1 INTRODUCTION 394 395 395 396 396 399 Plasma and tissue protein binding of drugs is a major factor that affects both pharmacokinetics and pharmacodynamics of the drug. It is usually the free (unbound) form of the drug that can exert pharmacological activity, while the bound form of the drug is usually pharmacologically inactive (Ascenzi et al., 2014). Many drugs can bind to plasma proteins to form a drugprotein complex, the binding is usually reversible, and the unbound (free) form of the drug exists in equilibrium with the bound form (Li et al., 2015). Drugs bind mainly with plasma proteins such as albumin, alpha-1-acid glycoprotein, lipoproteins, and other biological moieties, e.g., red blood cells (RBCs) (Pellegatti et al., 2011). The reversible binding of drugs to proteins has a significant impact on many pharmacokinetic parameters such as volume of distribution and clearance of the drug (Berezhkovskiy, 2010). Since the drugprotein complex has a large size, this will limit its ability to leave the vascular space and enter into cells thus restricting its distribution, while the unbound (free) drug can readily diffuse into cells. Also, the drugprotein complex is usually too large to be filtered by the glomeruli, and only the unbound drug can be filtered and excreted by the kidney. Thus, plasma protein binding also affects clearance of the drug by the kidney, and sometimes if the drug has a higher affinity for the plasma proteins than the liver enzymes, the drug will not be available for metabolism and clearance by the liver. Hence, only the unbound drug will be metabolized (Han et al., 2010). 11.2 BINDING KINETICS The reversible binding of drugs to proteins is governed by the law of mass-action according to Eq. (11.1): D ½1 P½ k1 " k2 ½ DP (11.1) where [D] is the unbound (free) drug concentration, [P] is the unoccupied protein binding concentration and [DP] is the drugprotein complex concentration, and k1 and k2 are the association and dissociation constants of the drugprotein complex, respectively.
The equilibrium between the unbound [D] and bound [DP] drug forms is established rapidly (usually in milliseconds), and at equilibrium, the association constant Ka is used to describe the affinity of the drug for the protein to which it binds according to Eq. (11.2). Ka 5k1 k2 5 ½DP D ½3½P (11.2) The association constant Ka describes the affinity by which a drug binds to a protein to form a drugprotein complex, the higher the association constant Ka value, the stronger the binding of a drug to the protein and more the drugprotein complex forms and vice versa. However, in practice, it is usually the dissociation constant Kd that is more commonly used, as shown in Eq. (11.3)(Musteata, 2012). Kd 51Ka (11.3) It is important to remember that the dissociation constant Kd is the inverse of the association constant Ka. The lower the dissociation constant Kd value, the stronger the binding of a drug to the protein and the more the drugprotein complex forms and vice versa (note that in this aspect Kd is similar to the MichaelisMenten constant km, which describes the affinity of binding of a ligand to an enzyme). Two common ways are used to express the plasma protein binding of drugs. The first is to describe it as the unbound fraction in plasma (fup), which is the ratio of the unbound drug concentration [D] and the total drug concentration in the system [D]1[DP], as shown in Eq. (11.4). The second way is to express it as a percentage of the bound drug concentration [DP] to the total drug concentration [D]1[DP] in the system, as shown in Eq. (11.5). fup 5 Cfree Ctotal 5 %PPB5 ½ D D ½1½DP Cbound Ctotal 3100 (11.4) (11.5) The total protein concentration [Pt] is the sum of the unbound protein concentration [P] and the bound protein concentration [PD], as shown in Eq. (11.6). Pt ½5 P½1½PD By rearranging Eq. (11.6) and substituting into Eq. (11.2) we get Eq. (11.7): ½PD ½Pt5 Ka½D 11Ka½D (11.6) (11.7) The ratio [PD]/[Pt] which is the moles of drug bound per moles of total protein is designated as r, and Eq. (11.7) can be written in the form of Eq. (11.8). r 5Ka ½D 11Ka½D
(Since the dissociation constant (Kd) is used more often than the association constant (Ka), and Kd is 1/Ka, then Eq. (11.8) can be written as Eq. (11.9): r 5½D (11.9) Kd 1½D Eq. (11.9) assumes that the drug binds only to one independent binding site in the binding protein (i.e., one mole of the drug binds to one mole of the binding protein). In cases when there are more than one independent binding site for the drug in the binding protein, then Eq. (11.10) can be used. r 5n½ D Kd 1½D (11.10) In Eq. (11.10), n represents the number of identical binding sites available for the drug in the binding protein. If the protein contains more than one type of binding sites (i.e., not identical binding sites) or if the drug binds to more than one protein in a given system, then Eq. (11.11) is used. r 5n 1K1½D 11K1½D1 n2K2½D 11K2½D (11.11) In Eq. (11.11), the numbers associated with n and K represents the different types of binding sites which the drug binds to where n represents the number of binding sites per mole of protein and the K accounts for the binding constant associated with each type of different binding site. It should be noted that these equations assume that the drug binds independently to the different binding sites and the binding of drug molecules to a binding site does not alter the affinity of other binding sites to the drug molecules. However, some drugs may show cooperativity phenomena while binding to proteins. When binding of a drug molecule to one binding site on a binding protein affects the subsequent binding affinities of drug molecules to other binding sites, this is known as the cooperativity phenomena. An example of the cooperativity is the binding of oxygen molecules (O2) to hemoglobin, in which the binding of the first O2 molecule to the first binding site in hemoglobin increases the affinity of binding for the second O2 molecule to the second binding site in hemoglobin, and in turn, the binding of the second O2 molecule enhances the affinity of binding of the third O2 molecule to hemoglobin (Sinko, 2011; Shargel and Andrew, 2015). Binding of drugs to plasma proteins is usually linear (concentration-independent) when the molar concentration of the unbound drug is lower than the dissociation constant (Kd), which means that [D] and PPB% will be constant and independent of changes in drug concentration. At higher concentration