Acs Jmedchem 8b01263

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Perspective
Clearance in Drug Design
Dennis Smith, Kevin Beaumont, Tristan S. Maurer, and Li Di
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01263 • Publication Date (Web): 03 Oct 2018
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Page 1 of 48 Journal of Medicinal Chemistry
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3 Clearance in Drug Design
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8 Dennis A. Smith1, Kevin Beaumont2, Tristan S. Maurer2, Li Di3*
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4 The Maltings, Walmer, Kent, CT14 7AR, UK.
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15 Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Cambridge, MA 02139, USA
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Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, CT 06340, USA
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24 Invited Perspectives
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26 Journal of Medicinal Chemistry
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31 September 28, 2018
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Journal of Medicinal Chemistry Page 2 of 48
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3 Abstract
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6 Due to its implications for both dose level and frequency, clearance rate is one of the most
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8 important pharmacokinetic parameters to consider in the design of drug candidates. Clearance
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10 can be classified into three general categories, namely metabolic transformation, renal excretion
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and hepatobiliary excretion. Within each category, there are a host of biochemical and
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15 physiological mechanisms that ultimately determine the clearance rate. Physiochemical
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17 properties are often indicative of the rate-determining mechanism, with lipophilic molecules
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tending toward metabolism and hydrophilic, polar molecules tending toward passive or active
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22 excretion. Optimization of clearance requires recognition of the major clearance mechanisms
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24 and use of the most relevant in vitro and in vivo tools to develop structure-clearance
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26 relationships. The reliability of methods to detect and predict human clearance varies across
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29 mechanisms. While methods for metabolic and passive renal clearance have proven reasonably
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31 robust, there is a clear need for better tools to support the optimization of transporter-mediated
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33 clearance.
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3 Introduction
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6 The importance of pharmacokinetics (PK) in the design of medicines has been well established1-
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8 . An effective drug needs to have an acceptable delivery route, sufficient access to the site of
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10 action and a residence time sufficient for the desired duration of action. Modulation of the
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potential PK of a candidate series is a key aspect of the modern medicinal chemistry strategy.
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15 Previously in this series, the importance of half-life and volume of distribution in drug
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17 disposition have been discussed4, 5. This perspective will examine the basic theoretical and
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practical considerations relevant to rational drug design aspects of clearance.
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24 Definition of Clearance
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26 Clearance is an important PK parameter to consider in both pharmaceutical research and clinical
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29 practice. It quantitates the irreversible removal of a drug from the measured matrix (typically
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31 blood or plasma). The key word in this definition is ‘irreversible’ which separates distribution
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33 into tissues (which is reversible) from clearance, where there is a permanent change to the
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molecule or removal from the body. The clearance parameter (CL) is one which links the
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38 measured concentration of the drug to the rate of elimination (Equation 1). The units of CL are
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40 in volume per time, reflecting the volume of blood or plasma from which drug is completely
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eliminated per time. While CL is typically constant, the rate of drug elimination is concentration
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45 dependent. This is the basis for the typical exponential (log-linear) decline in drug concentration
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47 with time.
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52 Rate of Elimination = CL × Concentration (Equation 1)
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58 3
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3 A typical plasma concentration versus time curve following intravenous (IV) administration is
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6 shown in Figure 1. This exemplifies the information required to calculate clearance. By
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8 measuring the concentration of drug in plasma over a time course, the area under the plasma
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10 concentration versus time course (AUC) can be calculated. This can be converted to clearance
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(CL) by relating to the dose through Equation 2.
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17 CL = Dose/AUC (Equation 2)
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22 In this theoretical example, the dose was administered by the IV route. Under these
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24 circumstances, the entire dose has been placed into the systemic circulation, which is where the
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26 PK is measured. Consequently, the clearance calculated from this experiment is often quoted as
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29 systemic clearance (CLs). In addition, the matrix of analysis was (as often) plasma and so can
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31 also be quoted as plasma clearance (CLp). If blood were measured, then clearance would be
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33 blood clearance (CLb). The latter distinction is important since, depending upon the blood-to-
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plasma ratio (Rb) of a compound, clearance can be different when measured in plasma or blood.
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38 This reflects the ratio of concentration in whole blood (blood cells + plasma) versus that in
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40 plasma alone. It is not to be confused with the concentration in blood cells versus that in plasma.
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The relationship between CLb and CLp is given by Equation 3.
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47 = ⁄ (Equation 3)
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52 As such, when Rb is unity (i.e., the drug distributes evenly between the blood cells and plasma),
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54 the CLp equals CLb. Certain drugs, such as UK-2246716, can specifically bind to components
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58 4
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3 within the erythrocyte and drive to high Rb. In these cases, the concentration of drug in blood is
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6 significantly higher than in plasma and CLb is lower than CLp. Therefore, blood is a more
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8 appropriate matrix to measure clearance than plasma. In the case of UK-224671, 89% of the
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10 compound resides in the blood cell and blood clearance is significantly lower than plasma
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clearance. Conversely, many acidic molecules are significantly more highly bound in plasma
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15 than in the blood cells as blood cells do not expressed high concentrations of acid binding
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17 albumin. Therefore, even though the unbound drug will distribute into the blood cell (assuming
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passive membrane permeability), the acid will predominantly reside on the albumin meaning that
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22 Rb values will collapse to the hematocrit (e.g., ~ 0.6). In these cases, CLb can be up to 2-fold
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24 higher than CLp.
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29 The analytical matrix chosen to measure during PK experiments is an important consideration.
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31 Measurement of plasma clearly entails discarding of the blood cell fraction, which is appropriate
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33 if Rb approaches unity. When drug extensively distribute into blood cells, then the majority of
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the drug in the blood sample will be discarded if plasma is the matrix of measurement. In these
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38 cases it is important to correct for Rb to account for CLb, since it is blood that circulates through
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40 organs, not plasma. If Rb is significantly greater than 1, measurement of blood rather than plasma
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should be considered. When Rb approaches 1 or below, it is recommended to measure plasma
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45 and correct the CLp to CLb using Rb.
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49 The route of drug administration is critical for clearance assessment. As stated above, the IV
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52 route guarantees that all of the administered dose reaches the systemic circulation (or the blood
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54 compartment) where it can be measured. For all other routes of measurement, there are potential
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3 barriers which can prevent drug molecules from reaching the systemic circulation. This is the
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6 case for oral administration (PO), where the dose is applied to the gastrointestinal (GI) tract and
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8 has to be absorbed and avoid first pass extraction by the gut wall and liver prior to reaching the
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10 systemic circulation. The AUC following PO is often lower than that following IV of the same
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dose due to incomplete absorption, first-pass intestinal extraction and/or hepatic extraction. The
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15 corollary to this is that, clearance following PO (CLpo) is most often higher than CLs. The
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17 relationship between oral clearance and systemic clearance relies on an understanding of oral
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bioavailability (F) and is explained by Equation 4.
