In Order to Exert a Therapeutic Effect, a Drug Should Have High Affinity and Selectivity

DRUG ABSORPTION

In order to exert a therapeutic effect, a drug should have high affinity and selectivity for its intended biological target (for example a protein or a protein complex in or on a particular cells type) and also reach a sufficient concentration at this site. In general, to match this goal, the drug has to be released from the delivery system, transported from the site of application to the site of action, biotransformed and finally eliminated (metabolism) from the body. Thus, the whole process can be divided into the pharmaceutical phase (where drug liberation takes place) and the pharmacokinetic phase describing the course of the drug in the body and its metabolism. Undoubtedly, both the first and the second phase are strongly dependent on mass transport and that is why it plays a central role in determining delivery system reliability and effectiveness.

1Oral Drug Absorption

1.1 Introduction

Oral drug formulations are non-invasive, convenient, easy, and flexible, and thus they usually meet better patient compliance.

Figure 1.1. Schematicrepresentation of drug absorption by GI tract. Solubility and permeability steps are underlined.

They also make easier to provide dose-to-dose and batch-to-batch reproducibility. For these reasons, the oral route continues to be the dominant and preferred way of dosing both existing and new drugs[i],[ii].

The crucial point for the success and reliability of a whatever formulation is represented by drug bioavailability, defined as the rate and extent to which the active drug is absorbed from a pharmaceutical form and becomes available at the site of drug action[iii]. Despite drug dissolution, drug permeability through the intestinal wall, metabolism, lumen stability and gastrointestinal (GI) physiological factors highly affect drug absorption rate by living tissues, bioavailability mainly depends on drug solubility and permeability. Indeed, the drug has to dissolve in the hydrophilic GI fluid and then it has to cross the lypophilic cellular membrane (Fig. 1).

1.2.3Pharmacokinetics

In order to exert a therapeutic effect a drug should have high affinity and selectivity for its intended biological target (= receptor) and also reach a sufficient concentration at this site[iv]. A receptor is defined as the protein or a protein complex in or on a cell that specifically recognizes and binds to a compound acting as molecular messenger. In a broader sense, the term receptor is often used as a synonym for any specific drug binding site, as opposed to non- specific interactions, e. g. to plasma proteins. Factors which influence the possibility of the drug to reach this receptor have to be considered. This process is affected by the liberation of the drug from its formulation environment, its subsequent transport from the site of application to directly adjacent compartment (= absorption), its transport to deeper compartments (= distribution), its biotransformation and its elimination from the body[v]. These steps together determine the percentage of drug available in the circulation for further distribution to the macromolecular receptor site (= bioavailability). The extent to liberate from the formulation the incorporated drug in a sufficient amount herein of a particular importance (= pharmaceutical availability). Thus, the whole process determining the bioavailability of a drug can be divided into two phases – first, into a pharmaceutical phase where drug liberation takes place, and second into so called pharmacokinetic phase which describes the course of the drug in the body. As a result of these two processes a biological response (= biological activity) leading to a therapeutic effect may be obtained.

Pharmacokinetics is the study of the time course of drug absorption, distribution, metabolism and excretion (ADME), and how these ADME processes are related to the intensity and time course of the pharmacological (therapeutic and toxic) effects of drugs[vi].

The oral bioavailability (F) of drugs is defined as the fraction of the ingested dose that is available to the systemic circulation[vii]. Thus, both absorption and elimination processes determine the oral bioavailability of a given drug. A general representation of oral bioavailability is described in figure 1.3 and can be estimated as:

F = Fa * Fg * Fh * Fl(1.1)

where F refers to systemic oral bioavailability, Fa is the fraction absorbed across the intestinal wall, and Fg * Fh * Fl is the product of the fractions escaping clearance by the gastrointestinal tract, liver and lung.