of the drug or when the binding affinity for the protein is high (low Kd), the drug will display nonlinear (concentration-dependent) protein. As the drug reaches its site of action, it will interact with its target (which could be a receptor, enzyme, etc.) which would result in a pharmacological effect. Binding is usually reversible and can be written as shown in Eq. 11.12: D ½1 T½ k3 " k4 ½DT ( where [D] is the unbound (free) drug concentration, [T] is the target protein for the drug and [DT] is the drugtarget protein complex concentration. k3 and k4 are the association and dissociation constants of the drugtarget protein complex, respectively. Since the drug at the site of action may show a binding affinity for other proteins, which would result in a decrease in the pharmacological effect of the drug since these other proteins compete with the target protein for the free drug concentration. But in cases where the drug has a much higher binding affinity for its target than the other proteins (i.e., K3 *[T] is much larger than K4 *[P]), then the drug will bind preferentially to its target than to the other proteins (Schmidt et al., 2010). 11.2.1 Graphical Plots Used to Determine Binding Constants Several graphical methods can be used to determine binding constants between the drug and protein. A direct plot can be constructed if the receptor concentration is known, by plotting r which is the moles of drug bound to the total protein concentration against the free drug concentration [D], which gives a hyperbolic curve as shown in Fig. 11.1 which is a graphical representation of Eq. 11.10. By taking the reciprocal of Eq. 11.8, it is possible to obtain the number of binding sites and the dissociation constant with good precision. Eq. 11.13 is the reciprocal of Eq. 11.8,and by plotting a graph of 1/r versus 1/[D], a straight line is obtained as shown in Fig. 11.2,this graph is called a double reciprocal plot. The number of binding sites can be obtained from the y-intercept which is 1/n,andnKa value can be obtained from the slope of the curve which is 1/nKa. In cases where the graph obtained by plotting 1/r versus 1/[D]doesnot give a straight line, then this can be a result of the presence of more than one independent binding site (each with its own association constant Ka)(Shargel and Andrew, 2015).1 r 51 1 nKa½D1 n (11.13) Another commonly used graphical method is the Scatchard plot. By rearranging Eq. (11.8), Eq. (11.14) is obtained, and by plotting, r/[D](or[DP]/[D]) versus r gives a straight line as shown in Fig. 11.3. The association constant Ka can be obtained from the slope which is 2Ka and number of binding sites n is obtained from the y-intercept. FIGURE 11.1 General graphical representation of a direct plot.
The Scatchard plot gives a straight line if there is one type of binding site and no cooperativity, while a curvature in the curve is observed if there is more than one type of binding site (Zhivkova, 2015). r ½D5nKa2rKa 11.3 OVERVIEW OF PLASMA PROTEINS (11.14) The human plasma contains many different proteins that are responsible for various functions such as transport of endogenous biomolecules, immunity, blood coagulation, etc. However, some of these plasma proteins are capable of binding to drug molecules, and the binding is usually reversible by noncovalent interactions such as electrostatic interactions and hydrophobic interactions. Although the plasma is known to have more than 60 different proteins, only a few of them are considered important in the drug binding phenomena (Putnam, 2012). The human serum albumin which constitutes about half of the total plasma proteins is of particular importance in drug binding; in addition to albumin, alpha-1-acid glycoprotein and lipoproteins also have a specific significance in drug binding in plasma. Acidic drugs bind to albumin while basic and neutral drugs bind to alpha-1-acid glycoprotein (although there are exceptions). The characteristics of these three-major drug-binding proteins are summarized in Table 11.1. It should be noted that other plasma proteins such as transthyretin and antibodies can also participate in drug binding, but usually, they have affinities for specific drugs only (Howard et al., 2010; Han et al., 2010). 11.3.1 Albumin Human serum albumin is the major component of plasma proteins that is synthesized by the liver and has many essential functions, for instance, it acts as a transporter protein for various endogenous molecules and metals such as fatty acids, bilirubin, and calcium ions. Also, it plays a significant role in the maintenance and regulation of plasma colloidal pressure (Fanali et al., 2012). Human serum albumin can also be used as a biomarker for the diagnosis of various diseases. Structurally, albumin is a single polypeptide chain composed of 585 amino acids with a molecular weight of 66.7 kDa, usually nonglycosylated. The 3D structure of albumin has been determined using X-ray crystallography which can be seen as a globular heart-shape and the structure consists of three homologous domains (designated as domain I, II, and III); each of these three domains contains two subdomains (designated as A and B) as shown in Fig. 11.4. The structural organization of albumin is responsible for its high ability to bind a wide range of molecules, and it can bind nine equivalents of fatty acids. There are mainly two major binding sites in albumin that bind to drugs with high affinity (in addition to the presence of other binding sites that bind drugs with low affinity), these sites are named siteI(alsocalledwarfarinbindingsite)andsiteII(alsoknownasbenzodiazepambindingsite).AnotherbindingsitethatisnotentirelyelucidatedissiteIII,whichcanspecificallybindtodigoxin.Humanserumalbuminhasbeenshowntobindmainlyacidicandneutraldrugs,butitmayalsobindbasicdrugssuchasdiazepam.Table11.2showssomeTABLE11.2ImportantDrugsthatBindAlbuminwithTheir,BindingSite,BindingConstant,andChemicalStructureRespectively important drugs that bind albumin binding sites along with their respective binding constants (Yamasaki et al., 2013; Anguizola et al., 2013). Nowadays albumin is also utilized as a polymer to formulate nanobased drug delivery system (Tekade et al., 2015). An important factor to consider regarding the binding process of drug molecules to albumin and other plasma protein, in general, is the stereoselective binding of chiral drugs (Shen et al., 2013). Since albumin and other plasma protein are the chiral molecules, they can have different binding affinities to chiral drug molecules (Li and Hage, 2017). For example, during the study of albumin binding to the (S)-and (R)-enantiomers of the drug amlodipine, it was found that albumin binds the (S)-enantiomer of amlodipine with higher extent than binding with the (R)-enantiomer of amlodipine (Maddi et al., 2010). Finally, the ability of albumin to bind different drugs can be utilized to extend the drug’s half-life by adjusting its binding to albumin. Extension of a drug’s half-life can be beneficial for various purposes such as enhancing the patient compliance (Sleep et al., 2013). 