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24 CLpo = CLs/F (Equation 4)
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29 Importance of Clearance in Drug Disposition
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31 Clearance is typically the most important PK parameter for a medicinal chemist to modulate in a
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33 chemical series. This is because clearance is a determinant of every other PK parameter of
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relevance in design, including half-life, oral bioavailability and efficacious dose.
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38 (a) Effect of Clearance on Half-life
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40 Together with volume of distribution (Vd), clearance governs the elimination rate (kel) of
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a drug and ultimately half-life (t1/2). Equations 5 and 6 provide a useful approximation of
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45 half-life from Vd and CL (which is exact in the case of a one compartmental PK model
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47 with linear clearance). From this relationship, a medicinal chemistry strategy that lowers
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49 systemic clearance within a series whilst maintaining volume of distribution should
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52 increase elimination phase half-life in a series.
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3 CL ≅ V × k (Equation 5)
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6 t/! ≅ ln2 × V ⁄CL (Equation 6)
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(b) Effect of Clearance on Oral Bioavailability
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13 Many small molecule drugs are administered by the oral route. If solubility and/or
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15 permeability are not optimal, absorption of the administered dose may be incomplete.
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17 During absorption, drugs are exposed to a host of metabolizing enzymes in the GI tract
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20 which can further extract a portion of the administered dose. Finally, the liver can also
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22 extract a portion of the administered dose by metabolism and/or transport due to the fact
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24 that drugs directly enter the liver via the hepatic portal vein before reaching systemic
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circulation. This process is termed hepatic first-pass extraction.
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31 If a drug is cleared from the blood by the liver, then the hepatic extraction is a key
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component of both oral bioavailability and clearance (Equations 7 and 8), where the
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36 terms are defined as: F, fraction bioavailable; Fa, fraction absorbed; Fg, fraction escaping
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38 gut wall extraction; Eh, hepatic extraction ratio; and Q, hepatic blood flow. As clearance
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40 of a drug approaches hepatic blood flow, hepatic extraction ratio approaches 1 and oral
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43 bioavailability approaches zero. Consequently, minimizing hepatic extraction by
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45 lowering clearance is a key component in optimizing oral bioavailability. Other
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47 parameters, such as solubility, permeability and transporter affinity, affect oral
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bioavailability.
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54 F = Fa × Fg × (1 - Eh) (Equation 7)
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3 Eh = CL / Q (Equation 8)
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8 (c) Effect of Clearance on Efficacious Dose
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10 The importance of clearance in the relationship between steady-state concentration and
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dose was recognized early by Wagner in 1965 (Equation 9)7, where Css,av is the average
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15 concentration at steady state (related to potency and target concentration) and τ is dose
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17 interval. Unlike the other determinant parameters, clearance optimization is expected to
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20 pay dividends in both the numerator (decreased CL) and denominator (increased F).
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22 Consequently, modulation of clearance has a direct effect on efficacious dose within a
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24 particular chemical series.
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29 Dose = Css,av x CL x τ / F (Equation 9)
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First Concepts and the Well-Stirred Model
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36 In the evolution of PK, the concept of clearance was an important milestone. Borrowing heavily
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38 from engineering, a relatively simple and generally relevant mathematical model was developed
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to describe the removal of drugs from the body. A single organ of clearance such as the liver can
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43 be envisaged as having a flow of drug through which is defined by the blood flow to the organ
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45 (Q), the intrinsic ability of the organ to clear drug independent of flow or binding limitations
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47 (CLint,u) and the unbound fraction (fu) of drug available for clearance (Equation 10)8-10. This is
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50 known as the well-stirred model of drug clearance. Although other mathematical models of
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52 clearance (e.g., parallel-tube and dispersion models) have been described, the well-stirred model
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3 has been shown to be broadly relevant and is perhaps the most commonly used and conceptually
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6 useful of the available models11, 12.
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10 CL = #Q × f% × CL&'(,% *,#Q + f% × CL&'(,% * (Equation 10)
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15 For an orally delivered and hepatically cleared small molecule, the well-stirred model equation
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17 (Equation 10) can be combined with the dose and oral bioavailability equations (Equations 7 and
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20 9, respectively) to produce a dose equation that neatly describes the fundamentals of clearance
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22 modulation in small molecule drug discovery (Equation 11).
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27 Dose = Css,av,u x CLint,u x τ / Fa / Fg (Equation 11)
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31 This equation relates dose to steady state unbound average concentration (Css,av,u), intrinsic
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34 clearance (CLint), dose interval (τ), fraction absorbed (Fa) and fraction escaping gut-wall
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36 extraction (Fg). In order to reduce the dose within a small molecule chemical series for orally
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38 administrated drugs, one can modulate one or a combination of the following properties:
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41 1) Increase the potency against the pharmacological target (decrease target unbound
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43 concentration, Css,av,u).
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45 2) Lower the rate of metabolism and/or uptake clearance by the liver (lower CLint,u)
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48 3) Give the compound more often (lower τ)
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50 4) Increase oral absorption (Fa) or fraction escaping intestinal extraction (Fg).
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52 For the purposes of this perspective on clearance, we have arrived at the fundamental parameter
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55 of unbound intrinsic clearance (CLint,u) that is the major driver for compound optimization in
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3 small molecule drug discovery. CLint,u is best described as the rate of removal of unbound drug
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6 by an organ (in this case the liver) in the absence of any blood flow or protein binding
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8 limitations. Equation 11 also points to two very important aspects of modulation of dose to
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10 improve drug disposition in a small molecule series. Firstly, CLint,u is an unbound parameter and
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so any assay designed to be used to investigate this parameter needs to correct for any binding in
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15 that experiment. Secondly, there is no factor in Equation 11 for fraction unbound in plasma,
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17 despite its presence in the equations used in the derivation. Simply put, fraction unbound has no
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impact on unbound steady-state average concentrations and therefore is not expected, generally
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22 speaking, to impact dose requirements (i.e., protein binding is not a parameter to optimize13).
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24 This aspect will be discussed in detail later. Overall, in order to improve dose via hepatic
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26 clearance within a chemical series, medicinal chemists should only focus on reducing CLint,u.
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31 Mechanisms of Clearance
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33 In order to be able to reduce CLint,u in a particular series, it is important to understand the
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mechanisms by which molecules within the series can be removed from the blood. The liver and
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38 the kidney are the two major organs of drug clearance. Passive and active processes, such as
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40 metabolism by enzymes and uptake/efflux by transporters, are the major mechanisms for drug
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clearance.
