**2.** Protein binding: Binding to proteins is inevitable in the case of drugs particularly lipoproteins, glycoproteins, and β-globulins. The extent of binding depends on the affinity of the drug molecule with the protein, and the maximum affinity could be 99 % also.

**3.** Lipid solubility: Lipid solubility is a major factor affecting the extent of drug distribution, particularly to the brain, where the *blood–brain barrier* restricts the penetration of polar and

**4.** Molecular size: Molecular size is a factor affecting the distribution of extremely large

**5.** Distribution depends upon the ionization of drug, whereas unionized drugs can go

**1.** Tissue differences in rates of uptake of drugs: Blood flow and capillary permeability

**2.** Differences in tissue/blood ratios at equilibrium: Dissolution of lipid-soluble drugs in adipose tissue, binding of drugs to intracellular sites, and plasma protein binding

The volume of distribution (Vd), also known as apparent volume of distribution, is a pharma‐ cological, theoretical volume that the total amount of administered drug would have to

If the amount of drug (*X*) and the resulting concentration (*C*) are known, then the volume of

*X =VdC*, *where X* = amount of drug in body, *Vd* = volume of distribution, and *C* = concentration

Lipid-insoluble drugs are mainly confined to the plasma and interstitial fluid; most do not enter the brain following acute dosing. Lipid soluble drugs reach all compartments and may accumulate in fat. For drugs that accumulate outside the plasma compartment, Vd may exceed

*ion trapping*.

following:

molecules.

anywhere into the body.

**3.** Apparent volume of distribution (Vd)

provide the same concentration as it is in blood plasma.

distribution (*Vd*) can be calculated using the simplified equations:

**11.1. Volume of distribution**

in the plasma.

the total body volume.

of *cardiac output* received by the organs.

124 Basic Pharmacokinetic Concepts and Some Clinical Applications

Unbound drug diffuses in the liquids surrounding the cells.

**Reasons for the variation in concentration of drug distribution are:**

ionized molecules. Highly lipid-soluble drug can enter the tissues.

In our body, various structures are acting as reservoir for storage of drug substance.

Multiple paths of drug distribution in the blood stream are shown in Figure 3.

**Figure 3.** Multiple paths of drug distribution in the blood stream

#### **11.2. Compartment models in kinetics of drug distribution**

Compartment models are hypothetical structures used to describe the fate of a drug in a biological system after its administration into the body. Various compartment models in pharmacokinetic are:

*One-compartment model:* Following drug administration, the body is depicted as a kinetically homogeneous unit.

*Two-compartment model:* The two-compartment model resolves the body into a central com‐ partment and a peripheral compartment.

*Multicompartment model:* In this model, the drug distributes into more than one compartment and the concentration–time profile shows more than one exponential [9, 15, 26-29].

Various body compartments and the drug distribution is shown in Table 1.

**Table 1.** Body compartment and the drug distribution

#### **12. Biotransformation/Metabolism**

Biotransformation or drug metabolism is the enzyme-catalyzed conversion of drugs to their metabolites. Metabolism makes the drug less polar; lipid-soluble substance makes it more polar as well as water soluble, thus facilitating their excretion by the kidney. If a drug is already highly polar and water soluble, then it may not get metabolized and may get excreted as such. Liver is the chief organ for biotransformation of most drugs, but drug-metabolizing enzymes are found in many other tissues, including the gut, kidneys, brain, lungs, and skin. Lipophilic drug is converted to a hydrophilic one by extensive metabolism in the liver.

Drug metabolism is traditionally carried out by phase I and phase II processes.

Cytochrome P450 system has an important role and occupies a pivotal role in drug clearance in phase I.

Phase I: First step in biotransformation is the formation of product susceptible to phase II conjugative reaction. The phase I also involves unmasking a functional group like OH, NH2, and SH and conversion to more polar products which may be mostly inactive, less active, and modified activity.

Phase II: Coupling of drug or its oxidized metabolite to endogenous conjugating agent derived from carbohydrate, protein, or sulfur sources; generally products are more water-soluble and more readily excreted in urine or bile. Phase II involves conjugation reactions with glucuronic acid, sulfuric acid, acetic acid, and amino acid.

