Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oral bioavailability of rats and humans

Review

10.1517/17425225.3.4.469 © 2007 Informa UK Ltd ISSN 1742-5255 469

1. Introduction

2. Concepts and

theoretical calculations

of oral bioavailability

3. Determinants of

oral bioavailability

4. Expert opinion

Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oral bioavailability of rats and humans Susan Hurst , Cho-Ming Loi , Joanne Brodfuehrer & Ayman El-Kattan †

Pfi zer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA

The onset, intensity and duration of therapeutic response to a compound depend on the intrinsic pharmacological activity of the drug and pharmacokinetic factors related to its absorption, distribution, metabolism and elimination that are inherent to the biological system. The process of drug transfer from the site of administration to the systemic circulation and the interspecies factors that impact this process are the scope of this review. In general, the factors that influence oral drug bioavailability via absorption and metabolism can be divided into physicochemical/biopharmaceutical and physiological factors. Physicochemical and biopharmaceutical factors that influence permeability and solubility tend to be species independent. Although there are significant differences in the anatomy and physiology of the gastrointestinal tract, these are not associated with significant differences in the rate and extent of drug absorption between rats and humans. However, species differences in drug metabolism in rats and humans did result in significant species differences in bioavailability. Overall, this review provides a better understanding of the interplay between drug physicochemical/biopharmaceutical factors and species differences/similari-ties in the absorption and metabolism mechanisms that affect oral bioavail-ability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.

Keywords: absorption , bioavailability , biopharmaceutical factors , first-pass effect , metabolism , physicochemical factors , species differences

Expert Opin. Drug Metab. Toxicol. (2007) 3(4):469-489

1. Introduction

The discovery and development of therapeutically efficacious drugs is a time consuming, resource intensive and complicated process. Recent estimates show that developing a new drug in the US requires 10 – 15 years to reach the marketplace with an average cost of $800 million [1] . Most of the drugs that are available in the marketplace are administered via the oral route, which is a convenient and cost effective route of administration [2,3] . Oral dosing, unlike intravenous dosing, does not require administration by trained medical personnel and is not associated with the potential risk of infection [3,4] . Thus, oral bioavailability is one of the key factors frequently evaluated in drug discovery when considering a new chemical entity (NCE) for further development. It is well established that poor oral bioavailability is

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Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oral bioavailability of rats and humans

470 Expert Opin. Drug Metab. Toxicol. (2007) 3(4)

one of the major causes of therapeutic variability [5] . Hellriegel et al. reported a significant inverse relationship between the oral bioavailability of drugs from several therapeutic classes and the coefficient of inter-individual variability in their oral bioavailability [6] . The increased variability associated with poor oral bioavailability may predispose the patient to a higher risk of exposure to toxic or subtherapeutic plasma drug concentrations and increase the risk for drug–drug or food–drug interactions [7] . This is particularly important for drugs with a narrow therapeutic window or potential for resistance development, such as cytotoxic drugs and antibiotics [8] .

The preclinical evaluation of an NCE’s pharmacokinetic profile is routinely performed during drug discovery. This data, in conjunction with that obtained from in silico and in vitro studies, is commonly used to project the pharmacokinetic profile in humans. Typically, the in vivo pharmacokinetic evaluations, including oral bioavailability determination, are performed in rodents because they have short lifespan, which allows for the growth of a larger number of animals in a short period of time and the conduction of pivotal in vivo pharmaco-kinetics studies. In addition, NCEs are synthesized in small quantities, which are insufficient to perform such studies in larger animal species. Results of such evaluations are then used to project the bioavailability in humans. However, projection of human oral bioavailability sometimes cannot be accurately extrapolated from an in vivo rat study. For instance, Cao et al. studied the oral bioavailability of 48 drugs with different physicochemical properties in rats and humans and found no correlation in the oral bioavailability values between the two species [9] . This clearly illustrates the challenge of extrapolating human pharmacokinetic profile from rat data, which may result in erroneous conclusions regarding the pharmacokinetic properties of the NCE being drawn and/or misguided decisions being made regarding the potential of the NCE as a viable therapeutic agent [10-12] . This report provides a critical review in three areas: i) discussion of the theoretical basis of factors that affect oral bioavailability; ii) an overview of the species similarities and differences in the absorption and metabolism mechanisms that affect oral bioavailability of NCEs in rats and humans; and iii) reviews the physicochemical/biopharmaceutical factors that influence the mechanisms of drug absorption and metabolism in both rats and humans. Overall, this review provides a rational perspective on the interplay between drug physicochemical/biopharmaceutical properties and species differences/similarities in the absorption and metabolism mechanisms that affect oral bioavailability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.

2. Concepts and theoretical calculations of oral bioavailability

Bioavailability (F%) is the extent to which an active moiety is absorbed from a pharmaceutical dosage form and becomes

available in the systemic circulation [1] . As a parameter, there are two types of bioavailability: i) absolute bioavailability; and ii) relative bioavailability [5] . Absolute bioavailability refers to the fraction of the extravascular (e.g., oral) dose that reaches the systemic circulation unchanged in reference to an intravenous dose [5] . It is usually determined by calculating the respective plasma drug exposure assessed as the total area under the drug plasma concentration versus time curve (AUC) after oral and intravenous administration (Equation 1).

(1)

Absolute bioavailabilityAUC

AUC

Dose

Doseoral

IV

IV

oral

� �

In cases where intravenous drug administration is not practical, the bioavailability of a drug is determined by comparing the fraction of a dose of drug reaching the systemic circulation relative to a reference standard [5,13] . This parameter is termed relative bioavailability and usually calculated as in Equation 2.

(2)

Relative bioavailabilityAUC

AUC

Dose

Dosetest

ref

ref

test

� �

In general, determinants of oral drug bioavailability include fraction of dose absorbed in the gastrointestinal (GI) tract and fraction of dose that escapes elimination by the intestinal tract, liver and lung. Thus, oral bioavailability can be defined mathematically by Equation 3 [14] .

(3)F F F F Fabs g h l� ⋅ ⋅ ⋅

For Equation 3, F is the oral drug bioavailability, F abs is the fraction of the dose that is absorbed from the intestinal lumen to the intestinal enterocytes, F g is the fraction of the dose that escapes pre-systemic intestinal first pass elimination, F h is the fraction of the dose that passes through the liver and escapes pre-systemic liver first pass elimination and F l is the fraction of the dose that passes through the lung and escapes pre-systemic pulmonary first pass elimination. It should be pointed out that F l contributes to the oral bioavailability of a drug only if the oral plasma exposure is compared with the plasma exposure following intra-arterial not intravenous administration. In this case, F l cancels out because, in a similar manner to the oral dose, the intravenous dose should first pass through the lung before reaching the systemic circulation [14] . Consequently, the oral bioavailability of a drug that is compared with its intravenous administration is a product of only F abs , F g and F h (Equation 4).