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47 Clearance by the Kidney
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49 The major purpose of the kidney is to produce urine in order to excrete the by-products of
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52 intermediary metabolism. Renal clearance is also route for excretion of drugs and metabolites.
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54 Urine is produced by filtration of blood at the Bowman’s capsule (glomerular filtration) which
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3 allows any molecules with molecular weight of less than 50 KDa to pass through into the
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6 proximal convoluted tubule. Any useful products (such as glucose) are reabsorbed from the
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8 proximal convoluted tubule and the balance of water and ions is modulated through the Loop of
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10 Henle, before the urine passes through the ureter to the bladder. Any drug that is not bound to
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plasma protein will also be filtered into the proximal convoluted tubule. The rate of filtration is
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15 dependent on the flow to the glomeruli (rate of glomerular filtration, GFR ~ 1-2 ml/min/kg in
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17 humans) and the unbound fraction of drug in blood (fu,b) as given by Equation 12.
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22 CLr = fu,b x GFR (Equation 12)
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26 In accordance with Fick’s law, passive reabsorption of filtered drug is driven by permeability
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29 and the chemical potential created by the near complete (~99%) reabsorption of filtrate over
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31 length of the nephron. As such, polar, poorly permeable drugs may be cleared at rates
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33 approximating the product of GFR and fu,b. At the other extreme, highly permeable drugs may
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be cleared at rates approximating 1% of the product of GFR and fu,b. Compounds can also be
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38 actively secreted to renal tubules by transporters (OATs, OCTs, P-gp) expressed at the proximal
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40 convoluted tubule that can remove drugs from the renal blood flow and deposit them into the
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urine for excretion. Depending upon the extent of reabsorption, this can result in CLr value
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45 exceeding the product of fu,b and GFR, with a theoretical upper limit approximating renal blood
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47 flow (RBF ~ 16 ml/min/kg in humans).
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52 Clearance by the Liver
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3 The liver is the major drug clearance organ. It is highly perfused receiving approximately 25% of
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6 the cardiac blood output. The architecture of liver allows for maximal mixing of the blood with
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8 the liver cells (hepatocytes). The hepatocytes possess an array of uptake/efflux transporters (e.g.,
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10 OATPs, P-gp, BCRP, and MRPs) and many drug metabolizing enzymes (e.g., CYPs and UGTs)
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that can promote hepato-biliary secretion or metabolic clearance of a drug (Figure 3).
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15 Hepatobiliary clearance is generally an active process, whereby a compound with relatively poor
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17 passive membrane permeability is recognized by a specific transporter on the sinusoidal (blood)
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side of the hepatocyte. Uptake of the drug into the hepatocyte is promoted by the transporter. The
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22 drug can then be actively secreted into the bile by bile canalicular transporters. By virtue of this
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24 mechanism, drugs can be rapidly cleared from blood and excreted in the bile.
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29 Alternatively, once a compound enters the hepatocyte (by active or passive processes) they can
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31 be subject to metabolism by CYPs, UDP-glucuronyl transferases or one of the many drug
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33 metabolising enzymes present in the hepatocyte. The metabolites formed by these enzymes can
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then be secreted into the bile (and excreted) or removed into the blood for excretion in urine.
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38 Due to the importance of the liver as a drug clearance organ, much work on optimizing in vitro
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40 assays using sub-cellular fractions has been completed. Liver obtained post mortem from
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animals and humans can be treated in different ways to provide a variety of cellular and sub-
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45 cellular fractions that can be used to study metabolism and transport in vitro (Figure 4). A simple
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47 collagenase digest of liver tissue will liberate hepatocytes which can be cryopreserved or treated
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49 in a variety of ways to study drug metabolism and hepatic uptake. Homogenization of liver tissue
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52 in buffer followed by a slow speed (9,000g) spin produces a pellet containing mitochondria and
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54 lysosomes. The supernatant (or S9 fraction) contains the majority of the Phase 1 and 2 drug
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3 metabolizing enzymes of the hepatocytes (in a relatively dilute solution). A further high speed
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6 (100,000g) spin of the S9 fraction in the presence of calcium produces a supernatant containing
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8 all of the soluble enzymes present in the cytosol of the hepatocyte. The pellet contains the
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10 remnants of the cellular endoplasmic reticulum which in the presence of calcium have snapped
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back onto themselves to form spheres of membranes called microsomes. As such, microsomes
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15 do not occur in nature, but are artefacts of hepatic sub-cellular fraction preparation. They have
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17 been a major driver of fully understanding hepatic metabolic clearance in vitro, since they
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contain all the membrane bound proteins, such as CYPs and UGTs.
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24 Hepatocytes and microsomes form the basis of two very useful in vitro reagents for study and
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26 prediction of metabolic intrinsic clearance. Ideally, study of the Michaelis-Menten kinetics of
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29 any metabolic step would be completed at optimized enzyme and substrate concentrations.
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31 However, this would represent a considerably time consuming experiment to complete for one
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33 compound let alone a series of compounds in a drug discovery project. To make this experiment
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more amenable for use in a drug discovery setting, some compromises have been made. The
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38 disappearance of a compound by metabolism in an in vitro incubation can be completed at a set
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40 enzyme and substrate concentration. When the substrate concentration is below the Km
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(Michaelis constant) for the metabolic enzyme, then disappearance will be a first-order process.
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45 The plot of substrate concentration versus time will be an exponential decline and a natural log
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47 transform will produce a straight line (Figure 5). The slope of this line is related to the Vmax/Km
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49 (Vmax, maximum reaction rate) of the molecule and can be transformed into the apparent intrinsic
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52 clearance (CLint,app) of that molecule accounting for cell number (for hepatocytes) or protein
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54 concentration (for microsomes) used in the incubation. (Equation 13 and Figure 6).
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58 13
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5 1.345 89 -.:;0/-<.
6 -./,0 = /6/7
× :=99 # <? [?</=-.] (Equation 13)
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11 This methodology has been extensively explained previously14, 15
. Here it is important to
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13 remember that this is an apparent value which needs to be corrected for the unbound fraction in
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16 the in vitro incubation (fu,inc) in order to arrive at the unbound intrinsic clearance (CLint,u,
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18 Equation 14). This is an important consideration because highly bound compounds may have a
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20 low CLint,app by virtue of a low fu,inc, leading to an underestimation of the clearance rate.
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23 Ultimately, CLint,u is the appropriate value to incorporate into the well-stirred model equation
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25 (Equation 10) to predict the human hepatic clearance of that compound.
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CLint,u = CLint,app / fu,inc (Equation 14)
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34 The value of CLint,u at this point still has the units associated with the incubation (µL/min/mg
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37 protein for microsomes and µL/min/million cells for hepatocytes). In order to be useful for
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39 predicting in vivo CL, it needs to be scaled to the entire body by multiplying by the number of
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41 cells (or the microsomal protein concentration) per gram of liver and the number of grams of
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44 liver per kg body weight in the species of interest (Figure 6). This generates a predicted unbound
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46 intrinsic clearance value (units mL/min/kg) which can be used in the Well-Stirred Model to
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48 predict the CL of the molecule in that particular series.