11.3.2 Alpha-1-Acid Glycoprotein The alpha-1-acid glycoprotein is considered to be another major plasma protein that can bind many drugs, in spite of the fact that it has the lower concentration than albumin. An alpha-1-acid glycoprotein is a glycoprotein that is composed of a single polypeptide chain that is 183 amino acids length, with a molecular weight of about 41 kDa; the alpha-1-acid glycoprotein is extensively glycosylated with B45% of its components being carbohydrates. This high degree of glycosylation gives the alpha-1-glycoprotein a net negative charge at pH B7 and a high degree of water solubility. The 3D structure of alpha-1-acid glycoprotein is shown in Fig. 11.5. An alpha-1-acid glycoprotein is an acute phase protein, which means its concentration increases in response to conditions such as inflammation, infection, and injuries (Tesseromatis et al., 2011; Huang and Ung, 2013). The functions of alpha-1-acid glycoprotein are not completely well-understood. Although it has been shown that it acts as immunomodulation agent, it also acts as platelets aggregation inhibitor. Clinically, it can be used as a biomarker for some diseases (Bachtiar et al., 2010; Ren et al., 2010). Alpha-1-acid glycoprotein polymorphism can play a significant role in drug binding to alpha-1-acid glycoprotein which accounts for individual variations in drug binding level. Alpha-1-acid glycoprotein binds neutral and basic drugs,
examples of such drugs include diazepam, disopyramide, and chlorpromazine. Although alpha-1-acid glycoprotein binds mainly to basic and neutral drugs, studies have shown that it is capable of binding acidic drugs as well in some cases (Ascenzi et al., 2014). 11.3.3 Lipoproteins Since lipids are relatively insoluble in the aqueous media of plasma, they are usually transported in association with proteins in complexes called lipoproteins. Lipoproteins consist of both hydrophilic and hydrophobic portions. The hydrophilic portion typically consists of apoproteins and the hydrophilic part of phospholipids and cholesterol points outwards, interacting with the surrounding aqueous media. The hydrophobic portion consists of triglycerides and cholesteryl esters and is buried away from the aqueous media. Thus, lipoproteins act as soluble transporters of lipids in the circulation, and they also play an important role in lipid metabolism (Bishop et al., 2010, Voet and Voet, 2011). The general structure of lipoproteins is shown in Fig. 11.6. Based on their density, lipoproteins can be classified into four groups, namely chylomicrons, very low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs). Table 11.3 shows the general characteristics of lipoprotein classes (Voet, and Voet, 2011). As expected, lipoproteins bind mainly lipophilic substances. Drugs that bind to lipoproteins can sometimes show selective binding only to specific classes of lipoproteins, such as amphotericin B which binds only to LDL and eritoran which binds to HDL. Other drugs such as halofantrine can bind both HDL and LDL, while some drugs such as amiodarone can bind to all classes of lipoproteins (Ascenzi et al., 2014). Because of the characteristics of lipoproteins such as their biocompatibility, circulation half-life, particle size, and the ability to incorporate hydrophobic substances into them, they can be utilized as drug delivery systems. It is also possible to attach a tethering molecule to the protein portion of the lipoprotein which can lead the molecule to specific cells (e.g., cancer cells) to selectively target such type of cells (Huang et al., 2015; Thaxton et al., 2016; Jia et al., 2012). 11.4 TISSUE BINDING The tissue binding of drugs is as important as the plasma proteins binding and has a significant impact on the pharmacokinetics and pharmacodynamics of the drug as it affects both the distribution and elimination processes of the drug in addition to affecting the pharmacological effect of the drug. Generally, the greater the free fraction of the drug, the higher the rate of elimination, while extensive tissue binding will result in a lower elimination rate and a high volume of distribution. The pharmacodynamics of the drug is affected because only the unbound fraction of the drug can distribute to its target site and interact with its target to give a pharmacological effect. Following drug distribution phase, equilibrium will be established between the drug concentration in the plasma and the concentration in tissues. The ratio of tissues concentration to plasma concentration will be equivalent to the unbound (free) fraction of the drug in plasma (fup) to the unbound (free) fraction of the drug in tissue (fut) ratio (Shargel and Andrew, 2015). Tissue binding of drugs is different according to the type of tissue because drugs usually have different affinities for different kinds of tissues. And sometimes, binding of a drug in a particular organ is not homogenous, i.e., the drug concentration in a specific site in an organ is greater than the concentration in another site in the same organ. The liver is the organ with which most of the drugs show highest binding affinities regarding tissues binding, other organs with which drugs are found to bind to a lesser extent are the kidney and the lungs. There are many different components of tissues with which a drug may interact including macromolecules of the cell such as proteins, e.g., tubulin, actin, and myosin. Measurement of tissue binding of drugs is more difficult than the measurement of plasma protein binding, because of the difficulty of measuring drug unbound and bound concentrations inside the tissues, as this usually requires invasive techniques. However, various new methods have been developed to estimate drugs tissue binding such as in vivo microdialysis which as a semi-invasive method used to determine drug tissue binding (Kotsiou and Tesseromatis, 2011).