Figure 1.3. Schematic representation of the process ofdrug absorption following oral administrationof drugs. Drugs are absorbed into the portalcirculation (Fa), with the fraction reaching theportal circulation being Fa . Fg, where Fg isthe fraction not removed by the gut. Thefraction of dose entering the hepatic vein isthe fraction escaping liver clearance and canbe estimated as Fa*Fg*Fh. The arterialbioavailability of the drug is the product ofdrugs escaping the intestinal, liver and lungclearance and can be estimated from Fa*Fg*Fh*Fl.

The product of fraction available after gut and liver extraction (Fg * Fh) following oral administration primarily determines the oral clearance of the drug, although the contribution of lung clearance should also be considered.

Clearance

The best estimate for drug elimination can be obtained by determining the ‘total clearance’ (Cl) of the drug. Organ clearance is defined as the volume of blood that must be cleared of drug in a unit of time in order to account for the rate of drug elimination. Thus, clearance is the ratio of elimination rate of the drug to the drug concentration in blood entering the organ. The total clearance is the sum of all individual organ clearances of the drug. However, it should be noted that not all organ clearances are additive. Because drugs are eliminated by various tissues, including gastrointestinal wall, liver, lungs and kidneys, the anatomical arrangement of organs is quite important. When the eliminating organs are arranged in parallel (such as the liver and the kidneys), the total clearance of the drug can

be estimated by additive clearance from individual eliminating organs. When the eliminating organs are arranged in series (e.g. gastrointestinal tract and liver), a different approach using the products of organ clearance is required for the estimate of total clearance[viii][ix]. Because clearance plays an important role in both drug elimination and oral bioavailability, its prediction is of utmost importance in estimating PK properties of drugs[x]. Most drugs are eliminated primarily by the liver and/or the kidney. Hence, prediction of hepatic and renal clearances are of prime importance in predicting PK properties of NCEs. It is well known that total systemic clearance (Cls) of a drug is estimated as the ratio of dose to area under the curve (AUC) following intravenous administration of the drug:

Cls = Dose(iv) / AUC(iv) (1.2)

where iv is intravenous. Following oral administration, Cloral is defined as:

Cloral = Dose(oral) / AUC(oral) (1.3)

Combining the relationships for clearance and oral bioavailability, Cloral can also be estimated from:

Cloral = Cls / (Fa * Fg * Fh * Fl) (1.4)

where Fa, Fg, Fh and Fl are the fraction of drug absorbed into the portal vein, and fractions not subject to elimination by the gut, liver and lung, respectively. Assuming negligible gut and lung clearance, eq.(1.3) can be reduced to:

Cloral = Cls / (Fa * Fh) (1.5)

If drugs are cleared by both liver and kidneys,

Cls = Clh + Clr(1.6)

where Clh is hepatic clearance and Clr is renal clearance. Thus,

Cloral = (Clh + Clr) / (Fa * Fh) (1.7)

Thus, it is evident from eq.(1.7), that if one were able to predict renal and hepatic clearance, as well as the fraction of drug absorbed into the portal circulation and fraction of drug escaping hepatic elimination, the estimates of oral clearance and oral bioavailability would be quite accurate.

The prediction of renal clearance for humans has been quite successful using interspecies allometric scaling approaches[xi],[xii],[xiii] (allometry is the study of relationships between body size and the structural and functional capacities of organs). Although allometric scaling has successfully predicted renal clearance in humans, it requires experimentation in four to five species, thus limiting the practical value of such an approach. A simpler approach for predicting human renal clearance is to use the ratio of glomerular filtration rate (GFR) between rats and humans[xiv]. With the latter approach, the ratios of renal clearance for various drugs in rats and humans were roughly similar to the ratio of GFR between these two species. Thus, knowledge of renal clearance in rats allows the approximate estimation of human renal clearance using the GFR ratios. Because of the reasonable accuracy of prediction of these two approaches, in vivo urinary excretion data in rats and other species has been used to estimate renal clearance of drugs eliminated by the kidneys.