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53 Clearly, the most important parameter to modulate in human in vitro metabolism experiments is
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55 the CLint,u as reducing this parameter will directly lead to improvements in requisite dose level
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58 14
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3 and frequency. Herein lays the paradox with respect to clearance optimization in modern drug
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6 discovery. Medicinal chemists have become very adept at reducing metabolic intrinsic clearance
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8 within chemical series. This has led to a number of challenges, perhaps the most significant of
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10 which is a rise in the awareness and relative significance of other clearance mechanisms such as
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hepatobiliary transport.
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17 Evolution of Liver Clearance Concept from Metabolism to Enzyme-Transporter Interplay
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Decades ago in drug discovery, there was limited DMPK input into the optimization and
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22 selection of compounds for drug development. Compounds were largely optimized for potency
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24 against the pharmacological target. Since potency can (in part) be driven by lipophilicity, these
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26 early candidates tended to be large and significantly lipophilic. Such compounds tended to be
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29 good substrates for CYPs and significant development candidate attrition was due to
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31 inappropriate PK (too short a half-life or too low oral bioavailability or poor exposure)16.
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33 Starting in the 1990s, significant scholarship on the effect of physicochemical properties on
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PK17-19 was completed and the in vitro metabolic assays (such as microsomal and hepatocyte
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38 stability) became available15, 20. For the latter, there were a significant number of CYP substrates
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40 with human clearance values that validated the use of such assays in prediction of human
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clearance.
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47 The concepts of physicochemical properties (notably the positive correlation between
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49 lipophilicity and CLint,u) and availability of clinically validated in vitro metabolic clearance
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52 assays enabled optimization in drug discovery which now took into account metabolic intrinsic
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54 clearance in addition to target potency. There was a clear reduction in human PK attrition in the
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58 15
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3 following decade16. Subsequently, further scholarship on physicochemical properties driving
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6 toxicology led to an even greater emphasis on lipophilicity reduction21. At the extreme,
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8 reductions in lipophilicity led to a reduction in membrane permeability and uncovered series of
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10 compounds where entry to the hepatocyte (either by passive or active processes) rather than
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metabolism is the rate determining step in defining the clearance of these molecules. This is
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15 underlined in a comparison of clearance in hepatocytes and microsomes22. As expected,
16
17 compounds predominately metabolized by CYP-mediated mechanisms gave comparable intrinsic
18
19
clearance values in microsomes and hepatocytes when passive permeability was high. However,
20
21
22 when hepatic passive permeability was low, intrinsic clearance in microsomes was faster than
23
24 that in hepatocytes, due to limited access to the enzymes within the cell. For certain non-CYP
25
26 metabolism (e.g., aldehyde oxidase, reductases) that is not present microsomes, intrinsic
27
28
29 clearance in hepatocytes will be higher. Some of the non-CYP enzymes have high extrahepatic
30
31 expression making it challenging to develop quantitative in vitro-in vivo correlation and
32
33 accurately predict human PK due to lack of appropriate tools and species differences. Medicinal
34
35
36
chemists need to pay attention to the impact of extrahepatic clearance in lead optimization.
37
38
39
40 In addition, early observations indicated that application of the previously described scaling
41
42
approach to CLint,u values derived from hepatocytes tended to under-predict the clearance of drug
43
44
45 known to be substrates of uptake transport mechanism (e.g. OATPs) due to the lower activity of
46
47 these transporters in hepatocyte preparations optimized for metabolic clearance estimation23.
48
49 These observations have led to an emphasis on the study of hepatic uptake as a potential rate
50
51
52 determining step in the clearance on molecules. The validation of in vitro assays for predicting
53
54 hepatic uptake limited clearance has been limited by the relative paucity of compounds for which
55
56
57
58 16
59
1
2
3 hepatic uptake is proven to be the rate determining step in human clearance. Hepatocytes can be
4
5
6 grown in culture under conditions where they will express drug uptake transporters and potential
7
8 form bile ducts (e.g., plated hepatocytes or sandwich culture hepatocytes)24-27. The intrinsic
9
10 clearance values determined for uptake substrates tend to be much lower than would be required
11
12
13
to explain the hepatic clearance of these compounds in vivo (for the few compounds where the
14
15 full dataset is available)28, 29. Thus, extensive empirical scaling factors are currently required for
16
17 prospective human clearance prediction28, 29
. On a mechanistic level, the interplay between
18
19
metabolism and transport is captured by what is called the extended clearance concept. This
20
21
22 concept expands the hepatic CLint,u parameter into its component parts to provide a better
23
24 understanding of the rate determining mechanisms of clearance (Equation 15, which assumes all
25
26 parameters represent the unbound values for the sake of simplicity). More specifically, in this
27
28
29 concept, the rate determining mechanism of clearance is depicted as a complex function of
30
31 passive clearance rate (PSinf,passive), active uptake rate (PSinf,active), metabolic intrinsic clearance
32
33 (CLint,met), active sinusoidal clearance (PSeff,active), and biliary intrinsic clearance (CLint,bile). It is
34
35
36
clear from this equation that the rate determining mechanism is not dependent upon the absolute
37
38 value of any one rate, but rather the rates of these individual mechanisms in relation to one
39
40 another.
41
42
43
44
45 Hepatic CLint = (PSinf,active+PSinf,passive) x (CLint,met+CLint,bile) /
46
47 (PSeff,active+PSeff,passive+CLint,met+CLint,bile) (Equation 15)
48
49
50
51
52 Given the complexity of sorting the relative values of each process, classification systems which
53
54 are based on experimentally obtainable data and/or physiochemical properties (e.g., ionization
55
56
57
58 17
59
1
2
3 state) have been proposed as a means for predicting the likely rate-determining clearance
4
5
6 mechanisms and consequently directing researchers to the most appropriate systems by which to
7
8 predict the composite clearance rate. The clearance parameters of the extended clearance model
9
10 can be determined experimentally through a variety of approaches including:
11
12
13
(a) PSinf,active and PSinf,passsive in suspension or plated human hepatocytes with and without
14
15 uptake transporter inhibitors or at 4°C
16
17 (b) CLint,met in human liver microsomal or hepatocyte incubations
18
19
20 (c) CLint,bile in human sandwich-cultured hepatocyte incubations (generally assuming that
21
22 metabolism in this system is negligible).