Biotransformation occurs somewhere between absorption and excretion; some may occur in the gut (digestion, decomposition in gastric acidity).

Role of enzymes in the biotransformation are drug metabolism; conversion of prodrug to active forms; synthesis of steroidal hormones, cholesterol, and bile acids; and finally formation and excretion of bilirubin.

Biotransformation is mediated by cellular enzymes in the sarcoplasmic reticulum, mitochon‐ dria, cytoplasm, lysosomes, and nucleus.

Drug-metabolizing enzymes are classified into:

**1.** Microsomal (inducible)

Various body compartments and the drug distribution is shown in Table 1.

Extracellular water = 0.2 Larger water-soluble drugs

Fat = 0.2 – 0.35 Lipid-soluble drugs

Bone = 0.07 Certain ions

**Table 1.** Body compartment and the drug distribution

126 Basic Pharmacokinetic Concepts and Some Clinical Applications

**12. Biotransformation/Metabolism**

acid, sulfuric acid, acetic acid, and amino acid.

the gut (digestion, decomposition in gastric acidity).

Total body water = 0.6 (extracellular and intracellular)

Blood = 0.08 Plasma = 0.04

in phase I.

modified activity.

excretion of bilirubin.

**Body compartments (L/kg body weight) Drug distribution in the body compartments**

Small water-soluble drugs

Biotransformation or drug metabolism is the enzyme-catalyzed conversion of drugs to their metabolites. Metabolism makes the drug less polar; lipid-soluble substance makes it more polar as well as water soluble, thus facilitating their excretion by the kidney. If a drug is already highly polar and water soluble, then it may not get metabolized and may get excreted as such. Liver is the chief organ for biotransformation of most drugs, but drug-metabolizing enzymes are found in many other tissues, including the gut, kidneys, brain, lungs, and skin. Lipophilic

Cytochrome P450 system has an important role and occupies a pivotal role in drug clearance

Phase I: First step in biotransformation is the formation of product susceptible to phase II conjugative reaction. The phase I also involves unmasking a functional group like OH, NH2, and SH and conversion to more polar products which may be mostly inactive, less active, and

Phase II: Coupling of drug or its oxidized metabolite to endogenous conjugating agent derived from carbohydrate, protein, or sulfur sources; generally products are more water-soluble and more readily excreted in urine or bile. Phase II involves conjugation reactions with glucuronic

Biotransformation occurs somewhere between absorption and excretion; some may occur in

Role of enzymes in the biotransformation are drug metabolism; conversion of prodrug to active forms; synthesis of steroidal hormones, cholesterol, and bile acids; and finally formation and

drug is converted to a hydrophilic one by extensive metabolism in the liver. Drug metabolism is traditionally carried out by phase I and phase II processes.

Plasma protein-bound large drugs

**2.** Nonmicrosomal (non-inducible)

#### **12.1. Microsomal enzymes (inducible)**

Microsomes are artificial spheres obtained from the endoplasmic reticulum by homogeniza‐ tion and fractionation, and they possess various drug-metabolizing enzymes.

**1.** Mixed-function oxidases (monooxygenases) cytochrome P-450, cytochrome P-450 reductase, and NADPH

Reactions catalyzed by monooxygenase are N-dealkylation, O-dealkylation, aromatic ring oxidation, side-chain oxidation, sulfoxide formation, N-oxidation, N-hydroxylation, deami‐ nation of primary and secondary amines, and desulfuration (S replacement by O2).

**2.** Glucuronyl transferase for conjugation

The drugs containing phenols, alcohols, and carboxylic acids are metabolized by conjugation method. The conjugates are mostly inactive and excreted in the bile and urine by anion carrier mechanism and enter into enterohepatic cycling (β-glucuronidase and sulfatase in the gut).

**3.** Some enzymes are involved in reduction and hydrolysis

The modification of enzyme activity such as enzyme induction and enzyme inhibition was observed.