(4)F F F Fabs g h� ⋅ ⋅

As shown in Equation 4, the fraction of the dose that escapes first-pass elimination across the intestines (F g )

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Hurst, Loi, Brodfuehrer & El-Kattan

Expert Opin. Drug Metab. Toxicol. (2007) 3(4) 471

and liver (F h ) can be estimated experimentally via the comparison of systemic exposures (AUC ratios) where the dosing routes are selected to isolate the contribution by a particular organ.

F g can be estimated for a compound when doses are given orally and via a cannulated hepatic portal vein (h.p.v.) when the fraction absorbed (F abs ) is either assumed to be complete or is known and can be accounted for as in Equation 5.

(5)

F FAUC

AUC

Dose

Doseabs goral

h p v

h p v

oral

⋅ � �. . .

. .

Similarly, F h can be estimated for a compound when doses are given via a cannulated h.p.v. and intravenously as in Equation 6.

(6)

FAUC

AUC

Dose

Dosehh p v

i v

i v

h p v

� �. . .

. .

. .

. . .

In addition, F g and F h can be calculated for a compound via the organ extraction ratio (E) using Equations 7 and 8.

(7)F Eg g� �1

(8)F Eh h� �1

The hepatic extraction ratio can also be estimated using the hepatic blood clearance (CL h ) and hepatic blood flow (Q h ) using Equation 9, where CL h is calculated either via the scaling of in vitro data or from in vivo data (given an understanding of the relationship of CL h to the systemic clearance for a given compound) [15,16] .

(9)

ECL

Qhh

h

�

Following oral administration, the drug plasma concentration (C p ) and AUC of a drug following a linear one-compartment model can be expressed as in Equations 10 and 11.

(10)

CF Dose

Ve eP

a

d a e

t te a��

�� K

K KK K⋅ ⋅ ⋅ ⋅

( )( )

(11)

AUCF Dose

CL�

⋅

For Equations 10 and 11, K a and K e are the first order rate constants for absorption and elimination, respectively, and V d is the volume of distribution of the drug molecule [13,17] .

The rate of absorption and elimination have a significant impact on two pharmacokinetic parameters which are C max (defined as the highest drug concentration in the systemic circulation following oral dosing) and T max (defined as the length of time required to achieve the maximum concentration of the drug in the systemic circulation following oral dosing).

Equations 10 and 11 clearly demonstrate that the drug plasma concentration following oral dosing is determined not only by the absorption rate constant (K a ), but also by the drug elimination rate constant (K e ), bioavailability (F) and distribution (V d ). In contrast, the extent of plasma exposure is assessed by AUC and is only a product of drug dose, clearance (CL) and bioavailability. Thus, species differences in drug absorption and metabolism following oral dosing can lead to significant differences in the plasma exposure of a drug following oral dosing.

3. Determinants of oral bioavailability

3.1 Absorption Drug intestinal permeability and solubility in the biological fluid in which it is dissolved are related to the rate of absorption (absorptive flux), which can be expressed using Fick’s first law (Equation 12) [18] .

(12)J P C SAe� ⋅ ⋅∆

In Fick’s first law, J is the drug absorptive flux across a homogeneous intestinal membrane, P e is the effective intesti-nal permeability (cm/s), ∆ C is the concentration gradient across intestinal mucosa ‘ drug solubility in the luminal fluid ’ (mM/cm 3 ) and SA is the intestinal surface area available for oral absorption (cm 2 ). As shown in Equation 12, solubility and permeability are interrelated and affect the absorptive flux, which impacts the drug absorption rate and the fraction of oral dose absorbed [19] .

The factors that alter the rate and extent of oral drug absorp-tion can be divided into two main categories: physiological and physicochemical/biopharmaceutical factors [20-24] .

3.1.1 Physiological factors that impact oral drug absorption An extensive review of the anatomical and physiological differences in the GI tract between rat and human have been described previously and the following is a brief overview [25,26] .

3.1.1.1 Gastrointestinal anatomy and physiology In rats and humans, the GI tract consists mainly of the stomach, small intestine (the duodenum, jejunum and ileum) and large intestine (cecum, colon and rectum). The dimensions and surface areas of these components differ between the two species. Table 1 illustrates a comparison between the anatomical lengths and relative contributions of the

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major subdivisions of the GI tract in rats and humans [25] . Although the body weight of the human (70 kg) is significantly larger than that of rat (0.25 kg), the human GI tract (8.35 m) is only 5.5-fold longer than the rat GI tract (1.5 m). However, the relative size of the small intestine, which is considered the primary site of drug absorption, to the total length of the GI tract is 83% in rats versus 81% in humans. As for the large intestine, its relative size in rats and humans is 17 and 19%, respectively. It may also be pointed out that, in rats, the cecum, which is the major site of microbial digestion, forms ∼ 26% of the length of the large intestine in rats. On the contrary, it accounts for only 5% of the length of the human large intestine [27,28] .

Table 2 presents a comparison of the absolute surface areas of the GI tracts in rats and humans. Interestingly, the human small intestine surface area is 200-fold larger than the rat small intestine surface area [25] . This significant difference in the surface area is attributed to the fact that the human small intestine has three anatomical modifications that significantly increase the surface area of the human small intestine [29,30] , whereas the rat small intestine has only two of these. The human small intestine has grossly observable folds of mucosa (plicae circularis or folds of kerckring) that increase the surface area threefold and are absent in rat intestine. From the plicae circularis, microscopic finger-like pieces of tissue called villi project, which increase the surface area by 5- and 10-fold for rats and humans, respectively. Each villus is covered in microvilli, which increase the surface area by 20-fold. When the small intestine is normalized to the total body surface area, the relative surface area of the human small intestine is only fourfold larger than that of the rat [25] . Unlike the small intestine, the large intestine surface area does not have villi and is divided into geographical areas by transverse

furrows. In addition, the large intestine enterocytes differ slightly from that of the small intestine and its microvilli are less packed [26] . Overall, this significantly contributes to the smaller surface area of the large intestine in both rats and humans ( Table 2 ) and is consistent with the fact that small intestine is the major site of drug absorption in the two species.

3.1.1.2 Unstirred water layer Adjacent to the intestinal membrane is an unstirred water layer, which is a potential barrier for the absorption of various drug molecules across the intestinal membrane [31,32] . The thickness of this layer in rats is about 100 µm whereas in humans it is only 25 µm [33] . Chiou et al . quantitatively studied the impact of the unstirred water layer adjacent to the intestinal membrane on the rate and extent of absorption of passively absorbed drugs with different membrane absorption half-lives (10 – 300 min) in humans, dogs, rabbits, rats and mice. Results of this analysis suggested that the presence of the unstirred water layer is generally expected to have a relatively mild or insignificant effect on the rate of absorption and an insignificant effect on the extent of absorption [34] .

3.1.1.3 Gastrointestinal transit times The absorption rate of a drug molecule is generally a function of drug absorption through the GI tract, which is determined by the residence time and absorption in each GI segment [35] . In general, gastric transit time impacts the systemic exposure of rapidly dissolved and well absorbed drugs. However, intestinal transit time influences the absorption of drugs with limited mucosal permeability, carrier mediated uptake, drugs subject to intestinal degradation, or products whose dissolution is the rate limiting step for systemic absorption [36] . In contrast

Table 1. Comparison of the anatomical lengths of the intestinal tract and its major subdivision in rats and humans [25].