23
24
25
26
As shown previously, this methodology is based on a relatively new concept in the prediction of
27
28
29 the human clearance of transporter-mediated uptake substrate both with and without further
30
31 metabolism or biliary excretion (mixed clearance pathways). In contrast to purely metabolism
32
33 rate limited clearance, the absolute scalability of parameters estimated in this way is still under
34
35
36 investigation. More work is required to establish the scalability of these more complex
37
38 approaches covering non-metabolic or mixed mechanisms governing the rate of clearance.
39
40
41
42
43 The Importance of Physicochemical Properties in Determining Clearance Mechanism
44
45 Based on the above, providing the correct CLint,u data to estimate the clearance of a drug is
46
47 highly enabled if physicochemical properties are considered. Permeability through lipid
48
49
membranes determines not only the ability of a drug to be absorbed and reach an intracellular
50
51
52 target, it also governs the fate of a molecule. Drugs with high permeability will be reabsorbed in
53
54 the kidney tubule (following filtration at the Bowman’s capsule) and traverse the cell membranes
55
56
57
58 18
59
1
2
3 at high intrinsic passive flux rates with minimal influence of transporters. Under these
4
5
6 circumstances, clearance by the kidney can be discounted as such compounds will be cleared by
7
8 metabolism. As permeability declines, so the rate of membrane flux declines and the transfer of
9
10 the drug across lipid membranes is more impacted by transporters. Access to intracellular targets
11
12
13
becomes problematic and the clearance fate of the molecule becomes more complex with
14
15 potential for drug transporter involvement. Physicochemical measurements or calculations give
16
17 good guidance into the likely permeability characteristics of a molecule. Low permeability is the
18
19
result of low lipophilicity or high polar surface area (PSA). The role of physicochemical
20
21
22 properties in determining many aspects of drug clearance and performance is shown in Table 1
23
24 and Figure 7. Many of these concepts have been incorporated into a variety of drug disposition
25
26 classification systems30, 31
, some of which (e.g. BDDCS, ECCS, ECCCS) seek to define the
27
28
29 likely rate-limiting mechanisms of clearance (e.g. hepatic metabolism, hepatobiliary transport,
30
31 renal excretion, or mixed mechanisms) from physicochemical properties (e.g. ionization state,
32
33 molecular weight) and in vitro measures (e.g. membrane permeability, solubility, intrinsic
34
35
36
clearance). Each has strengths and weaknesses with regard to theoretical and practical aspects as
37
38 well as retrospective performance. While an extensive review of these classification systems is
39
40 beyond the scope of this work32, it is worth noting that the primary utility of these systems is to
41
42
direct early discovery research efforts intended to characterize, predict and optimize clearance of
43
44
45 lead matter.
46
47
48
49 Allometric Scaling of Clearance
50
51
52 Establishing in vitro to in vivo correlation in preclinical species for metabolic clearance and
53
54 extended clearance is important to reduce uncertainty for human translation from in vitro to in
55
56
57
58 19
59
1
2
3 vivo. In the absence of a simple in vitro assay to optimize and predict human clearance for
4
5
6 hepatic uptake substrates, the fall back approach is often to use preclinical species PK scaling to
7
8 predict human clearance. This practice has been known for many years and is termed allometric
9
10 scaling.
11
12
13
14
15 A true allometric scaling experiment requires IV administration to multiple preclinical species.
16
17 The clearance of the compound (expressed as mL/min) is plotted against body weight as shown
18
19
in Figure 8. When plotted versus body weight, the values for clearance in preclinical species can
20
21
22 be extrapolated to human body weight, to give a prediction of human clearance for that
23
24 compound. It has been well known for many years that physiological parameters (such as blood
25
26 flow, heart rate, liver weight) scale with an exponent of less than 1 when plotted versus body
27
28
29 weight33-36. The same is often true of clearance (i.e., it is not a one to one relationship). For this
30
31 reason, and the labour-intensive nature of generating multiple species PK profiles, some authors
32
33 have suggested single species scaling with a fixed allometric exponent of approximately 0.7, as a
34
35
36
pragmatic method for human clearance prediction of discovery compounds37. However, it must
37
38 be noted that fixing the allometric exponent is highly controversial38. Small deviations in the
39
40 exponent from the fixed value can lead to major mis-predictions of human clearance, due to the
41
42
large weight difference between some species (such as rodents) and human.
43
44
45
46
47 The accuracy in prediction of human clearance by allometric scaling is dependent upon the
48
49 clearance route and the extent of clearance. The best evaluation of allometric scaling prediction
50
51
52 was completed by Tang et al39, 40
. They examined 103 small molecule drugs with literature
53
54 citations containing IV PK studies in at least 3 preclinical species and human. They found that
55
56
57
58 20
59
1
2
3 errors in prediction of human clearance ranged from 0 (exact prediction) to greater than 3000%
4
5
6 (major mis-prediction). Prediction accuracy was improved for high clearance molecules by
7
8 metabolism and biliary secretion as well as molecules predominantly cleared by renal
9
10 elimination. Predictability was poor for lower clearance molecules, especially where metabolism
11
12
13
was a major player. This is not surprizing since renal elimination is most likely by passive
14
15 filtration at GFR and according to the well-stirred model, high CLint,u compounds will tend
16
17 towards hepatic blood flow clearance. Since blood flow scales well allometrically, compounds
18
19
where clearance is dependent upon blood flow will predict well by this method. When extraction
20
21
22 is towards the low level with respect to hepatic blood flow, clearance will be highly dependent
23
24 on CLint,u. It is well known that CLint,u values for a given compound in animals often are much
25
26 higher than in human, and so allometric scaling for metabolized compounds is likely to over-
27
28
29 predict the observed human clearance41-43. The same is likely true for hepatic uptake cleared
30
31 compounds as the drug transporters expressed in animals and humans are different44. Overall, the
32
33 use of animal PK experiments to predict human clearance is a useful approach. On a pragmatic
34
35
36
level, if the clearance in animals is low with respect to liver blood flow in that species, it is likely
37
38 that the human clearance will also be low. However, due to species differences in CLint,u, it is not
39
40 unusual for clearance in animals to be high relative to liver blood flow and low in humans, and
41
42
this approach may miss some important chemical series.
43
44
45
46
47 Pitfalls in Assessing Clearance
48
49 From the above discussion, it is understood that optimization of small molecule candidates for
50
51
52 acceptable human clearance is an essential part of the drug discovery process. It is also clear that
53
54 clearance is a complicated property to assess, with many potential ways a compound can be
55
56
57
58 21
59
1
2
3 cleared from blood and many potential species differences. Many oral drugs are required to be
4
5
6 dosed once daily, leading to a requirement for high oral bioavailability and a half-life that
7
8 matches the dose regimen. Consequently, the ability to assess and modulate human clearance
9
10 within a series is essential. There are some potential pitfalls that can be encountered in assessing
11
12
13
clearance in the drug discovery phase.