5 PLASMA AND TISSUE PROTEIN BINDING IMPLICATIONS ONPHARMACOKINETICS PARAMETERS The extent to which a drug binds to plasma protein will have major consequences on its pharmacokinetic profile. As only the unbound (free) drug is able to diffuse across cell membranes to enter inside the tissues, the drug bound to plasma proteins will not be able to diffuse into tissues, which means that the distribution process will be affected by protein binding of the drug, and the volume of distribution depends on both the plasma proteins and tissue binding of the drug. Since the elimination process of the drug happens inside tissues such as the liver and kidney, the clearance and the half-life of the drug will be affected by the binding of drugs to plasma protein. Other pharmacokinetic parameters such as bioavailability can also be affected by protein binding, although in some cases, protein binding does not seem to affect it. The Fig. 11.7 provides a schematic representation of the effects of plasma protein binding on the pharmacokinetics of the drug (Lambrinidis et al., 2015; Bohnert and Gan, 2013). 11.5.1 Bioavailability Bioavailability (F) is the extent of active drug that reaches the systemic circulation after administration of the drug into the body. Thus, the bioavailability of a drug is affected by several factors such as the route of administration. For example, drugs administrated intravenously will have 100% bioavailability because the active drug is introduced into the systemic circulation. While drugs administrated orally usually have lower bioavailability because an absorption process must take place prior to reaching the systemic circulation. Also, drugs administrated orally are prone to presystemic clearance. For example, they can be metabolized to inactive metabolites in the first-pass metabolism process in the liver or intestinal (gut) wall and consequently will have lower bioavailability. In cases where the first-pass metabolism in the liver is the predominant presystemic clearance mechanism (high extraction ratio drugs), the bioavailability (F) is determined by Eq. (11.15): F512E512 QH fup3CLint: (11.15) In Eq. (11.15), E is the hepatic extraction ratio for the drug, CLint. is the intrinsic clearance of the liver, fup is the unbound (free) fraction of the drug. Thus, the bioavailability is inversely proportional to the unbound (free) fraction of the drug, and in such cases, drugs having higher protein binding are expected to have higher bioavailability (assuming no differences in other factors affecting bioavailability). In cases where the drug has negligible first-pass metabolism by the liver (low extraction ratio drugs), the bioavailability is considered as independent from protein binding (Han et al., 2010). Recently many novel pharmaceutical formulations have been tried to increase the bioavailability problems associated with drug molecules (Rahul et al., 2017; Maheshwari et al., 2012). 11.5.2 Volume of Distribution The volume of distribution (Vd) can be defined as a theoretical volume that is used to relate the total amount of drug in the body and the plasma concentration of the drug. Since the volume of distribution is a theoretical volume, it does not have a real physiological volume, and it is used to estimate the drug distribution in the body. For drugs that diffuse readily across capillaries to extravascular space, their volume of distribution will be high, while for drugs that do not diffuse readily across capillaries and remain confined to intravascular space, their volume of distribution will be low. The factors that affect the volume of distribution include the ability of the drug molecules to diffuse to the extravascular space and entering the tissue cells which is mainly determined by the physicochemical properties of the drug such as lipophilicity, size, and charge. Lipophilic molecules can diffuse readily across cell membranes and consequently will have larger volumes of distribution while hydrophilic molecules usually do not diffuse easily across the cell membranes and will have lower volumes of distribution, although the availability of transporter proteins for some molecules is an exception to this process. Binding to plasma proteins is also an important factor that influences the volume of distribution. Since the drugprotein complex formed is significantly large to cross the cell membranes, binding to plasma proteins will restrict the bound molecule to the intravascular space and prevent their diffusion. Similarly binding to tissues will restrict the drug molecules from distributing back to plasma and confine them to the tissue space (Liu et al., 2011; Kotsiou, and Tesseromatis, 2011). The volume of distribution is affected by both the binding to plasma proteins as well as to tissues according to the Eq. (11.16): Vd5Vp1fup fut 3Vt (11.16) where fup is the unbound (free) fraction of the drug in the plasma, fut is the unbound fraction of the drug in the tissues, Vp and Vt are the plasma volume (B0.07 L/kg) and tissue volume, respectively. In cases where the drug can diffuse across the cell membranes, then as Eq. (11.16) describes, if fup is high, then the drug will distribute to tissue, and the volume of distribution will be large. And if the if fut is high, then the drug will distribute from the tissue back to the plasma and will have low volume of distribution. Assuming similar physical properties for a series of drugs (e.g., drugs from the same family having the same scaffold), the plasma protein binding can explain the differences in the volume of distribution for these drugs. Drugs that are highly bound to plasma protein will not diffuse across cell membranes and thus will have a lower volume of distribution, while drugs that are not bound to plasma proteins will diffuse to the tissues and will have a higher volume of distribution. For example, the difference in the volumes of distribution for a series of four cephalosporin drugs is explained by the difference in their plasma protein binding. The highest protein bound (lowest fup) drug is cefazolin, which has the lowest volume of distribution; on the other hand, the lowest protein bound (highest fup) drug is cefoperazone which corresponds to the highest volume of distribution among the four compared drugs (Shargel and Andrew, 2015). 11.5.3 Hepatic Clearance The liver is the major organ for the metabolism of drugs, and it contains enzymes that are responsible for the biotransformation of drugs, which results in inactive metabolites that are subsequently excreted (although in cases, some metabolites can be active or toxic). The hepatic clearance can be defined as the volume of blood (or plasma) that passes through the liver that is cleared from a substance per unit of time. The following Eq. (11.17) can be used to determine the hepatic clearance: CLhepatic 5 QH3fub3CLint: QH1fub3CLint: (11.17) In Eq. (11.17), QH is the hepatic blood flow, E is the extraction ratio, fub is the unbound (free) fraction of the drug in blood, and CLint. is the intrinsic clearance of the liver. The extraction ratio E is the fraction of drug that is cleared by the liver relative to hepatic blood flow. Generally, the extraction ratio for drugs can be divided into three classes, high extraction ratio (E.0.7), moderate extraction ratio (E50.30.7), and low extraction ratio (E,0.3). The other factor that is important in hepatic clearance is the hepatic blood flow QH, which is about 90 L/h for healthy individuals, but the hepatic blood flow is subjected to a decrease in some conditions such as liver diseases (e.g., cirrhosis) or diseases affecting blood circulation (e.g., heart failure). The intrinsic clearance represents the metabolic capacity of the liver enzymes which is a crucial factor in the hepatic clearance, but in some conditions, these enzymes can be inhibited by some drugs or other substances which would affect the hepatic clearance of drugs metabolized by these enzymes. Since the drugprotein complex formed in the plasma protein binding process is considered as large to diffuse across membranes and is unavailable for metabolism by other enzymes, it is assumed that only the unbound (free) fraction of the drug is metabolized by the liver. Thus, the protein binding is a factor that should be taken into consideration when determining the hepatic clearance (Schmidt et al., 2010; Liu et al., 2014).