Region of intestinal tract Human Rat

Length (m) % of total Length (m) % of total

Small intestine 6.80 81 1.25 83Large intestine 1.55 19 0.25 17Total intestinal tract 8.35 1.50

Table 2. Comparison of the absolute surface areas of the gastrointestinal tract in rats and humans [25].

Region of intestinal tract Human Rat

Absolute surface area (m2) Absolute surface area (m2)

Stomach 0.053 0.000624

Small intestine 200 1

Large intestine 0.35 0.034

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to gastric transit time, intestinal transit time is independent of the feeding conditions and the physical composition of the intestinal contents [37] . The rat and human intestinal transit time is ∼ 3 – 4 h [25,38] . Several studies suggested that in rat and human small intestine there is a gradient of velocity where the small intestinal transit in the proximal intestine was faster compared with the distal intestine [38,39] . In rats, the transit time in the large intestine is around 15 h and the transit time in human large intestine can vary in the range of 8 – 72 h [25,40,41] .

3.1.1.4 The gastrointestinal pH The extent of ionization plays a pivotal role in determining the drug dissolution rate and passive permeability across the GI tract. Therefore, it becomes clear that the pH at the absorption site is a critical factor in facilitating or inhibiting the dissolution and absorption of various ionizable drug molecules [25,42] . It should be stressed that the pH of the luminal content (chyme) is altered by the luminal secretions. Figure 1 demonstrates the pH values of the GI tract of rats and humans [36] . In rats, the pH of chyme in the stomach is ∼ 3.3; in humans, the pH of chyme is more acidic and can be as low as 2.3. When the chyme arrives in the duodenum, it is quickly neutralized by the secretion of the pancreatic bicarbonate and bile. The pH values of chyme become progressively more alkaline in the distal portion of the small intestine in rats and humans. However, the pH of chyme in the large intestine is generally more acidic than the pH observed in the small intestine in both species, possibly due to the fermentation mediated by the microbial flora [26] .

3.1.1.5 Bile fl uid Bile is produced by hepatocytes and is drained through the many bile ducts that penetrate the liver [25] . During this process, the epithelial cells add a watery solution that is rich in bicarbonates which increases the alkalinity of the solution. In humans, the bile is stored and concentrated up to five times its original potency in the gall bladder. However, rats lack a gall bladder which suggests that the bile enters continuously as a dilute solution in the duodenum. It is to be noted that the human gall bladder secrets bile at a rate of 2 – 22 ml/kg/day. On the other hand, the rat liver directly secrets 48 ml/kg in the duodenum each day [26] .

In rats and humans, bile acts as a detergent to emulsify fats by increasing the surface area to help enzyme action, and thus aid in their absorption in the small intestine. In addition to bicarbonate solution, bile is composed of bile salts, such as the salts of taurocholic acid and deoxycholic acid, which are combined with phospholipids to break down fat globules in the process of emulsification by associating their hydrophobic side with lipids and the hydrophilic side with water. Emulsified droplets are then organized into many micelles which increases their absorption. Because bile increases the absorption of fats, it also plays a pivotal role in the absorption of the fat-soluble vitamins and steroids [43,44] .

3.1.1.6 Bacterial microfl ora In rats and humans, bacterial microflora exists in most of the GI tract and become an important component of the luminal content. In rats, there is significant number of bacterial microflora in the stomach as well as small and large intestines. In humans, there is no bacterial microflora in the stomach and upper small intestine. This is mainly attributed to the low pH of the gastric content. However, a large number of bacterial microflora populates the human’s distal small and large intestines [45] .

These bacterial microflora play a role in the metabolism of various chemicals and xenobiotics through hydrolysis, dehydroxylation, deamidation, decarboxylation and reduction of azide groups [45-47] . Among these reactions, hydrolysis of the glucuronide conjugates is the most important metabolic reaction that is mediated by the β -glucronidase enzyme and produced by the bacterial microflora found in the GI tract of rats and humans. It should be stressed that β -glucuronidase enzyme levels are significantly higher in the rat GI tract compared with that found in the human GI tract [26] . Furthermore, bacterial microflora are known to downregulate CYP isoforms [26] .

In regards to in vivo models for human intestinal microflora metabolism, conventional laboratory animals are limited. A germ-free rat model with associated human colonic bacteria has been used as an in vivo model for human projections [48,49] .

3.1.1.7 Lymphatic absorption The intestinal lymphatic route plays a key role in the absorption of drugs that are highly lipophilic. It has many advantages, such as increase in the oral bioavailability of highly lipophilic drugs by avoiding hepatic first pass effect, direct targeting of lymphoid tissue, indirect targeting of specific sites associated with low-density lipoprotein receptors, and alteration in the rate of oral drug input to the systemic circulation thereby providing opportunity for controlled release drug formulation [50-52] .

There is little information on species differences in the drug lymphatic uptake. This is attributed to the bias shown with the current types of approaches that are used to assess drug lymphatic uptake in animals [53] . However, similarities and differences in drug lymphatic uptake are determined by the lymph flow to various absorption sites, mechanisms of drug lymphatic absorption, mechanism of lipid digestion, and bile composition and secretion pattern [36] .

3.1.1.8 Mechanism of oral absorption Following oral dosing, drug molecules can cross the luminal membrane through various mechanisms that involve passive diffusion or active transport. Passive diffusion is comprised of two pathways: the paracellular pathway, in which drug diffuses through the aqueous pores at the tight junctions between the intestinal enterocytes; and the transcellular (lipophilic) pathway, which requires drug diffusion across the

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lipid cell membrane of the enterocyte. The active transport pathway is mediated by transporters and is divided into active drug influx and efflux. It is important to note that the relevance of each route is determined by the compound’s physicochemical properties and its potential affinity for various transport proteins [1] .

3.1.1.9 Passive diffusion In paracellular diffusion, drug molecules are absorbed by diffusion and convective volume flow through the water-filled intercellular space [54] . In general, drugs that are absorbed through this pathway are small molecules (e.g., molecular weight [MW] < 250 Da) and hydrophilic in nature (Log P < 0). Because the junctional complex has a net negative charge, positively charged molecules pass through more readily, whereas negatively charged molecules are repelled [55] . Furthermore, the paracellular pathway offers a limited window for absorption since it accounts for < 0.01% of the total surface area of intestinal membrane. In addition, the tight junctions between cells become tighter traveling from the jejunum towards the colon. There are no significant species differences between the absorption of drugs by the paracellular route in both rats and humans. For example, He et al. compared the absolute bioavailability for hydrophilic and metabolically stable compounds, which are predominantly excreted unchanged in the urine following intravenous administration. They reported a good correlation between the bioavailability of these drug molecules, which includes acyclovir, atenolol, cimetidine, nadolol and hydrochlorothiazide,

in humans and rats, with rats tending to slightly underpredict the bioavailability in humans. The group concluded that rat is a good model for predicting human bioavailability of paracellularly absorbed compounds [56] .