14
15
16
17 Tips to Address Pitfalls in Clearance Assessment
18
19
(a) Understanding extent of binding of molecules
20
21
22 As shown above, only compound that is unbound in an in vitro incubation is available to
23
24 be metabolized or transported into hepatocytes to generate the true unbound intrinsic
25
26 clearance (CLint,u). Compounds that are highly bound under the incubation conditions
27
28
29 (binding to microsomes and hepatocytes) will tend towards low values for CLint,app, but
30
31 are very likely to be susceptible to rapid metabolism of the unbound drug due to high
32
33 lipophilicity. Consequently, optimization of a series in the absence of understanding the
34
35
36
extent of binding in the incubation, will lead to apparent low in vitro intrinsic clearance
37
38 that leads to high CLint,u in vivo.
39
40
41
42
Similar issues apply to in vivo studies. PK studies exclusively measure total drug
43
44
45 concentrations in blood or plasma and CLs is a total parameter. However, only unbound
46
47 drug is available to exert a pharmacological effect and, according to the Well-Stirred
48
49 model, CLint,u is the major driver of exposure to unbound drug. Consequently, a high
50
51
52 plasma protein binding (low fu) will affect CLs but will not affect CLint,u, meaning that it
53
54 is possible to generate a low CLs with a highly plasma protein bound compound, but
55
56
57
58 22
59
1
2
3 exposure to unbound drug will be low. In general, driving CLs lower by increasing
4
5
6 plasma protein binding is not a good strategy, unless CLint,u is lowered or the compound
7
8 has a high degree of pharmacological potency (i.e., low dose). The converse is also true.
9
10 Lowering plasma protein binding in a highly bound chemical series does not improve
11
12
13
exposure to unbound concentration, unless CLint,u is also lowered. Many groups have
14
15 claimed to have improved drug properties by lowering plasma protein binding45-47, but in
16
17 all these cases the strategy to lower plasma protein binding was to lower lipophilicity
18
19
with a consequent reduction in CLint,u. The effect of plasma protein binding on clearance
20
21
22 and dose properties has been extensively studied13, 48, 49 and in almost all circumstance,
23
24 plasma protein binding modulation is not a viable method to improve a compound series.
25
26
27
28
29 (b) Using the correct in vitro assay
30
31 As noted above, there are numerous mechanisms that can drive the clearance of any
32
33 particular chemical series (e.g., metabolism, uptake, renal). The in vitro assays that have
34
35
36
been developed often separate these mechanisms, such that one assay tends to focus on
37
38 one particular mechanism. During the process of optimization of the clearance of a
39
40 particular small molecule series, it is important to ensure that the in vitro assay used
41
42
reflects the true mechanism of clearance of that series. If the assay does not have the
43
44
45 appropriate mechanism, e.g., optimizing for metabolism when uptake is the issue; or
46
47 optimizing CYP when UGT is the main pathway, the translation to in vivo studies will be
48
49 flawed. Also, in the process of compound optimization, if the structural changes made
50
51
52 within a series drive to a different clearance pathway, then the in vitro assay used needs
53
54 to reflect this. For example, the most likely response to high CLint due to metabolism is to
55
56
57
58 23
59
1
2
3 lower lipophilicity in a chemical series. At some point, lowering lipophilicity will drive
4
5
6 to poor membrane permeability and the rate determining step could change to hepatic
7
8 uptake or renal clearance rather than metabolism.
9
10
11
12
13
Another issue with in vitro assays is often detection limits. Under certain circumstances
14
15 even compounds that are cleared at the lower limit for the assay will scale to predicted in
16
17 vivo clearance values that are higher than required for the optimal dose regimen. Good
18
19
examples of this are the in vitro assays for metabolic clearance. Hepatocyte and
20
21
22 microsomal incubations are limited by the length of incubation time and ability to detect
23
24 disappearance of the parent compound. Low clearance does not mean no clearance. A
25
26 novel approach to increasing the incubation time for hepatocyte assays has been proposed
27
28
29 as a method to lower the limit of determination and enable investigation of CLint values
30
31 that are 10-fold lower than the standard methods used50-52.
32
33
34
35
36
(c) Focus on low dose
37
38 In progressing to in vivo PK studies, care must be taken to maintain as low a dose as
39
40 possible. When clearance is an active process (such as metabolism or hepatic uptake) it
41
42
will obey Michaelis-Menten principles. This means that clearance will only be dose- and
43
44
45 concentration-independent at unbound concentrations that are significantly below the Km
46
47 for the clearance mechanism. High dose studies (such as those used in toxicity studies)
48
49 are not appropriate studies to generate PK parameters (such as clearance) as this
50
51
52 requirement cannot be met and clearance mechanisms will often be saturated.
53
54
55
56
57
58 24
59
1
2
3 (d) Use the correct terminology
4
5
6 Unfortunately the term clearance in its various forms is used incorrectly in many
7
8 discussions. Drugs with a short half-life are often termed to be too rapidly cleared,
9
10 whereas clearance may in fact be low, but the duration is compromised by a low volume
11
12
13
of distribution. In other discussions, clearance is confused with recovery of the total dose
14
15 (e.g.,14C labelled material) in urine and faeces.
16
17
18
19
Perspective
20
21
22 Clearance is the most important PK parameter for drug disposition since it impacts half-life (or
23
24 duration of action), oral bioavailability and dose. It can only be calculated from inspection of the
25
26 blood (or plasma) concentration versus time curve following IV administration. Measurement of
27
28
29 blood (or plasma) means that clearance is a parameter based on total (both bound and unbound)
30
31 drug. However, the unbound concentration of a drug is the driver of pharmacology and only
32
33 unbound drug is available for clearance from the blood. Modulation of the human clearance
34
35
36
within a small molecule chemical series is a key aspect of compound optimization during drug
37
38 discovery. For clearance optimization, medicinal chemists need to focus on the unbound intrinsic
39
40 clearance (CLint,u) which is effectively the maximal rate of clearance of drug by a clearing organ
41
42
in the absence of flow and binding limitations. The major organ of drug clearance in the body is
43
44
45 the liver. Sub-cellular hepatic fractions have been used to prepare in vitro assays for metabolic
46
47 clearance. These assays, along with understanding of the effect of physicochemical properties on
48
49 clearance, have enabled drug discoverers to minimize metabolic CLint,u in chemical series and on
50
51
52 the whole driven to molecules with lower clearance in human. However, by driving to lower
53
54 lipophilicity, the rate of membrane penetration has potentially become a rate-determining step in
55
56
57
58 25
59
1
2
3 clearance from the blood and the impact of transporters is more significant. With this trend,
4
5
6 improved reagents and an ever increasing understanding of the relevance of transporters in drug
7
8 disposition, more compounds are being identified as drug transporter substrates and uptake has
9
10 become a major player in clearance of small molecules. Whilst this shift has been recognized in
11
12
13
the thinking behind the extended clearance model, the in vitro assays required to lower CLint,u by
14
15 uptake transport are still in their infancy. The next evolution of modulation of clearance in drug
16
17 discovery will follow much greater experience in the application of these drug transporter assays.