Despite numerous attempts to predict human hepatic clearance from in vivo studies, interspecies scaling approaches for hepatic clearance have been less successful because of large inherent interspecies differences in the elimination processes[xv]. Rane et al.[xvi]first predicted in vivo hepatic clearance in rats from in vitro data from liver microsomes, taking into consideration hepatic blood flow and unbound fraction of the drug. Based upon the good correlation of predicted and observed hepatic extraction ratios (by isolated rat livers), the potential for in vitro–in vivo hepatic clearance prediction was identified. Models for prediction of hepatic clearance include the use of liver microsomes, isolated hepatocytes, 9000g supernatant (S9) fractions, recombinant (heterologously expressed) CYP isozymes, liver slices and in situ gastrointestinal/ liver single-pass perfusion preparations. All these approaches are reasonably predictive of hepatic clearance when liver metabolism is the predominant contributor to clearance, although they each have some limitations. Heterologously expressed CYPs are not predictive of intrinsic clearance because of contributions of several CYPs in the metabolism of many drugs and because metabolic rates differ extensively with the expression system used. Liver slices are not useful for kinetic predictions due to a lack of uniform diffusion of compounds into all the cells within the slice because of the tissue thickness (~260 µm)[xvii]. Recent advances in understanding the role of efflux pumps such as MDR1 and MRP have brought into question the ability of some of the in vitro approaches to predict hepatic clearance mediated by a combination of liver metabolism and biliary excretion. Liu et al.[xviii]have recently reported that hepatocytes cultured in a collagen-sandwich configuration for up to five days establish intact canalicular networks, maintain MRP2, re-establish polarized excretion of organic anions and bile acids, and represent a potentially useful in vitro model for studying hepatobiliary elimination of compounds. However, to date there are no significant data on the predictive ability of this approach.

Volume of distribution

The volume of distribution is a measure of the extent of drug distribution and is determined by the binding of the drug in plasma as well as tissues. Because it is assumed that the unbound drug can diffuse across membranes, it is implicit that the distribution to tissues is affected by plasma-protein binding. It is also important to understand that because of significant tissue binding for most drugs, the ‘apparent’ volume of distribution far exceeds the total body water (i.e. 58% of the adult human weight). As such, the volume of distribution is the proportionality constant relating the drug concentration in blood or plasma to the amount of drug in the body. Gibaldi and McNamara[xix] have shown that:

V = Vb + Vt . (fb/ft) (1.8)

where V is the volume of distribution, Vb is the blood volume, Vt is the extravascular tissue space volume, fb is the unbound fraction in blood and ft is the unbound fraction in tissues.

Allometric scaling approaches have been proposed for predicting the volume of distribution in humans. As seen from eq.(1.8), the estimation of volume of distribution requires knowledge of both blood-protein binding and tissue binding of compounds. The latter is methodologically quite difficult and few studies assess tissue binding of drugs. It has been reported by Fichtl et al.[xx] that when propranolol volume of distribution was corrected for the unbound fraction, the unbound volume of distribution was virtually identical for all species. These investigators suggested estimating the unbound volume of distribution in laboratory animals and in vitro plasma-protein binding for humans, and predicting human volume of distribution of drugs. However, Boxenbaum[xxi] has shown that the unbound volumes of distribution of many benzodiazepines are significantly different between dog and man. Obach et al.[xxii] have attempted to predict the human volume of distribution using four different methods. They found that allometric scaling across species, especially when excluding the interspecies protein-binding differences, was a poor predictor of human volume of distribution. When in vitro average fraction unbound in tissues of preclinical species was used with human plasmaprotein binding data, best estimates of human volume of distribution were obtained. Furthermore in vitro plasmaprotein binding data in dogs and humans along with intravenous PK in dogs was also a reasonable predictor of human volume of distribution in their analysis.