The transcellular pathway is the major route of absorption for most of drug molecules. In general, the rate of passive transcellular permeability is mainly determined by the rate of transport across the apical cell membrane, which is controlled by the physicochemical properties of the absorbed compound. Unlike the paracellular pathway, compounds that are absorbed through the transcellular pathway are unionised, with lipophi-licity of Log P > 0 and MW > 300 Da. In addition, the hydrogen-bonding capacity determined by the number of hydrogen bond donors and hydrogen bond acceptors is < 10 and 5, respectively [57,58] .

3.1.1.10 Active transport Functionally, intestinal membrane transporters can be classified as influx or efflux transporters [59] . A large number of influx transporters are expressed in the small intestinal mucosa and play a major role in the absorption of nutrients, vitamins, certain drugs and xenobiotics [59-61] . Examples of these influx transporters are di-/tripeptides (PEPT1), large neutral amino acids (system L), bile acids, nucleosides, monocarboxylic acid transporters and organic anion-transporting polypeptides (OATP). Drug molecules that are substrates for these influx transporters may exhibit an intestinal absorption higher than expected from their diffusion across intestinal cell membranes. PEPT1, which is expressed predominantly, but

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Figure 1. Comparative pH across the gastrointestinal tract of rat and human [36].

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not exclusively, in the small intestine, is a well-characterized influx transporter with many substrates, such as angiotensin-converting enzyme inhibitors, β -lactam antibiotics (both cephalosporins and penicillins), valacyclovir, PD-158473 and renin inhibitors [60,62,63] . Besides PEPT1, emerging data suggest that OATP is another important family of influx transporters expressed in the intestine. These influx trans-porters may exhibit substrate specificity. For instance, in a recent study, Glaeser et al. showed that multiple members of the OATP family of transporters including OATP1B1, -1B3, -2B1, and -1A2 are expressed in human duodenal tissues. However, among these OATP transporters, only OATP1A2 was capable of mediating the in vitro uptake of fexofenadine [64] .

In contrast to the influx transporters, efflux transporters function as an absorptive barrier that limits oral bioavailability of many drugs and xenobiotics. Most of the efflux transporters characterized to date belong to the ATP-binding cassette super-family of transporters and are expressed at the apical surface of the intestinal enterocytes. They include P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multi-drug resistance-associated protein (MRP) and organic ion trans-porters [60,62,63,65] . Expression of some of these transporters in the GI tract display regional distribution patterns [9,66] . Among the various efflux transporters, P-gp, which is a product of the multi-drug resistance (MDR1) gene, is considered the best-characterized member of the apical efflux transporters. This protein plays an important role in limiting the intestinal absorption of a large number of drugs such as digoxin, talinolol, UK-343,664, and ciclosporin [67-69] .

Of interest, Cao et al . characterized the species differences in gene expression of selected influx and efflux transporters in the rat and human duodenum and colon using GeneChip® analysis [9] . There was a moderate correlation in the transporter gene expression levels in the duodenum between rats and humans, whereas the correlation of transporter expression in rat and human colon is relatively poor. Many transporters (e.g., PEPT1, MRP2) display similar gene expression levels in both rat and human with regional-dependent expression patterns, and discrepancy was also observed for other transporters (e.g., MDR1, MRP3) in both the duodenum and colon of human and rat. Further understanding of species similarities and differences in drug influx and efflux transporters, their abundance and substrate specificity will likely enhance the ability to project bioavailability across different species.

3.1.1.11 Summary of physiological factors that impact oral drug absorption Significant species differences were observed in the anatomical and physiological factors that influence drug absorption in both rats and humans. For example, there are significant species differences in the dimensions and surface areas of the rats and human GI tracts. Furthermore, the unstirred water layer adjacent to the intestinal membrane tends to be four

times smaller in humans relative to rats. Notwithstanding all these differences, the extent of absorption of drug molecules as assessed by the fraction of drug absorbed tend to be similar between rats and humans. For example, Chiou et al . reported a highly significant correlation (p < 0.0001) between rat and human fraction of absorption for 64 drug molecules with different physicochemical and pharmacological properties with wide range of intestinal permeabilities (R 2 = 0.971) [70] . In a subsequent article, Chiou et al . attributed the similarity in the extent of oral drug absorption between rats and humans to the similarity in the intestinal transit time and rate of oral absorption in both species [71] . Although many studies have characterized the species differences in gene expression of drug transporters, further research is needed to evaluate the protein level and transporter activity with specific probe substrates in order to increase our understanding of the functional consequences of potential species differences in transporters and its impact on drug absorption.

3.1.2 Physicochemical and biopharmaceutical factors that impact oral drug absorption A more detailed discussion on the impact of the physico-chemical and biopharmaceutical properties on the drug permeability and solubility is outside the scope of this review and are well documented in a recent review [1] .

3.1.2.1 Lipinski’s rule of fi ve Drug physicochemical properties have a significant impact on the oral drug absorption. Lipinski et al . introduced the rule of five (RO5), which is one of the most widely used concepts to qualitatively predict oral drug absorption. The group analyzed 2245 compounds from the World Drug Index (WDI) database that were either considered for, or entered into, Phase II clinical trials. Results indicate that good oral absorption is more likely with drug molecules that have < 5 hydrogen bond donors (defined as NH or OH groups), < 10 hydrogen bond acceptors (defined as oxygen or nitrogen atoms, including those that are part of hydrogen-bond donors), MW < 500 and a calculated lipophilicity (cLog P) < 5 [58,72,73] .

3.1.2.2 Polar surface area Another widely used physicochemical factor to project oral drug absorption is the polar surface area (PSA) that is assumed to be related to the hydrogen bonding capacity of the drug molecule and defined as that part of the molecular surface (Van der Waals or solvent accessible) that is due to oxygen and nitrogen atoms or hydrogen atoms attached to them. Some workers also included sulfur and phosphorous and attached hydrogen as polar atoms [74] . PSA has been demonstrated to correlate with passive molecular transport through mem-branes, which enables the prediction of intestinal transport properties of various drug molecules. In 1996, Palm et al . showed a good correlation between the PSA of drug molecules and their permeability in two widely used in vitro intestinal

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permeability models (Caco-2 monolayers and rat intestinal segments). A homologous series of β -adrenoreceptor anta-gonists were used as model drug molecules. Excellent correlations were obtained between the PSA of these molecules and their permeabilities in Caco-2 cells and rat ileum (R 2 : 0.99 and 0.92, respectively) [75] . In a subsequent study, Palm et al . found an excellent sigmoidal correlation (R 2 : 0.94) between PSA and human F abs for 20 drug molecules that had a wide range of human F abs values (0.3 – 100 %) and physicochemical properties. These drug molecules were predominantly absorbed by passive diffusion [76] . Based on the regression analysis, the group concluded that drug molecules with a PSA values > 140 Å 2 would show poor oral absorption (< 10%) and drug molecules with PSA < 60 Å 2 could be predicted to show complete oral absorption (> 90%). Similar findings were reported by Kelder et al . who calculated the PSA for 2000 oral drugs that had reached Phase II trials. The group divided the compounds into oral CNS drugs (n = 776) and oral non-CNS drugs (n = 1590). They reported that the orally active non-CNS drugs that are transported passively through the transcellular route tend to display a PSA value of < 120 Å 2 . Alternatively, orally active CNS drugs tend to exhibit a PSA value that is < 60 – 70 Å 2 [77] .