18
19
20
21
22
23
24
25
26
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28
29
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31
32
33
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40
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42
43
44
45
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58 26
59
1
2
3 Biographical Sketch
4
5
6 Dr. Dennis A. Smith worked in the pharmaceutical industry for 32 years after gaining his Ph.D
7
8 from the University of Manchester. During this period he directly helped in the Discovery and
9
10 Development of eight marketed NCEs. More recently his roles include advisory boards or expert
11
12
13
panels with many drug research based organisations, both industrial and academic, including
14
15 Medicines for Malaria Venture and Cancer Research UK. His research interests and publications
16
17 span all aspects of Drug Discovery and Development particularly where drug metabolism
18
19
knowledge can impact on the design of more efficacious and safer drugs.
20
21
22
23
24 Mr. Kevin Beaumont has worked extensively in the discovery and development drug
25
26 metabolism field, throughout his 30 years in the pharmaceutical industry. His major area of
27
28
29 expertise is in the modulation of physicochemistry to affect drug disposition and prediction of
30
31 human PK. He is author on over 40 peer reviewed publications. Overall, Kevin has worked on
32
33 many drug discovery and development projects throughout his career. He has been responsible
34
35
36
for the DMPK input to at least 30 FIH studies as well as 10 Phase II compounds, including 1
37
38 marketed agent. Kevin now provides DMPK input to the Inflammation and Immunology and
39
40 Rare Disease Research Units, based in Cambridge Massachusetts.
41
42
43
44
45 Dr. Tristan S. Maurer received his PharmD from the University of Georgia in 1993 and his
46
47 PhD from the University of Buffalo, State University of New York in 1999. During his 18 year
48
49 tenure with Pfizer, his work has focused on the development and application of quantitatively
50
51
52 rigorous, biologically-based methods to predict human pharmacokinetics & pharmacodynamics
53
54 from preclinical data. He has co-authored over 60 manuscripts illustrating the utility of these
55
56
57
58 27
59
1
2
3 methods to drug design and early clinical development. Currently, Dr. Maurer sits on the
4
5
6 Medicine Design leadership team responsible for scientific and operational strategies spanning
7
8 from idea to loss of exclusivity. He also leads a modelling and simulation group responsible for
9
10 both computational chemistry and quantitative translational pharmacology across Pfizer’s small
11
12
13
molecule portfolio.
14
15
16
17 Dr. Li Di has about 20 years of experience in the pharmaceutical industry including Pfizer,
18
19
Wyeth and Syntex. She is currently a research fellow at Medicine Design Department, Pfizer
20
21
22 Global Research and Development, Groton, CT. Her research interests include the areas of drug
23
24 metabolism, absorption, transporters, pharmacokinetics, blood–brain barrier, and drug-drug
25
26 interactions. She has over 135 publications including two books and presented more than 85
27
28
29 invited lectures. She is a recipient of the Thomas Alva Edison Patent Award, the New Jersey
30
31 Association for Biomedical Research Outstanding Woman in Science Award, the Wyeth
32
33 President’s Award, Peer Award for Excellence and Publication Award.
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35
36
37
38
39 * Corresponding author
40
41 Li.Di@Pfizer.Com
42
43
44 860-715-6172
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58 28
59
1
2
3 Abbreviations
4
5
6 AUC, area under the curve; BCRP, breast cancer resistance protein; CL, clearance; CLb, blood
7
8 clearance; CLint,app, apparent intrinsic clearance; CLint,bile, biliary intrinsic clearance; CLint,met,
9
10 metabolic intrinsic clearance; CLint,u, unbound intrinsic clearance; CLp, plasma clearance; CLpo,
11
12
13
oral clearance; CLr, renal clearance; CLs, systemic clearance; Css,av, average concentration at
14
15 steady state; Css,av,u, unbound average concentration at steady state; CYPs, cytochrome P450s;
16
17 DMPK, drug metabolism and pharmacokinetics; Eh, hepatic extraction ratio; F, bioavailability;
18
19
Fa, fraction absorbed; Fg, fraction escaping gut wall extraction; fu, unbound fraction; fu,b, fraction
20
21
22 unbound in blood; fu,inc, unbound fraction in in vitro incubation; GFR, glomerular filtration rate;
23
24 GI, gastrointestinal; kel, elimination rate constant; Km, Michaelis constant; IV, intravenous;
25
26 MRPs, multidrug-resistance proteins; OATs, organic anion transporters; OATPs, organic anion
27
28
29 transporting polypeptides; OCTs, organic cation transporters, P-gp, P-glycoprotein; PK,
30
31 pharmacokinetics; PO, oral administration; PSinf,active, active uptake rate; PSeff,active, active
32
33 sinusoidal clearance; PSinf,passive, passive clearance rate; PSA, polar surface area; Q, hepatic blood
34
35
36 flow; Rb, blood to plasma ratio; RBF, renal blood flow; τ, dose interval; t1/2, half-life; UGTs,
37
38 uridine 5'-diphospho-glucuronosyltransferases; Vd, volume of distribution; Vmax, maximum
39
40 reaction rate.
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 29
59
1
2
3 References
4
5
6 1. Smith, D. A.; Allerton, C.; Kalgutkar, A. S.; van de Waterbeemd, H.; Walker, D. K.
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8 Pharmacokinetics and Metabolism in Drug Design. 3rd ed.; Wiley-VCH: Weinheim, Germany,
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10 2012.
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2. Di, L.; Kerns, E. H. Drug-Like Properties: Concepts, Structure Design, and Methods.
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15 Elevier: London, UK, 2016.
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17 3. Khojasteh, S. C.; Wong, H.; Hop, C. E. C. A. Drug Metabolism and Pharmacokinetics
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Quick Guide. Springer: New York, NY, 2011.
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22 4. Smith, D. A.; Beaumont, K.; Maurer, T. S.; Di, L. Volume of distribution in drug design.
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24 J. Med. Chem. 2015, 58, 5691-5698.
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26 5. Smith, D. A.; Beaumont, K.; Maurer, T. S.; Di, L. Relevance of half-life in drug design.
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29 J. Med. Chem. 2018, 61, 4273-4282.
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31 6. Beaumont, K.; Harper, A.; Smith, D. A.; Abel, S. Pharmacokinetics and metabolism of a
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33 sulphamide NK2 antagonist in rat, dog and human. Xenobiotica 2000, 30, 627-642.