1 Restrictive and Nonrestrictive Clearance Drugs that undergo hepatic clearance are classified as being either restrictively cleared drugs or nonrestrictively cleared drugs. For drugs that bind strongly to a protein, then only the unbound (free) fraction of the drug will be available for metabolism. Such drugs are classified as restrictively cleared drugs, these drugs usually have extraction ratio E that is smaller than their unbound (free) fraction, and the product of the unbound (free) fraction as well as the intrinsic clearance Clint. are significantly lower than the hepatic blood flow QH, which allows for the simplification of Eq. (11.17)toEq. (11.18): CLhepatic 5fup 3CLint: (11.18) Examples of restrictively cleared drugs include the oxicams such as isoxicam, tenoxicam, and piroxicam; these drugs have extraction ratio E lower than their unbound (free) fraction fup, and therefore they undergo restrictive hepatic clearance. On the other hand, for drugs which undergo hepatic clearance even though they are highly bound to proteins, they are classified as nonrestrictively cleared drugs. Drugs in this class have extraction ratio E higher than their unbound (free) fraction and the product of the unbound (free) fraction and the intrinsic clearance CLint. are significantly higher than the hepatic blood flow QH, which allows for the simplification of Eq. (11.17)toEq. (11.19): CLhepatic 5QH (11.19) The Eq. (11.19) shows that clearance of nonrestrictively cleared drugs is independent of protein binding and depends on the hepatic blood flow. An example of a nonrestrictively cleared drug is the beta blocker propranolol, which is highly protein bound but its extraction ratio E is higher than the unbound (free) fraction, which makes it a nonrestrictively cleared drug, it undergoes extensive hepatic metabolism (Schmidt et al., 2010). 11.5.4 Renal Clearance Renal clearance is the volume that is cleared from a substance by the kidney per unit of time. Renal clearance of drugs that are eliminated by the kidney can be calculated by Eq. (11.20): 12Fr ðÞ3Filtration rate1Secretion rate ðÞ CLrenal 5 Concentration in plasma (11.20) In Eq. (11.20), Fr is the reabsorbed fraction of the drug. In the case where no active secretion or tubular reabsorption takes place, and glomerular filtration is the only mechanism for clearance by the kidney, then Eq. (11.20) can be simplified to Eq. (11.21): CLrenal 5fup 3GFR (11.21) In Eq. (11.21), GFR is the glomerular filtration rate (generally B120 mL/min). Renal clearance decreases as protein binding increases because the drugprotein complex is too large to diffuse across the glomeruli capillary membranes. However, for drugs that are actively secreted, then the protein binding may be insignificant if the transporter has a higher affinity for the drug than the binding protein (Han et al., 2010).
11.5.5 Half-Life The half-life (t1/2) of a drug is the time required for the drug concentration to drop by one-half (50%). Two primary processes are important in determining the half-life of a drug which are the mechanism of elimination (metabolism and/or excretion) and the rate of drug movement from plasma into the tissue, and the t1/2 can be calculated by Eq. (11.22). t1=2 50:6933 Vd CL (11.22) As Eq. (11.22) shows, the half-life of a drug is dependent on both the volume of distribution and clearance, and since both these parameters are affected by protein binding, the half-life will be affected by protein binding as well. Thus, because of that, it is difficult to predict the effect of altered protein binding on half-life directly. In general, drugs with higher tissue binding will have higher half-lives (Kotsiou, and Tesseromatis, 2011). 11.5.6 Drug Plasma Concentration-Time Profile When the rate of drug input is equal to the rate of drug elimination, the drug has reached the steady state. The total average steady-state concentration (Css(total)) is determined by the bioavailability of the drug, the dosing interval, and the clearance. Eq. (11.23) can be used to calculate the total average steady-state concentration (Css(total)): CssðtotalÞ 5 F3D CL3τ (11.23) In Eq. (11.23), F is the bioavailability, τ is the dosing interval, D is the dose administered, and CL is the clearance. The unbound free average steady-state concentration (Css(free)) which is considered more important, can be calculated by using Eq. (11.24). CssðfreeÞ 5 fup 3F3D CL3τ (11.24) where fup is the unbound (free) fraction of the drug in plasma. It is apparent from Eq. (11.23) and Eq. (11.24) that the total and unbound (free) average steady-state concentrations depend on the route of administration (which affect bioavailability) and clearance. If the hepatic clearance is the primary elimination method for the drug, then the effect of protein binding on the total and free (unbound) average steady-state concentrations depends on whether the drug has high or low extraction ratio E. If the drug has a high extraction ratio and the drug is administrated parenterally, then protein binding will affect the unbound (free) average steady-state concentration as described by Eq. (11.25). While protein binding will be insignificant for high extraction drugs that are administrated orally as shown in Eq. (11.26). CssðfreeÞ 5 fup 3D CL3τ 5 D QH3τ CssðfreeÞ 5 fup 3Foral 3D CL3τ 5 D CLint: 3τ (11.26) If the renal clearance is the primary clearance method for the drug (assuming only glomerular filtration) then a decrease in protein binding (an increase in the unbound (free) fraction (fup) of the drug in plasma) will enhance the clearance of the drug which will result in a decrease in the total average steady-state concentration (Css(total)) as shown in Eq. (11.27). On the other hand, changing protein binding will not affect the free average steady-state concentration (Css(free)), because changing protein binding will not influence the clearance of free drug as depicted in Eq. (11.28)(Schmidt et al., 2010). CssðtotalÞ 5 CssðfreeÞ 5 F3D CL3τ 5 F3D fup 3GFR3τ fup 3Foral 3D CL3τ 5 F3D GFR3τ 11.6 FACTORS INFLUENCING PROTEIN BINDING (11.27) (11.28) Factors that can cause alterations in protein binding of drugs can be divided into three classes: physiologic conditions; pathologic conditions; and drug-induced changes. The alteration in protein binding can be caused by these physiologic and pathologic conditions that can occur by various mechanisms. The most common ones are changes in the binding protein concentration, changes in an endogenous substance concentration that binds the binding protein, or a change in the structure of the binding protein (e.g., glycosylation of albumin in diabetic patients) that would affect the binding affinity of the protein (Kotsiou and Tesseromatis, 2011; Joseph and Hage, 2010; Rondeau and Bourdon, 2011). Drug-induced change in binding can also occur by one of these mechanisms, but the most common one is by direct displacement of the drug form the binding protein by competing with it for the binding site of the binding protein (Heuberger et al., 2013). 