3.1.2.3 pH partition theory and pK a Most drug molecules are either weak acids or bases. When given orally, the extent of the ionization of these drugs is dependent on their p K a and pH gradient inherent to the GI tract. The interplay between these two interrelated parameters has a major impact on the drug permeability and solubility and ultimately the rate and extent of oral drug absorption. One of the original concepts that govern the oral drug absorption is the pH partition theory. This theory states that oral drug absorption takes place mainly by a passive diffusion of the un-ionized form of the drug molecule through the lipophilic intestinal membrane [78] . The fraction of the un-ionized form of a basic or acidic drug can be calculated by Henderson–Hasselbalch equation (Equations 13 and 14) [57] . Thus, for monoacidic compounds:

(13)

pH pIonized concentration

Un ionized concentrationa� �K log10 -

and for monobasic compounds:

(14)

pH pUn ionized concentration

Ionized concentrationa� �K log10

-

Accordingly, the oral absorption of a weak base will be favored in the intestine rather than the stomach, as the intestine has a higher pH and, as a result, a higher proportion of the un-ionized drug fraction will be available for oral

absorption ( Figure 2 ). On the contrary, weak acid absorption will be favored in the stomach owing to its lower pH ( Figure 3 ) [57] . However, the intestine continues to be the main site of absorption of both weak acids and weak bases because of the larger surface area available to absorption as compared to stomach and colon [79] .

In addition to its impact on drug permeability, drug pKa also influences the drug solubility. It is well recognized that both p K a and the intrinsic drug solubility (solubility of the unionized form) play a pivotal role in determining the drug solubility under the GI pH conditions [80,81] . It is of interest to note that a low p K a of an acidic drug or high p K a of a basic drug does not necessarily guarantee high drug solubility in the GI pH range unless the drug has high intrinsic solubility [82] .

3.1.2.4 Particle size Drug dissolution rate is an important parameter that affects oral drug absorption [83,84] . A drug is defined as being poorly soluble when its dissolution rate is so slow that dissolution takes longer than the transit time past its absorptive sites, resulting in incomplete oral absorption. Based on the Noyes–Whitney equation, many factors can affect the dissolution rate of a drug (Equation 15) [24,85,86] .

(15)

DRA D

hC Cs� �

⋅( )

For Equation 15, DR is the dissolution rate, A is the surface area available for dissolution, D is the diffusion coefficient of the drug, h is the thickness of the boundary diffusion layer adjacent to the dissolving drug surface, C s is the saturation solubility of the drug in the diffusion layer and C is the concentration of the drug in the bulk solution at time, t. As shown in Equation 15, the drug dissolution rate is directly proportional to the surface area of the drug particle, which in turn is increased with decreasing particle size. This can be accomplished by micronization or by the use of nano sus pension to reduce the particle size of the drug and, therefore, increases drug dissolution rate, which usually is associated with an increase in the extent as well as rate of oral absorption [82,83,87] . Examples of a drug for which reducing its particle size had significant impact on its dissolution rate is griseofulvin. This molecule has a particularly low solubility and was thus studied as a micronized powder with a median particle size of 3 µm [88,89] . Measurement of the amount dissolved in water versus time using a micronized powder showed that the rate of dissolution depended on the area of contact, which is related to the particle size. Increasing this area was an effective way of increasing the rate of dissolution of this drug [83] .

3.1.2.5 Salt form As noted in Section 3.1.3.3, many drug molecules can be classified as either weak acids or bases that tend to form strong ionic interaction with an oppositely charged counter-ion and

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maintain that interaction through crystallization. The resulting solid comprises charged drug molecules and their associated oppositely charged counter-ions and is usually referred to as salt. The use of salt forms as active pharmaceutical ingredients is well established in the literature [81,90,91] . A salt form of a drug molecule changes the coulombic attraction between the drug molecule and its counterion and alters the potential energy of the solid state. This is usually associated with alteration of the pH of the diffusion layer at the surface of the dissolving solid, and, therefore, significantly increases the solubility of the parent drug molecule (C s ), in that layer over its inherent solubility at the pH of the dissolution medium (C). In general, these changes can result in a significant increase in the dissolution rates and higher apparent solubility of the drug molecules in physiologically relevant timescales. Overall, if other relevant factors, such as chemical stability, permeability, and intestinal and liver metabolism, remain constant, the dissolution rate of a compound should determine the rate of build-up of blood levels with time and the maximal levels achieved [81,90-92] .

In summary, the drug salt form usually alters the drug dissolution rate by modifying the diffusion layer pH at the surface of the dissolving solid [90] . Nelson was the first to report this phenomenon in which the salts of acidic theophylline with high diffusion layer pH’s had greater in vitro dissolution rates than those exhibiting a lower diffusion layer pH. In fact, the rank order of dissolution rates of theophylline was closely correlated with the clinical blood exposure. This report led

many additional studies that demonstrated the influence of the salt form on drug dissolution and the benefit of changing nonionized drug to salts [81,82,90-97] .

3.1.2.6 Polymorphism and drug amorphous form Polymorphs of a drug substance are chemically identical. However, due to the differences in their molecular packing, they have different physical properties such as crystal shape, molecular density, melting temperature, hygroscopicity and enthalpy of fusion [82,94] . Despite these differences, the various polymorphs tend to have comparable solubility profile. Pudipeddi and Serajuddin evaluated the effect of various polymorphs of drug molecules reported in the literature on their solubility profiles. The group reported that the solubility values of various polymorphs for these drug molecules did not differ more than two-fold. This difference in the solubility value is not expected to have a profound impact on the compound biopharmaceutical profile depending on the doses used, particle sizes and solubility values [98] . However, polymorphism may influence the physical and chemical stability of various drug molecules by influencing the rate and mechanism of decay [99-101] . Examples are carbamezepine [100] , indomethacin [96] , furosemide [102] and enalapril maleate [103] .

There are significant differences between crystalline polymorphs and the amorphous form of a drug. In general, the amorphous form tends to have significantly higher dissolution rate and solubility compared with their crystalline forms, which may significantly increase their rate and extent of

0Solution pH

Base pKa = 3.3Base pKa = 6.6

Base pKa = 9.9

0.0

0.2

0.4

0.6

Fra

ctio

n o

f d

rug

un

-io

niz

ed 0.8

1.0

1.2

2 4 6 8 10 12 14

Figure 2. Schematic diagram to illustrate the relationship between the fraction of drug un-ionized, and pH and pKa of bases.

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oral absorption. However, the amorphous form is generally less chemically stable due to the lack of a three dimensional crystalline lattice, higher free volume and greater molecular mobility. The chemical stability of amorphous systems has been discussed in detail elsewhere [99,104-106] .