Majority of the drugs however are metabolized by the nonmicrosomal enzymes resulting in their activation, inactivation, or modification. The reactions are:

Drug metabolism is affected by various factors. The diseases that are categorized as acute and chronic liver diseases (reduces metabolism), liver cancer, cardiac diseases limiting blood flow to the liver, pulmonary diseases reducing hydrolysis of procainamide, and hyperthyroidism where metabolism are affected. And also metabolism increases t1/2 and hypothyroidism reduces metabolism t1/2 [14, 30-36].

#### **12.2. Metabolism methodologies**

#### *12.2.1. In vitro methods*

In vitro techniques are well suited for the study of biochemical toxicology, cytotoxicity, irreversible drug protein binding, drug metabolism, and enzyme regulation. Induction of drug-metabolizing enzymes can have a dramatic impact on the disposition, toxicology, and metabolic profile of the agent under study.

Primarily hepatic enzymes from animals and humans are used for drug metabolism studies. Other enzymes from the intestine and brain are also being used in the metabolism studies. In human, cytochrome P450 is used primarily, whereas its subfamilies such as CYP1A, CYP2C, CYP2D, CYP2E, CYP3A, and CYP4A are also being used.

*Enzyme systems:* Single or isolated enzyme systems are a powerful technique for the study of enzymatic process due to easy maintenance and manipulation in the substrate, enzyme, and cofactor concentrations. Interested enzyme from animal or human tissue can be isolated by extraction and purification and reconstituted to study the drug metabolisms. Single-enzyme system is useful in the study of enzyme kinetics, specificity, and mechanism. Other enzymes such as cytochromes CYP450, flavin-containing monooxygenases, glucuronyltransferases, sulfotransferases, epoxide hydrolases, glutathione S-transferases, and N-acetyltransferases are also used in the drug metabolism studies.

*Subcellular fractions*: Microsomes as subcellular fraction is frequently utilized as in vitro model. These subcellular components, composed of endoplasmic reticulum, contain most of the oxidative drug-metabolizing enzymes, such as the cytochromes P450 and flavin monooxyge‐ nases, glucuronyltransferase, epoxide hydrolases, alcohol dehydrogenases, esterases, and methyltransferases, that can be separated by cell disruption and differential centrifugation.

*Cellular systems*: Cell culture system is utilized to study both drug metabolism and toxicology within a physiological environment due to manipulation of its enzyme concentrations and cofactors under appropriate conditions. These systems can be used to evaluate multiple aspects of drug metabolism, drug transport across cell membranes, enzyme induction, and cytotoxicity from such organs as the kidney, intestinal mucosa, and liver.

*Liver slices*: Organ slices were extensively used to study a variety of biochemical process because of the ability to produce uniform-cut organ slices by commercial tissue slicers and improved organ culture conditions. The slices have been isolated from many different species including human, and several organs such as the liver, brain, heart, and kidney are used.

*Organ perfusion*: Organ perfusion is used to measure the toxicological and pharmacokinetic events and parameters because of its close approximation to the tissues. This perfusion method offers several advantages over other in vitro methods such as preservation of organ architec‐ ture and ability to regulate perfused flow rate; two sampling sites are available for determi‐ nation of substrate and metabolite concentrations.

But the limitation is that only one experiment can be performed per animal.

#### *12.2.2. In vivo methods*

**12.2. Metabolism methodologies**

metabolic profile of the agent under study.

128 Basic Pharmacokinetic Concepts and Some Clinical Applications

also used in the drug metabolism studies.

CYP2D, CYP2E, CYP3A, and CYP4A are also being used.

from such organs as the kidney, intestinal mucosa, and liver.

nation of substrate and metabolite concentrations.