11.6.1 Physiologic Factors Influencing Protein Binding Physiologic factors that can influence drugprotein binding include age, gender, pregnancy, and others. Since concentrations of both albumin and alpha-1-acid glycoprotein in neonates are lower than adults and the higher concentrations of fatty acids and bilirubin which are both normal substances that bind albumin, drug-binding to albumin and alpha1-acid glycoprotein is lower in neonates. Albumin concentration in the elderly is also lower compared to normal adults. Also, a higher percentage of glycosylated albumin is found in the elderly which accounts for the observed decreased binding of albuminbinding drugs. Another physiologic factor is gender, the concentration of both albumin and alpha-1-acid glycoprotein are found to be lower in adult females (nonpregnant) as compared to adult males which explains the slight decrease in the bound fraction of some drugs. During pregnancy (mainly in the third trimester), alterations in the binding of albumin-binding drugs can occur. Since albumin concentration is lower and there is an increase in the concentration of free fatty acids, the binding of albumin-binding drugs is reduced, and higher unbound fractions are observed (Fanali et al., 2012; Hanley et al., 2010; Tesseromatis et al., 2011). 11.6.2 Pathologic Factors Influencing Protein Binding Many pathologic conditions can influence drugprotein binding. Several of these cause a change in the concentration of a binding protein(s), e.g., conditions characterized by a lowered plasma albumin concentration, such as hypoalbuminemia or analbuminemia, cause an increase in the unbound fraction of albumin-binding drugs, while hyperalbuminemia (which is relatively rare) is associated with increased plasma albumin concentration and thus an increase in the bound fraction of albumin-binding drugs. On the other hand, pathologic conditions can alsocauseanincreaseinanendogenoussubstance concentration that binds the binding-protein, such as the increased bilirubin concentration in hyperbilirubinemia, where bilirubin binds to albumin and causes an increase in the unbound fraction of albumin-bound drugs (Gatta et al., 2012; Dagnino et al., 2011). In a similar fashion, unbound fractions of alpha-1-acid glycoprotein-binding drugs will be affected by pathologic conditions that alter the concentration of alpha-1-acid glycoproteins, e.g., hepatitis, hepatic cirrhosis, pancreatic cancer, and nephrotic syndrome are diseases associated with a decrease in alpha-1-acid glycoprotein concentration and thus, an increase in the unbound fraction of alpha-1-acid glycoprotein-binding drugs. On the other hand, since alpha-1-acid glycoprotein is an acute phase protein, conditions that can result in an acute phase response will cause an increase in alpha-1-acid glycoprotein, which may result in reduced unbound fraction of alpha-1-acid glycoprotein-binding drugs that may require a dose adjustment, such pathological conditions include cancer and myocardial infarction (Stangier et al., 2010; Vivekanandan-Giri et al., 2011). Drugs binding to lipoproteins can also be affected by pathologic conditions, as changes in free fractions of lipoproteins-binding drugs have been observed in dyslipidemia (Anger and PiquetteMiller, 2010; Franssen et al., 2011). 11.6.3 Drug-Induced Changes in Protein Binding The coadministration of one drug can affect the protein binding of another administrated drug and thus change its unbound fraction. The mechanism by which one drug can alter the binding of another drug to a plasma protein is through direct displacement of the drug from the binding site. If the competing drug has a higher affinity for the protein, then it will displace the other one from the binding site and thus increase its unbound fraction which may lead to toxicity. For example, in patients taking the anticoagulant drug warfarin, the coadministration of phenylbutazone that binds to the same binding site of warfarin in albumin will cause displacement of warfarin from the binding site, which in turn may result in an increased free fraction of warfarin and an increase in prothrombin time with increased risk of bleeding. Another example is the coadministration of sulfonamides with tolbutamide which is a hypoglycemic agent, because the sulfonamides displace the hypoglycemic agent tolbutamide, increasing the free fraction. An increased hypoglycemic effect was observed in patients taking tolbutamide in addition to sulfonamides. However, mechanisms other than protein binding alterations have been observed in explaining the drugdrug interactions between these drugs, such as alterations of the metabolism and excretion. The displacement of a drug can occur by the coadministrated drug or by one of its metabolites. Other mechanisms by which one drug can affect the protein binding of another drug is by causing a conformational change in the binding protein. In this case, the drug binds to an allosteric site in the binding protein and causes a conformational change in the protein which will modify the active site shape, thus reducing the affinity of binding to another drug (Fanali et al., 2012; Ansari, 2010; Hines and Murphy, 2011). 11.7 PLASMA PROTEIN BINDING DETERMINATION METHODS Various analytical methods and techniques are used to determine plasma protein binding, each of these methods have its own advantages and disadvantages, and they also vary in their cost, ease of use, and the ability to measure a little fraction of the unbound (free) fraction of the drug. The most common methods used in determining plasma protein binding are equilibrium dialysis, ultrafiltration, and ultracentrifugation, all of these of methods are conducted in vitro. Equilibrium dialysis is the most commonly used method and is regarded as the gold standard method for determining the plasma protein binding of drugs. The choice of method to use depends on various factors such as the discovery stage of the drug, as drugs in advanced development stages require more accurate details regarding their plasma protein binding. For example, determining the initial dose in clinical trials in phase I requires taking the plasma protein binding of the drug candidate into consideration and thus, sufficient data regarding plasma protein binding is required before clinical studies. Other factors affecting the choice of method to imply when studying the binding of a drug to plasma protein include the physicochemical properties of the drug because some properties can pose problems in measuring leading to inaccurate results. For example, compounds with high absorptivity can adsorb to various parts of the device being used which would give false results regarding their unbound (free) fraction. Another important property is the solubility of the compounds to be analyzed, as compounds with low solubility in the medium being used can pose a problem in the measurement process. Some of these problems can be alleviated by the proper choice of the method. There are also various software products commercially available that can predict the plasma protein binding of drugs. Usually these software predict the ADMET properties of the compounds which include the plasma protein binding, although there are software that are designed specifically to predict plasma protein binding only, and some are designed to predict the binding to a specific protein only, such as binding to albumin (Zhang et al., 2012; Howard et al., 2010).