3.1.2.7 Drug complexation The drug complexes of interest are generally divided into two major categories based on the energy of attraction between the components of the complexes. They are: i) covalently linked complexes; and ii) ionic/inclusion complexes. It is interesting to note that the energy of attraction of covalently linked complexes is ∼ 100 kcal/mol, whereas in the latter type of complexes it is < 10 kcal/mol. Examples on covalently linked complexes are prodrugs that are prepared by chemical modification of the drug through the addition of a labile moiety, such as ester group [107] . This approach is widely used to increase drug solubility/permeability and thus improving drug bioavailability. The labile groups are usually broken by enzymatic action, and the parent drug is freed to produce its pharmacological action. The prodrug approach has been widely used in the development of bacampicillin, chloramphenicol, pivampicillin and enalapril [107-109] .

Inclusion compounds, which form the second category of complexes, result more from the architecture of molecules than from their chemical interaction. One of the constituents of the complex is trapped in the cage-like molecular structure of the other to yield a stable arrangement. Cyclodextrins have been most widely used for this purpose because they can trap lipophilic drugs in their molecular envelope and form a complex having a comparatively more hydrophilic character [110] . It is well established in the literature that a complex formation of a drug with cyclodextrin is known

to improve drug solubility or dissolution rate, and, thereby, oral bioavailability [95,110-112] .

It should be stressed that the drug molecules can also form complexes that may adversely affect their oral bioavail-ability. One widely reported example is the complexation of tetracycline with aluminum, calcium or magnesium ions to form an insoluble complex that cannot be absorbed [113,114] . Before the complexation phenomenon was known, the administration of antacids with tetracycline was suggested to minimize the GI disturbance (nausea and vomiting) caused by the antibiotic [115] . As most antacids contain aluminum or magnesium hydroxide and/or calcium carbonate ions, such coadministration have greatly reduced the bioavailability of the antibiotic. However, complexation can also arise due to the calcium ions present in milk and other dairy products [116] . For example, for democycline, only 13% was absorbed when administered with milk. Doxycycline has been reported to be less prone to complexation with dairy products, yet only 10% was absorbed when coadministered with aluminum hydroxide gel [115] .

3.1.2.8 Summary of physicochemical and biopharmaceutical factors that impact oral drug absorption The physicochemical and biopharmaceutical properties of drug molecules have a profound impact on oral bioavailability by influencing both drug permeability and solubility [21,57,117] . These properties are inherent in the molecules and thus are largely species independent. The physicochemical factors of drug molecules affect the permeability and solubility of NCEs in an opposite direction. Therefore, discovery scientists should closely evaluate the effect of these factors on the oral

Solution pH

Acid pKa = 3.3Acid pKa = 6.6Acid pKa = 9.9

Fra

ctio

n o

f d

rug

un

-io

niz

ed

0

1.2

1.0

0.8

0.6

0.4

0.2

0.0

2 4 6 8 10 12 14

Figure 3. Schematic diagram to illustrate the relationship between the fraction of drug un-ionized, and pH and pKa of acids.

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absorption of drug molecules and seek to establish a balance between solubility and permeability.

3.2 Metabolism In addition to absorption, one of the most important factors that influence oral bioavailability is drug metabolism. Metabolism of an orally administered drug that occurs between the GI tract (site of administration) and the systemic circulation (site of measurement) as it passes for the first time through organs of elimination (liver and intestine) is called first-pass metabolism [118] .

Even for drugs that are completely absorbed from the GI tract, absolute oral bioavailability can be low due to the impact of first-pass metabolism particularly for moder-ate to high hepatic extraction ratio drugs (0.3 ≤ E h ≤ 1.0) (e.g., lidocaine) [119-121] . The relationship of absolute bioavailability to F h and F g for a compound undergoing complete absorption (F abs = 1) can be seen in Figure 4 . First-pass metabolism can occur in the intestines, the liver or both. The clinical relevance of intestinal first-pass metabolism has been observed in studies using midazolam [122] and ciclosporin A [123] . In a similar manner to drug absorption, first-pass metabolism is also influenced by both physiological (involving metabolizing enzymes) and physicochemical/biopharmaceutics factors.

3.2.1 Physiological factors that impact drug metabolism When deciding if preclinical data is appropriate to use for human projections, it is critical to understand, in vitro , the rate and the metabolic fate of the compound in both species and determine if the intrinsic metabolic clearances or extraction ratios are comparable. The following section focuses on cross-species comparison examples for two major drug metabolizing enzyme families.

3.2.1.1 Species differences in metabolic enzymes 3.2.1.1.1 Cytochrome P450s CYP enzymes are a superfamily of drug-metabolizing enzymes that require NADPH as a cofactor. The CYP superfamily is divided into families (i.e., CYP1, CYP2, etc.; > 40% amino acid identity), subfamilies (i.e., CYP2C, CYP2D; > 55% identical) and finally identified by individual enzyme (i.e., CYP3A4, CYP3A5) [124,125] . In animals as well as humans, CYPs are found in virtually every organ including the liver, small intestine, kidney, brain, skin, lung, nasal epithelium and testis. However, the predominant sites of CYP drug elimination are the liver and intestine [126,127] .

The most important human CYP enzymes involved in drug metabolism are CYP1A2, -2C9, -2C19, -2D6 and -3A4 with CYP2B6, -2C8 and -3A5 playing a role in the metabolism of certain therapeutic agents [128-135] . CYP2A6 and -2E1 play only a minor role in xenobiotic metabolism. Although CYP2D6, -3A4 and -2C9 constitute ∼ 50% of the total hepatic CYP protein, these three enzymes metabolize nearly 80% of drugs available in market [127,136] .

The CYP1A subfamily consists of CYP1A1 and -1A2 in rats and humans. Between humans and rats there are significant regions of similarity and highly conserved regions for both CYP1A1 and -1A2 [137] . CYP1A2 is constitutively expressed in the liver of both humans [137,138] and rats [139] . CYP1A1 is constitutively expressed at very low levels in rat and human liver. CYP1A1 is present in the small intestine [140-142] , lung [143] and placenta [144,145] . CYP1A1 expression levels are variable in human small intestine and have been shown to be inducible by cigarette smoking and ingestion of chargrilled meats [146,147] . Furafylline inhibits both rat and human CYP1A2. Interestingly, the potency is 1000-fold less for rats than humans. This species difference in furafylline inhibition suggests a major difference in the active site topography between rat and human orthologs of CYP1A2 [148] .

In humans, the CYP2C family consists of CYP2C8, -2C9, -2C18 and -2C19 [125] . CYP2C8, -2C9, -2C18 and -2C19 are polymorphically expressed in humans [149] . The order of relative abundance of the CYP2C mRNA determined in the liver and small intestine of 15 patients undergoing gastrec-tomy or pancreatoduodenectomy were CYP2C9 > CYP2C8 > CYP2C19 > CYP2C18 [150] . The order of relative abundance of CYP2C protein was the same as that observed for the mRNA (except for CYP2C18 which was not detected in either liver or small intestine) with the overall abundance of CYP2C protein greater in the liver than small intestine. The onset of CYP2C hepatic activity occurs during the first week after birth in humans which correlates with mRNA and protein levels [151] .