In vitro techniques are well suited for the study of biochemical toxicology, cytotoxicity, irreversible drug protein binding, drug metabolism, and enzyme regulation. Induction of drug-metabolizing enzymes can have a dramatic impact on the disposition, toxicology, and

Primarily hepatic enzymes from animals and humans are used for drug metabolism studies. Other enzymes from the intestine and brain are also being used in the metabolism studies. In human, cytochrome P450 is used primarily, whereas its subfamilies such as CYP1A, CYP2C,

*Enzyme systems:* Single or isolated enzyme systems are a powerful technique for the study of enzymatic process due to easy maintenance and manipulation in the substrate, enzyme, and cofactor concentrations. Interested enzyme from animal or human tissue can be isolated by extraction and purification and reconstituted to study the drug metabolisms. Single-enzyme system is useful in the study of enzyme kinetics, specificity, and mechanism. Other enzymes such as cytochromes CYP450, flavin-containing monooxygenases, glucuronyltransferases, sulfotransferases, epoxide hydrolases, glutathione S-transferases, and N-acetyltransferases are

*Subcellular fractions*: Microsomes as subcellular fraction is frequently utilized as in vitro model. These subcellular components, composed of endoplasmic reticulum, contain most of the oxidative drug-metabolizing enzymes, such as the cytochromes P450 and flavin monooxyge‐ nases, glucuronyltransferase, epoxide hydrolases, alcohol dehydrogenases, esterases, and methyltransferases, that can be separated by cell disruption and differential centrifugation.

*Cellular systems*: Cell culture system is utilized to study both drug metabolism and toxicology within a physiological environment due to manipulation of its enzyme concentrations and cofactors under appropriate conditions. These systems can be used to evaluate multiple aspects of drug metabolism, drug transport across cell membranes, enzyme induction, and cytotoxicity

*Liver slices*: Organ slices were extensively used to study a variety of biochemical process because of the ability to produce uniform-cut organ slices by commercial tissue slicers and improved organ culture conditions. The slices have been isolated from many different species including human, and several organs such as the liver, brain, heart, and kidney are used.

*Organ perfusion*: Organ perfusion is used to measure the toxicological and pharmacokinetic events and parameters because of its close approximation to the tissues. This perfusion method offers several advantages over other in vitro methods such as preservation of organ architec‐ ture and ability to regulate perfused flow rate; two sampling sites are available for determi‐

But the limitation is that only one experiment can be performed per animal.

*12.2.1. In vitro methods*

*Radionuclides*: Formation and excretion of metabolites can be easily monitored by attaching radiotracer tag on a drug candidate. Radiotracer tag is placed at chemically and metabolically stable site. Tritium (3 H) and carbon14 (14C) are the most commonly used radionuclides used as tracer tag in drug metabolism studies [37].

#### **13. Clearance (Elimination)**

Drug clearance (CL) is defined as the volume of plasma in the vascular compartment cleared of drug (only free, i.e., not protein bound) per unit time by the processes of metabolism and excretion. Clearance is related to the concentrations of the drug present in blood after admin‐ istration. Clearance of drug occurs by the perfusion of blood to the organs of extraction. Extraction is the ratio of the clearance process (*E*) referring to the proportion of drug presented to the organ which is removed irreversibly (excreted) or altered to a different chemical form (metabolism) from the organ.

Hepatic clearance (ClH) and renal excretion (ClR) are generally involved in the extraction of the drug from the body. The overall value for systemic clearance (CIS) can be calculated by

$$\mathrm{Cl}\_{\mathrm{s}} = \mathrm{Cl}\_{\mathrm{H}} \star \mathrm{Cl}\_{\mathrm{R}}$$

The amount of drug in the circulation is related to the volume of distribution, and therefore elimination rate constant (*k*el) can be calculated by

$$k\_{cl} = \text{Cl} / V\_d$$

Clearance for a drug is constant if the drug is eliminated by first-order kinetics.

Half-life: The time required to reduce the plasma concentration to one half its initial value is defined as the *half-life* (*t*1/2).

Zero-order reaction: The reaction proceeds at a constant rate and is independent of the concentration of drug present in the body.

First-order reaction: The reaction proceeds at a rate that is dependent on the concentration of drug present in the body.

*Excretory organs*:

Major routes: kidneys, liver, and lungs.

Minor routes: sweat, saliva, tears, and breast milk.

Urine: It helps to quantitate the amount of drug excreted and is the most important excretory route for nonvolatile drugs and their metabolites (drug not bound to plasma proteins), proximal tubular active secretion, and passive tubular reabsorption.