11.7.1 Equilibrium Dialysis Method The equilibrium dialysis method is the most frequently used method for determining the plasma protein binding of drugs. This method depends on the physical separation of the bound and unbound (free) fractions of the drug using a semipermeable membrane and then measuring the unbound (free) fraction of the drug by using a proper analytical method. The device used in equilibrium dialysis consists of two chambers separated by a semipermeable membrane as shown in Fig. 11.8. The solution containing the protein (usually serum) and the drug to be analyzed is added to one chamber, while a buffer solution is added to the other chamber. The semipermeable membrane allows the passage of the free drug molecules, while it is impermeable to the protein or the drug molecules bound to the protein. Thus, only the free drug molecules can freely diffuse across the membrane separating the two chambers. After equilibrium is established, the unbound (free) drug molecules concentration will be equal in both chambers, while the bound drug molecules are restricted to the chamber containing the protein solution. After measuring the total drug concentration in the chamber containing the protein solution and measuring the free drug concentration in the chamber containing the buffer solution, it is possible to calculate the bound drug concentration. The time required to reach equilibrium is different for each compound, generally higher molecular weight compounds require a longer time to reach equilibrium, compounds that are highly bound to proteins also tends to require a longer time to reach equilibrium. Long times required to reach equilibrium can pose problems which can lead to errors in the results, e.g., long times can lead to bacterial growth in the medium which can interfere with the binding or change the pH of the medium. One way to reach equilibrium faster can be done by adding the compound to the solution containing the protein and agitating it. Other disadvantages of the equilibrium dialysis method include nonspecific binding of drug molecules, leakage of the protein molecules through the semipermeable membrane, and volume shifts. The nonspecific binding of drug molecules occurs when the drug molecules bind to some parts of the device being used or bind the semipermeable membrane which would result in a lower value for the unbound (free) fraction of the drug. The nonspecific binding can be alleviated by the proper choice of the material of the device being used, as some materials can reduce nonspecific binding of drug molecules, however, in cases where nonspecific binding is too high, a different method should be considered which uses a different technique for separation, such as ultracentrifugation. The leakage of protein molecules occurs when the some of the protein molecules diffuse across the semipermeable membrane, this will happen if the membrane integrity is damaged. This would lead to a higher value of the unbound (free) fraction of the drug. Another potential problem is the volume shifts, which occurs when part of the buffer solution is transferred to the protein solution chamber because of the colloidal osmotic pressure, resulting in a diluted protein solution and causing an alteration in the equilibrium. A long time is required to reach an equilibrium which can potentiate volume shift. This problem can be alleviated by considering it during the calculation of the unbound (free) fraction of the drug. An alternative way is to add dextran to the solution of a buffer, which would counteract the effect of colloidal osmotic pressure. However, binding of a drug to dextran can give false results, thus it should be confirmed that dextran does not interfere with the binding of the drug. In cases where the volume shifts are of low values, they can be considered negligible (Howard et al., 2010; Ye et al., 2017; Vuignier et al., 2010; Van Liempd et al., 2011). 11.7.2 Ultrafiltration Method The ultrafiltration method is another method that is frequently used in measuring protein binding of drugs, it shares several characteristics of the equilibrium dialysis method but offers numerous advantages over the equilibrium dialysis method. The ultrafiltration method uses a device composed of two chambers, the upper and the lower chamber, with a semipermeable membrane in-between them, as shown in Fig. 11.9. The solution containing the drug and the protein is added to the upper chamber of the device (the total concentration of the drug in the solution is determined prior to addition to the upper chamber) and then the solution is allowed to be filtered through the membrane to the lower chamber of the device. The driving force for filtration of the solution is usually positive pressure or centrifugation. The unbound (free) drug passes through the membrane to the lower chamber and is then measured, then the bound fraction of the drug can be calculated from the total drug concentration and the unbound (free) drug concentration in the lower chamber. The advantages that the ultrafiltration method has in comparison to the equilibrium dialysis method include the ease of use, as this technique is more simple and rapid than equilibrium dialysis method. Also, several problems encountered in equilibrium dialysis such as the passage of proteins across the semipermeable membranes and volume shifts are reduced using the ultrafiltration method. The disadvantages of the ultrafiltration methods include the difficulty to control the temperature and pH during the experiment, possible protein leakage across the semipermeable membrane and the permeability of the membrane to the drug and the plasma water. As in some cases, the permeability of the membrane for the water is different than the permeability for the drug molecules. For example, highmolecular-weight drugs may pass across the membrane at a lower rate than the water molecules which can result in a lower value of the unbound (free) fraction of the drug. The most significant disadvantage associated with the ultrafiltration method is the nonspecific binding of the drug molecules, as drug molecules can bind to the semipermeable membrane or the lower chamber which would result in a lower value of the unbound (free) fraction of the drug during measurement. Also, this issue is found to be more significant with the more lipophilic drugs. Various solutions to reduce nonspecific binding of drugs have been proposed, e.g., the pretreatment of the semipermeable membrane with Tween 80 (when the drug being tested is acidic or neutral) or with benzalkonium chloride (when the drug being tested is basic), this can substantially reduce nonspecific binding. Another way is the proper choice of the semipermeable membrane material, as some materials show reduced nonspecific binding in comparison with others (Wang and Williams, 2013; Howard et al., 2010). 11.7.3 Ultracentrifugation Method The ultracentrifugation method does not depend on the separation of the unbound and bound drug fractions by a semipermeable membrane as seen with the equilibrium dialysis and the ultrafiltration methods. Instead, the ultracentrifugation method uses centrifugation force to separate the unbound and the bound drug fractions. The plasma sample containing the drug is allowed to be centrifuged, which would divide the plasma sample into three layers. The upper layer contains VLDLs and chylomicrons, the middle layer is the aqueous layer, and the lower layer contains the plasma proteins such as albumin and alpha-1 acid glycoprotein, in addition to the lipoproteins (HDL and LDL). The drug is measured in each of these layers which were extracted prior to analysis if necessary by methods such as liquidliquid extraction. The drug in the lipoproteins and plasma protein layers represents the bound fraction of the drug, while the drug in the aqueous (middle) layer represents the unbound (free) fraction of the drug. The total drug concentration in the plasma is determined before centrifugation. From these measurements, it is possible to calculate the plasma protein bound drug percentage. The main advantage of the ultracentrifugation method is the avoidance of nonspecific binding of drug molecules that is seen with methods using a semipermeable membrane for separation such as equilibrium dialysis and ultrafiltration which makes the ultracentrifugation method as the method of choice for compounds with high absorptivity. A disadvantage of the ultracentrifugation method is the high cost of the device, and also the device is considered more complex than the devices used for the equilibrium dialysis and the ultrafiltration method. Another disadvantage is the limited number of samples that can be tested at the same time. Also, it is hard to maintain physiological conditions during centrifugation, e.g., even if the pH at the beginning of centrifugation is 7.4, an increase in this value is observed during centrifugation (Zhang et al., 2012). 