In rats, the CYP2C family consists of several isoforms, including CYP2C6, -2C7, -2C11, -2C12, -2C13, -2C22 and -2C23. There are sex and developmental stage-dependent differences in the expression of the CYP2C family in rats [152,153] . However, sex differences in CYP2C metabolism have not been observed in humans [154,155] .

In humans, CYP2D6 consists of one isoform and is expressed in various tissues including liver and small intestine [127,142,156] . CYP2D6 exhibits polymorphism with > 80 identified alleles within the human population [156] . In contrast, there are six CYP2D isoforms in rats [125] . The rat isoforms are CYP2D1, -2D2, -2D3, -2D4, -2D5 and -2D18. In evaluating the enzymatic activity of expressed CYP2D isozymes, all evaluated rat (CYP2D1, -2D2, -2D3 and -2D4) and human recombinant CYP2D isoforms possessed activity for the 1 ′ -hydroxylation of bufuralol [84] . However, the 1 ′ 2 ′ -ethenylation of bufuralol was catalyzed only by rat CYP2D4 and human CYP2D6, indicating a difference between the rat and human CYP2D catalytic properties. The inhibition profile is also different for rat CYP2D as compared with human. It is interesting to note that human (not rat) CYP2D is inhibited by quinidine, whereas rat CYP2D is weakly inhibited by quinine [157,158] .

In humans, CYP3A consists of four isozymes (CYP3A4, -3A5, -3A7 and -3A43). CYP3A4 and -3A5 are expressed in both liver and intestines. CYP3A4 is considered the most

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abundant CYP3A enzyme in the liver. CYP3A5, which is polymorphically expressed, may be the predominant enzyme in some individuals [127,135,159,160] . CYP3A catalytic activity in the intestines was the greatest in the jejunum followed by duodenum and ileum [161] . CYP3A7 is considered a fetus specific form of CYP3A; however, the CYP3A7*1C polymorphism causes the persistence of enzymatic activity of CYP3A7 during adult life [162] . CYP3A43 is minimally expressed in liver with low catalytic activity compared to CYP3A4 [163,164] . Sex differences in CYP3A4 activity have been observed using the probe substrate midazolam. It should be emphasized that the overall differences in the systemic exposure that are related to the sex differences were of negligible clinical importance [165] .

In rats, CYP3A consists of several isoforms, including CYP3A1, -3A2, -3A9, -3A18 and -3A62. There are sex- and developmental-stage-dependent differences in the expression of the CYP3A family in rats. CYP3A2 and -3A18 are male-specific forms, whereas CYP3A9 is a female dominant form [166,167] . CYP3A1 and -3A2 were only detected in the liver, CYP3A9 and -3A18 were detected in both liver and intestines, and CYP3A62 is a predominant form in the intestines [168] . GeneChip analysis of both rat and human intestine showed no correlation between the enzyme expression level of CYP3A4 and -3A9 in either duodenum or colon [9] .

In regards to the catalytic ability of CYP3A, nifedipine, a known probe substrate for CYP3A4, is not metabolized by rat CYP3A1, but by rat CYP2C [169] . Rat CYP2C is also the rat

isoform involved in the metabolism of another CYP3A4 human substrate sildenafil [170] . In contrast, CYP3A is involved in the metabolism of midazolam in both rats and humans [171] . Species differences have also been observed in induction where rifampin is a strong inducer in humans than rats [172] . Conversely, dexamethasone is a stronger inducer in rats than humans [172] .

3.2.1.1.2 Uridine 5 ′ -diphosphate-glucuronosyltransferases Uridine 5 ′ -diphosphate (UDP)-glucuronosyltransferases (UGTs) are a superfamily of drug metabolizing enzymes that require UDP-glucuronic acid as a cofactor. The UGT superfamily is divided into two families (UGT1 and UGT2) with members of the UGT1 subfamily sharing > 50% identity to each other, but they are < 50 % identical to members of the UGT2 subfamily [173] . The most common human UGT enzymes involved in drug metabolism are UGT1A1, -1A4 and -2B7 [174] .

In rats and humans, UGTs are located in various tissues with tissue specificity dependent on the isoform. In humans, UGT1A1, -1A3, -1A4, -1A6, -1A9, -2B4, -2B7, -2B15 and -2B17 have been identified in the liver based on mRNA levels [175] . UGT1A1, -1A3, -1A6, -1A8 and -2B7 have been identified in human small intestine based on mRNA levels [175] . In rats, the UGTs identified in liver based on mRNA levels include UGT1A1, -1A5, -1A6, -2B1, -2B2, -2B3, -2B6 and -2B12 [176] . UGT1A1, -1A2, -1A3, -1A6, -1A7, -2B3, -2B8 and -2B12 have been identified in rat intestines based on mRNA levels [176] .

Fg = 1

Fh

F (

abso

lute

bio

avai

lab

ility

)

00

0.2

0.2

0.1

0.4

0.4

0.3

0.6

0.6

0.5

0.8

0.8

0.7

0.9

1

1

Fg = 0.9

Fg = 0.75

Fg = 0.5

Fg = 0.25

Fg = 0.1

Figure 4. Relationship of absolute bioavailability to varying values of Fg and Fh following complete absorption (Fabs = 1). Fabs: Fraction of the dose that is absorbed from the intestinal lumen to the intestinal enterocytes; Fg: Fraction of the dose that escapes pre-systemic intestinal fi rst-pass elimination; Fh: Fraction of the dose that passes through the liver and escapes pre-systemic liver fi rst-pass elimination.

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In regards to species differences, species-dependent region-selectivity in glucuronidation has been suggested to be caused by different UGT isoforms responsible for the meta-bolism in each species. Species-dependent region-selectivity has been observed for the glucuronidation of AZ-1193714, luteolin and quercetin for rats and humans [177,178] .

3.2.1.2 Species differences in the fi rst-pass metabolism and its impact on oral bioavailability In addition to understanding the species differences in the metabolic profile of a NCE (including the specific enzymatic pathways), an understanding of the species differences in the rate and extent of first-pass metabolism is crucial for the appropriate projection of human bioavailability from preclinical data. This is illustrated using the examples of atomoxetine and indinavir.

Atomoxetine is a CYP2D6 substrate with an absolute human oral bioavailability of 94 and 63% in poor and extensive metabolisers of CYP2D6, respectively [179] . The moderate-to-high human oral bioavailability suggests nearly complete oral absorption of atomoxetine. On the other hand, the absolute oral bioavailability of atomoxetine in rat is only 4% [180] . In the case of atomoxetine, a second preclinical species was also evaluated. The oral bioavailability of atomoxetine in dog was 74% [180] . Overall, the disposition of atomoxetine is similar in rats, dogs and humans with a primary oxidative metabolite of 4-hydroxyatomoxetine that is subsequently conjugated to form 4-hydroxyatomoxetine- O -glucuronide. In a radiolabeled study in rats administered 14 C-atomoxetine, the atomoxetine AUC following oral administration accounted for only 2% of the total 14 C AUC as compared with 30% of the 14 C AUC following intravenous administration, indicating extensive first-pass metabolism in rats [180] . In a corresponding radiolabeled study in dogs administered 14 C-atomoxetine, the atomoxetine AUC following oral administration accounted for 33% of the total 14 C AUC as compared with 39% of the 14 C AUC following intravenous administration, indicating less pronounced first-pass metabolism in dogs [180] . This example clearly illustrates the importance of understanding not only the species differences in the drug metabolic fate, but also the extent of species differences in the first-pass metabolism when using preclinical data to project human oral bioavailability.