*Renal excretion*: Small molecules with low molecular weight will appear in urine through glomerular filtration. Through tubular carrier systems (tubular secretion), a drug can be transported against the concentration gradient from the blood capillaries to the nephron lumen to be excreted in the urine.

*Lipophilicity in drug clearance*: Reduction in lipophilicity is observed when compared to the parent molecule during administration. For hydrophilic drugs (log *D*7.4 below 0), renal clearance is the predominant mechanism, whereas the drugs with log *D*7.4 values are above 0, renal clearance decreases with lipophilicity. Metabolic clearance increases with increasing log *D*, and this becomes the major clearance route of lipophilic compounds. The lowest clearance (negligible) is observed below log *D*7.4 values of 0 by combined renal and metabolic processes (log *D*7.4 Logarithm of the distribution coefficient (*D*) at pH 7.4).

*Lipophilicity and reabsorption by the kidney*: The degree of reabsorption (all along the nephron) depends on the physicochemical properties (degree of ionization and intrinsic lipophilicity) of the drug. After absorption, the equilibrium is reestablished in the kidney where the unbound drug in the urine and unbound drug in plasma are present on both sides of the membrane. The water-soluble drugs are absorbed easily, but lipophilic drugs will be reabsorbed by diffusion due to concentration gradient.

*Effect of charge on renal clearance*: Tubular pH is often more acidic (pH 6.5) than plasma; hence, acidic drugs are reabsorbed more extensively than basic. Greater rates of excretion/clearance can occur for these charged moieties due to the tubular active transport proteins.

*Renal clearance:* The unbound drug will be cleared by filtration, and the protein-bound drug will be cleared slowly as it dissociates after a long time. Drugs with increasing plasma protein binding have increased lipophilicity, which decreases the renal clearance.

*Renal clearance in drug design*: Small molecules with relatively simple structures (molecular weights below 350) can successfully combine paracellular absorption and renal clearance.

*Liver and biliary excretion*: Liver is the organ where maximum metabolism takes place. The unabsorbed drugs and the metabolized drugs are excreted through fecal matter. Enzyme cytochrome is having a pivotal role in drug clearance by various oxidation reactions such as aromatic hydroxylation, aliphatic hydroxylation, *N*-dealkylation, *O*-dealkylation, *S*-dealkyla‐ tion, *N*-oxidation, *S*-oxidation, and alcohol oxidation. Hepatic and renal clearance process is shown in Figure 4.

Lungs: The lungs are an important route for the excretion of gaseous anesthetics, alcohol, iodine, and iodates.

Other excretion routes are sweat, saliva, and tears which are generally pH dependent that mediate drug excretion by passive diffusion of lipophilic drugs.

**Figure 4.** Hepatic and renal clearance process

Milk: Milk is more acidic than plasma; hence, basic drugs tend to accumulate due to ionic trapping, whereas concentration of acidic drugs is lesser than in the plasma. Nonelectrolytes (ethanol, urea) enter milk in a pH-independent manner.

Hair and skin: Toxic metal may be excreted (murder, suicide) [8, 14, 29, 38-42].

#### **14. Conclusion**

Urine: It helps to quantitate the amount of drug excreted and is the most important excretory route for nonvolatile drugs and their metabolites (drug not bound to plasma proteins),

*Renal excretion*: Small molecules with low molecular weight will appear in urine through glomerular filtration. Through tubular carrier systems (tubular secretion), a drug can be transported against the concentration gradient from the blood capillaries to the nephron lumen

*Lipophilicity in drug clearance*: Reduction in lipophilicity is observed when compared to the parent molecule during administration. For hydrophilic drugs (log *D*7.4 below 0), renal clearance is the predominant mechanism, whereas the drugs with log *D*7.4 values are above 0, renal clearance decreases with lipophilicity. Metabolic clearance increases with increasing log *D*, and this becomes the major clearance route of lipophilic compounds. The lowest clearance (negligible) is observed below log *D*7.4 values of 0 by combined renal and metabolic processes