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7. Wagner, J. G.; Northam, J. I.; Alway, C. D.; Carpenter, O. S. Blood levels of drug at the
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38 equilibrium state after multiple dosing. Nature 1965, 207, 1301-1302.
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40 8. Pang, K. S.; Rowland, M. Hepatic clearance of drugs. I. Theoretical considerations of a
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45 blood cell binding, and the hepatocellular enzymatic activity on hepatic drug clearance. J.
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47 Pharmacokinet. Biopharm. 1977, 5, 625-653.
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49 9. Rowland, M.; Benet, L. Z.; Graham, G. G. Clearance concepts in pharmacokinetics. J
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3 10. Wilkinson, G. R.; Shand, D. G. Commentary: a physiological approach to hepatic drug
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6 clearance. Clin Pharmacol Ther 1975, 18, 377-390.
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8 11. Chiba, M.; Ishii, Y.; Sugiyama, Y. Prediction of hepatic clearance in human from in vitro
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10 data for successful drug development. AAPS J. 2009, 11, 262-276.
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12. Hallifax, D.; Foster, J. A.; Houston, J. B. Prediction of human metabolic clearance from
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15 in vitro systems: retrospective analysis and prospective view. Pharm. Res. 2010, 27, 2150-2161.
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17 13. Smith, D. A.; Di, L.; Kerns, E. H. The effect of plasma protein binding on in vivo
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efficacy: misconceptions in drug discovery. Nat. Rev. Drug Discovery 2010, 9, 929-939.
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22 14. Houston, J. B. Relevance of in vitro kinetic parameters to in vivo metabolism of
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24 xenobiotics. Toxicol. in Vitro 1994, 8, 507-512.
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26 15. Obach, R. S. Prediction of human clearance of twenty-nine drugs from hepatic
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29 microsomal intrinsic clearance data: an examination of in vitro half-life approach and
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31 nonspecific binding to microsomes. Drug Metab. Dispos. 1999, 27, 1350-1359.
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33 16. Kola, I.; Landis, J. Opinion: can the pharmaceutical industry reduce attrition rates? Nat.
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values. Drug Metab. Dispos. 2012, 40, 1860-1865.
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1
2
3 List of Tables and Figures
4
5
6 Table 1. Influence of physicochemical properties and permeability on factors determining drug
7
8 disposition
9
10 Figure 1. Typical IV plasma concentration versus time curve for a compound in human
11
12
13
Figure 2. Simple model of clearance for an organ
14
15 Figure 3. Schematic of the hepatobiliary system
16
17 Figure 4. Preparation of Hepatic Subcellular Fractions
18
19
Figure 5. In vitro metabolism experiment following parent depletion in linear and log scale
20
21
22 Figure 6. Scaling human liver microsomal and hepatocyte CLint,app to human blood CLb
23
24 Figure 7. Permeability controls the effects of transporter efflux or influx
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26 Figure 8. Basics of an allometric scaling experiment
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1
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3 Box 1. Key Messages
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5
6
7 1. Understand the binding of a molecule both in vitro and in
8
9 vivo, but do not optimize on binding. Rather optimize on
10
11 unbound intrinsic clearance.
12
13
14
2. Make sure the enzymes that metabolize the compound series
15
16 are present in the in vitro system used for optimization.
17
18 3. Pay attention to enzyme-transporter interplay and optimize
19
20
compound series based on rate-determining mechanisms.
21
22
23 4. Maintain unbound concentrations below the Km of the
24
25 enzymes/transporters clearing the series of molecules. Keep
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27 the dose/concentration relatively low.
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30 5. Understand the (many) terms used to describe clearance.
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1
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3 Table 1. Influence of physicochemical properties and permeability on factors determining
4
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6 drug disposition
7
8 Membrane Low Medium High
9
10 Permeability &
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12 Physicochemical Low Log D < 0 Log D >0 Log D >0
13
14
15
properties (PSA>75Å2) (PSA<75Å2)
16
17 Absorption Low unless MW < 250 Da Variable. Influenced High via transcellular
18
19 and absorbed by by permeability and route (propranolol)
20 paracellular route transporters
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22 (atenolol) (nelfinavir)
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24
25
Access to target Extracellular including cell Total body water, but Total body water, drug
26 proteins surface may be concentrated will show identical
27
28 or effluxed from unbound concentrations to
29 Rapid equilibrium with
30 intracellular cytosol that present in plasma
unbound drug in
31
32 circulation May have slower Rapid equilibrium with
33
34 equilibrium with unbound drug in
35
36
unbound drug in circulation
37 circulation
38
39
40
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42 Clearance Renal or Biliary (possible Uptake and efflux Metabolism
43
44 transporter involvement) transporters and
45
46
metabolism
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58 39
59
1
2
3 Figure 1. Typical IV plasma concentration versus time curve for a compound in human
4
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6 10000
7
Plasma Concentration
8
9 1000
10
11 (ng/mL)
12 100
13
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15 10 AUC
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18 1
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0 20 40 60
21 Time (hours)
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58 40
59
1
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3 Figure 2. Simple model of clearance for an organ
4
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7
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9
CLint
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13
Q Q
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Organ
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58 41
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1
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3 Figure 3. Schematic of the hepatobiliary system
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CYP P-gp
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9
UGT BCRP
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11 MRP2
12 Liver Bile
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Blood Passive OATP1B1
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26 Diffusion OATP1B3
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3 Figure 4. Preparation of Hepatic Subcellular Fractions
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1
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3 Figure 5. In vitro metabolism experiment following parent depletion in linear and log scale
4
5
6
7 100 100
8
9 80
Percent Remaining
Percent Remaining
10 60
11 10
12 40
13 20
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15 0 1
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
16 Time (hours) Time (hours)
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58 44
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1
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3 Figure 6. Scaling human liver microsomal and hepatocyte CLint,app to human blood CLb
4
5
6
7
8
9 In Vitro Incubation
10 Hepatocytes Cell density
11
Microsomes Protein concentration
12
13 In Vitro CLint,app
14
15 Binding in incubation
16
17 In Vitro CLint,u Hepatocellularity
18
19 Microsomal protein per liver weight
20
Scaling Liver weight and body weight
21
22 Predicted In Vivo CLint,u
23
24 Liver model Blood binding and blood flow
25
26 Predicted In Vivo Hepatic CL
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8 Figure 7. Permeability controls the effects of transporter efflux or influx
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1
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3 Figure 8. Basics of an allometric scaling experiment
4
5
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7
8 •
9
Log CL (mL/min) man
10
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13 •
14 dog
15
16 •
17 monkey
18 • rat
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20 •
21 mouse
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24 Log BWt
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3 Abstract Graph
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