Indinavir, a CYP3A4 substrate, is an HIV protease inhibitor for which variable bioavailability data were reported in preclinical studies. For example, the bioavailability of indinavir varied from 72% in dogs to 19% in monkeys and 24% in rats [181] . The differences in oral bioavailability across species were mainly attributed to species differences in the extent of hepatic first-pass metabolism. Chemical and immunochemical inhibition studies indicated the potential involvement of CYP3A isoforms in the metabolism of indina-vir in rats, dogs and monkeys [181] , which is consistent with the observation that CYP3A4 is the main isoform responsible for the oxidative metabolism of indinavir in human liver

microsomes [182] . The in vitro profile of indinavir metabolism was qualitatively similar across species [181] . In addition, an in vitro – in vivo correlation was established in rats using the in vivo hepatic clearance and hepatic first-pass extraction ratio obtained from in vitro rat metabolic data. Based on the in vitro – in vivo correlation established in rats, the in vitro intrinsic clearance of indinavir in human liver microsomes pro-jected a small first-pass metabolism in humans (E h = 0.25) [181] . This in vitro profile is consistent with the high oral bioavailability of indinavir (60 – 65%) that is observed in humans at clinically relevant doses [183] . This example depicts the importance of establishing an in vitro – in vivo correlation in tested preclinical species so as to use it as a basis to project human clearance and oral bioavailability.

3.2.1.3 Summary of physiological factors that impact drug metabolism Species differences in metabolic enzymes between rats and humans have been well characterized with the CYP enzymes and to a lesser extent with the UGT superfamily of enzymes. An important implication of this species differences is that the projection of bioavailability from rat data to human needs to be made with caution, as illustrated by the examples from atomoxetine and indinavir. Despite this potential limitation, there are approaches that can be used to improve the robustness of the projection of human oral bioavailability. For example, if an NCE demonstrates poor oral bioavailability in rats due to extensive first-pass metabolism, additional investigations in rat can be conducted to establish the validity of using rat in vitro data to project the rat in vivo first-pass metabolism. Once the in vitro – in vivo relationship for metabolic clearance has been established in rats, an understanding of the similarities and differences in the metabolic profile and metabolism rate between rat and human in vitro will provide additional insight regarding the appropriateness of extrapolating the in vivo rat bioavailability data or the human in vitro data to project human oral bioavailability.

3.2.2 Physicochemical factors that impact drug metabolism The relationship between lipophilicity and clearance is a key parameter for evaluation in early drug discovery during the critical stage of chemical template optimization [184] . For neutral drugs with LogD values > 0, the contribution of hepatic clearance to the overall clearance tends to increase with lipophilicity such that hepatic clearance can become the major route of elimination for highly lipophilic drugs [184] . This relationship between unbound hepatic intrinsic clearance and drug LogD has been observed for multiple chemical classes including both a series of β - adrenergic antagonist compounds and a series of calcium channel antagonists [184] . However, as a caveat, even highly lipophilic constituents need to have access to the active site of the metabolizing enzyme. Therefore, not only the lipophilicity

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of the subsistent but also its steric hindrance may impact metabolic clearance and should be considered when designing NCEs [185] .

4. Expert opinion

Oral bioavailability is a pivotal factor in the success of NCEs because low oral bioavailability is associated with higher interindividual variability and may compromise the therapeutic usefulness of the NCEs [6] . Typically, oral bioavailability is assessed in the rats during the early phase of drug discovery, with the data being used to project to that in humans. However, Cao et al . demonstrated a major limitation of this approach in that there is a poor correlation in bioavaila-bility value between rats and humans [9] . As reviewed in this paper, the key determinants of oral bioavailability involve a complex interplay of the physicochemical/biopharmaceutical properties of the NCEs and the biological processes involved in drug absorption and first-pass metabolism [79] . The physicochemical and biopharmaceutical properties of the NCEs are generally species independent, whereas many of the physiological factors that influence drug absorption and metabolism generally show species differences between rats and human. The latter factor renders extrapolation of the bioavailability data from in vivo rat study to humans challenging. Despite this limitation, it appears that the in vivo rat model can serve as a useful tool to assess drug absorption.

Early application of in silico computational models and in vitro ADME assays (such as lipophilicity, polar surface area, permeability, solubility and metabolic stability

determinations) can serve as useful guide to determine the factors that may influence oral bioavailability as well as to facilitate the optimization of desirable ADME properties to drive drug design prospectively. In instances where oral bioavailability is limited by extensive first-pass metabolism, additional evaluations employing in silico , in vitro and if practical, in vivo models would significantly improve the robustness of the projection. In addition, use of previous ADME knowledge with the evaluated chemical series, particularly if information from human is available, is essential to drive any structural optimization and design successfully.

Recent advances in the use of in silico computational models to predict ADME properties of compounds likely will continue to grow rapidly as the benefits they provide in throughput and early application in drug design are realized. In addition, there is an increasing range of models available that are focused on projecting various drug pharmacokinetic parameters including bioavailability [186] . This keen interest is now driving demand for developments in the component elements of model building, namely higher quality datasets, better molecular descriptors and more computational power, and the quality of models is improving rapidly [187-189] . Another area that will enhance the projection of oral bioavail-ability involves elucidation of the role that various drug influx and efflux transporters may play in absorption and clearance, as well as a firm understanding of species similarities/differences in drug influx and efflux transporters. Incorporation of the knowledge in this area into the in silico computational or in vitro models likely will enable a more robust extrapolation of a compound’s pharmacokinetic profile to humans.

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Affi liation Susan Hurst1 , Cho-Ming Loi2 , Joanne Brodfuehrer3 & Ayman El-Kattan † 4

†Author for correspondence 1 Senior Principal Scientist, Pfi zer Global Research and Development, Department of Pharmacokinetics, Dynamics and Metabolism, 2800 Plymouth Road, Ann Arbor, MI 48105, USA 2 Associate Research Fellow, Pfi zer Global Research and Development, Department of Pharmacokinetics, Dynamics and Metabolism, 2800 Plymouth Road, Ann Arbor, MI 48105, USA 3 Senior Director, Pfi zer Global Research and Development, Department of Pharmaceutical Sciences, 2800 Plymouth Road, Ann Arbor, MI 48105, USA 4 Senior Principal Scientist, Pfi zer Global Research and Development, Department of Pharmacokinetics, Dynamics and Metabolism, 2800 Plymouth Road, Ann Arbor, MI 48105, USA Tel: +1 734 622 2261 ; Fax: +1 734 622 4349; E-mail: Ayman.El-Kattan@Pfi zer.com, [email protected]

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