11.7.4 Important Considerations When Using In Vitro Methods Different parameters should be controlled during the measurement of plasma protein binding of drugs. These include the concentration of the drug and proteins, temperature, and pH, respectively. Also, the stability of the drug should be evaluated. Also, the drug displacement should be considered during measurement. In some cases, a slight change in one of these parameters can give different results, e.g., a slight shift in the value of the pH or the temperature of the experimental conditions can give substantial differences in the results of the bound drug concentration. Another factor that should be considered is the possibility of the presence of an active metabolite of the drug that has a high plasma protein bound fraction, in such a case the drug will have a high half-life value because of this metabolite, which means it is necessary to measure the plasma protein binding for the active metabolites of the drug as well. An example of such drugs is tasosartan which is an angiotensin receptor II antagonist. One of the active metabolites of tasosartan is enoltasosartan which was found to be highly bound to plasma protein, explaining a prolonged effect of tasosartan because enoltasosartan is much more tightly bound to the plasma proteins, the structures of tasosartan and its metabolite enoltasosartan are shown in Fig. 11.10 (Howard et al., 2010; Li et al., 2010). 11.7.5 In Vivo Methods The in vivo methods involve the sampling of the unbound (free) drug directly from a biological fluid through a blood vessel or tissues. The in vivo microdialysis is a commonly used in vivo method to measure the unbound (free) drug from the extracellular fluid of various tissues. The method uses a microdialysis fiber semipermeable membrane and depends on the passive diffusion of the unbound (free) drug molecules across the membrane down their concentration gradient. The microdialysis is performed by inserting the microdialysis probe that is composed of a hollow fiber semipermeable membrane into the extracellular fluid of the tissue of which the unbound (free) drug is to be measured. The microdialysis probe is attached to an inlet and outlet tubing through which a physiologic buffer perfuses at a slow rate, and as the fluid perfuses, the unbound (free) drug molecules diffuse across the semipermeable membrane down their concentration gradient and flow in the outlet tube, then the drug can be measured. The microdialysis method is an important procedure in drug research and development. However, it is not very practical to be used routinely for the measurement of unbound (free) drug concentration (Zhang et al., 2012; Bulik et al., 2010). 11.7.6 In Silico Methods Because of the effects of the plasma protein binding on the pharmacokinetic profile of drugs, it is important to estimate it when selecting lead compounds during the drug discovery process, as many developed drugs with good pharmacodynamics fail to make it to the market because of poor pharmacokinetic profiles. There are variously developed in in silico approaches which can be a beneficial tool to estimate the plasma protein binding of lead compounds during drug discovery. These can be helpful in deciding the compounds to be developed or help in the design of drugs with the appropriate plasma protein binding property. There are two in silico models for the prediction of the binding of drugs to a plasma protein, the structure-based model, and the ligand-based model, although the usage of a model of a combination of both is possible. In the structure-based model, the crystal structure of the protein is required to study the binding of the drug to it. The 3D crystal structure is obtained using X-ray crystallography or other methods. The major plasma protein, albumin, and alpha-1-acid glycoprotein have been crystallized, and their 3D structures have been solved and are available at the protein data bank. The two common methods used in the structure-based model are molecular docking, and molecular dynamics, both of them aim to predict the binding of the drug to the protein by using the 3D structure of the protein. Both methods tried to find the best pose of the drug molecule inside the active site of the protein, and can even give an estimation of the binding affinity and free energy. Additionally, it also provides information about the intermolecular interactions involved between the protein and the ligand which can be useful in modifying the structure of the ligand to adjust the binding to a protein. However, since plasma proteins such as albumin are not the target protein for the drug, it is necessary to make sure that modifying the structure of the drug to adjust its binding to albumin does not significantly affect the binding to its actual target protein.
The ligand-based model depends on the development of quantitative structure property relationship models (QSPRs) (Moroy et al., 2012; Vallianatou et al., 2013). The models require a dataset of compounds with known binding to the plasma proteins, then using molecular descriptors (e.g., lipophilicity, topographical features, etc.) a model can be constructed to predict the binding of various compounds. Many QSPR models have been developed to predict the binding of drugs to albumin, some of them have good predictive power (Ghafourian, and Amin, 2013; Zhivkova and Doytchinova, 2012; Li et al., 2011). It is possible to use two more methods in studying the binding of a drug to plasma protein. Each in silico method has its own advantages and disadvantages, and the use of two more methods together can provide a better understanding of the binding process (Zhivkova and Doytchinova, 2012). 11.8 CONCLUSION The binding of drugs to plasma and tissue proteins has significant consequences on the pharmacokinetic parameters of the drug and subsequently will affect the pharmacodynamics of the drug. The major plasma proteins which drugs mostly bind are albumin, alpha-1-acid glycoprotein, and lipoproteins, respectively. The drugprotein complex formed is unable to diffuse across the cell membranes which limits its ability to distribute into tissues, and as a result, highly protein bound drugs will have a low volume of distribution. The elimination process is also affected since the protein bound drug will not be available for metabolism in the case of restrictively cleared drugs, while protein binding is insignificant in the case of nonrestrictively cleared drugs. The half-life of a drug is also affected by protein binding because it depends on the volume of distribution and the clearance both of which are affected by protein binding of the drug. Different factors influence the protein binding of drugs which can be divided into three categories such as physiological factors, pathological factors, and drug-induced changes in protein binding respectively. Various methods have been used for the determination of protein binding of the drug, each has its strengths and weaknesses, and sometimes using more than one method is required. In summary, the binding of a drug to plasma and tissue proteins has major consequences on the pharmacokinetics and pharmacodynamics of the drug and should be taken into consideration in the drug discovery and development process. Acknowledgment The author Pran Kishore Deb acknowledge the internal Philadelphia University Research Grant, Jordan (Project ID: 46/34/100PU) as a start-up financial support to his research group for the development of selective inhibitors of cyclooxygenase-2 (COX-2) enzyme. The authors would like to acknowledge Science and Engineering Research Board (Statutory Body Established Through an Act of Parliament: SERB Act 2008), Department of Science and Technology, Government of India for the grant allocated to Dr. Rakesh Tekade for research work on gene delivery and N-PDF funding (PDF/2016/003329) for work on targeted cancer therapy. Disclosures: There is no conflict of interest and disclosures associated with the manuscript.