Elimination half time

The half-life (T1/2) of any drug is related to its apparent volume of distribution (V) and its systemic clearance (Cls) as:

T1/2 = 0.693 . (V/Cls) (1.9)

Thus, the half-life of any drug is a function of blood and tissue binding of the drug as well as its total clearance and is a derived parameter from Cls and V. For drugs with high clearance, the half-life is relatively independent of changes in intrinsic clearance, whereas for drugs with low clearance, increases in intrinsic clearance result in decreased half-life. Few reports are available on the prediction of drug half-life in humans. However, because half-life of drugs plays an important role in dosing frequency, it would be good to have estimates of half-life in humans during the drug-discovery phase. Obach et al.26 tried two approaches for predicting the human half-life for a set of proprietary compounds. One method relied on the direct correlations between animal and human half-life estimates, and the other method estimated individual volume of distribution and clearance, to yield T1/2 values. They reported for the set of compounds evaluated that more accurate predictions of clearance resulted in more accurate predictions of half-lives for humans. Ironically, they found that in vitro clearance methods that excluded estimates of protein binding provided better halflife predictions than when including the protein binding differences among species. In addition, allometric clearance prediction methods, when combined with volume of distribution predictions, provided less accurate predictions than the in vitro methods of estimating clearance. Overall their study indicated that relatively simple animal–human correlation methods based on preclinical intravenous PK data provided reasonably accurate predictions of human drug half-lives and did not warrant the development of complex techniques of extrapolating in vitro metabolism data or PK data from multiple species.

1.2.4Gastro-enteric absorption

Stomach

It is an important organ for drug absorption as the periodicity of its depletion affects the amount of drug feeding the intestine, the most important organ for drug absorption[xxiii] (figure 1.4). In fasted conditions (human beings), particles bigger than 2 mm are expelled from the stomach only during contraction phases that take place at regular intervals. In fed conditions, instead, contractions take place only at the end of the digestion process and this is the reason why drug experiences a longer stomach residence time if encapsulated in particles bigger than 2 mm. The transport to intestine of particles smaller than 2 mm and liquids is not affected by stomach contractions.

Stomach has specialised cells secreting cloridric acid that influences on the percentage of drug present in dissociated/un-dissociated form. Of course, this regards drugs that are weak acids or basis. Finally, the stomach contains particular enzymes (pepsin and gelatinase) and polysaccharides (mucus).

Figure 1.4. Schematic representation of stomach.

Stomach pH ranges between 1 and 2[xxiv]. When acid secretions achieve duodenum, they induce the pancreas to deliver an alkaline fluid (pH = 8) reach in anions HC. Together with the alkaline fluid secreted by intestinal cells and bile, it neutralises the acid. Usually, the upper stomach region (cardias region) is characterised by higher pH, while the inferior portion (pylori zone) is characterised by lower pH.

Intestine

Small intestine is made up by duodenum, jejunum and ileum and it is the main drug absorption site[xxv]. In human beings, duodenum measures approximately 20 cm, while jejunum and ileum are approximately 2.5 m long.

The innermost part of small intestine is made up by the sub-mucosa and mucosa tunica characterised by macroscopic folding. Mucosa tunica is constituted by endothelium, lamina propria and epithelial cells. Every macroscopic folding (figure 1.5) shows, in turn, villi (diameter 0.1 mm, height 0.5 – 1 mm) that have proper artery, veins and lymphatic capillaries. On their surface we can found enterocites that represents 90% of cells constituting villi[xxvi], devoted to food absorption.

Figure 1.5. Schematic representation of small intestine. The frame evidences the existence of a macroscopic folding.

Apical zone is characterised by the presence of a particular brushed structure devoted to absorption (microvilli, diameter 0.1 m, height 1 m). Microvilli are covered by a glycoproteic layer (glycocalix), 0.1 m thick,continuously produced by the enterocites. Probably, it serves for microvilli protection and for digestion facilitation.

Figure 1.6. This picture shows villi carrying, in the apical part, microvilli. Cellular membranes and glycocalix are also visible.