*Lipophilicity and reabsorption by the kidney*: The degree of reabsorption (all along the nephron) depends on the physicochemical properties (degree of ionization and intrinsic lipophilicity) of the drug. After absorption, the equilibrium is reestablished in the kidney where the unbound drug in the urine and unbound drug in plasma are present on both sides of the membrane. The water-soluble drugs are absorbed easily, but lipophilic drugs will be reabsorbed by

*Effect of charge on renal clearance*: Tubular pH is often more acidic (pH 6.5) than plasma; hence, acidic drugs are reabsorbed more extensively than basic. Greater rates of excretion/clearance

*Renal clearance:* The unbound drug will be cleared by filtration, and the protein-bound drug will be cleared slowly as it dissociates after a long time. Drugs with increasing plasma protein

*Renal clearance in drug design*: Small molecules with relatively simple structures (molecular weights below 350) can successfully combine paracellular absorption and renal clearance.

*Liver and biliary excretion*: Liver is the organ where maximum metabolism takes place. The unabsorbed drugs and the metabolized drugs are excreted through fecal matter. Enzyme cytochrome is having a pivotal role in drug clearance by various oxidation reactions such as aromatic hydroxylation, aliphatic hydroxylation, *N*-dealkylation, *O*-dealkylation, *S*-dealkyla‐ tion, *N*-oxidation, *S*-oxidation, and alcohol oxidation. Hepatic and renal clearance process is

Lungs: The lungs are an important route for the excretion of gaseous anesthetics, alcohol,

Other excretion routes are sweat, saliva, and tears which are generally pH dependent that

can occur for these charged moieties due to the tubular active transport proteins.

binding have increased lipophilicity, which decreases the renal clearance.

mediate drug excretion by passive diffusion of lipophilic drugs.

proximal tubular active secretion, and passive tubular reabsorption.

130 Basic Pharmacokinetic Concepts and Some Clinical Applications

(log *D*7.4 Logarithm of the distribution coefficient (*D*) at pH 7.4).

to be excreted in the urine.

diffusion due to concentration gradient.

shown in Figure 4.

iodine, and iodates.

Pharmacokinetics is the study of the time course of a drug within the body and incorporates the processes of absorption, distribution, metabolism, and excretion (ADME). The simplest pharmacokinetic concept is that based on concentration of drug in the biological matrix. Selective and sensitive bioanalytical method is required to quantify the concentration of the drug in the biological matrix. Most of the drugs are absorbed by passive diffusion process. The rate of drug diffusion by passive process depends upon the lipid solubility and the surface area available for absorption. The drug distribution is based on the plasma protein binding, molecular size, and lipid solubility. After distribution, the drug is metabolized into a metab‐ olite as either a pharmacologically active or inactive one. The liver plays a vital role in the drug metabolism. Metabolized drugs are cleared mainly by the liver and kidney. The drug discovery and development process required a large amount of clinical data for rapid screening, selection, and development of new compounds. Various mathematical models are developed to assess the pharmacokinetic parameters. Preliminary pharmacokinetic study results are very much useful to characterize the absorption, disposition profile, and drug metabolism, which are very much essential and important in the discovery and development of new therapeutic agents in areas of currently unmet medical needs.

#### **Author details**

S. Lakshmana Prabu1 , T.N.K. Suriyaprakash2\*, K. Ruckmani1 and R. Thirumurugan3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Dept. of Pharm. Technology, Bharathidasan Institute of Technology, Anna University, Tiruchirappalli, India

2 Dept of Pharmaceutics, Al Shifa College of Pharmacy, Kerala, India

3 School of Pharmacy, International Medical University, Malaysia

#### **References**

[10] Gunaratna C. (2000). Drug metabolism and pharmacokinetics in drug discovery: a primer for bioanalytical chemists, part I. *Current Separation,* 19, 17–23.

**Author details**

S. Lakshmana Prabu1

Tiruchirappalli, India

**References**

, T.N.K. Suriyaprakash2\*, K. Ruckmani1

2 Dept of Pharmaceutics, Al Shifa College of Pharmacy, Kerala, India

3 School of Pharmacy, International Medical University, Malaysia

1 Dept. of Pharm. Technology, Bharathidasan Institute of Technology, Anna University,

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