Pharmacology of Chiral Compounds: 2 ... - Ingenta Connect

Current Drug Metabolism, 2001, 2, 37-51

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Pharmacology of Chiral Compounds: 2-Arylpropionic Acid Derivatives M.F. Landoni* and A. Soraci** *Cátedra de Farmacología. Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata. Calle 60 y 118, cc 296 (1900) La Plata. Buenos Aires. Argentina **Cátedra de Toxicología. Facultad de Ciencias Veterinarias. Universidad Nacional del Centro de la Provincia de Buenos Aires. Campus Universitario. (7000) Tandil. Buenos Aires. Argentina Abstract: Molecules exist as three dimensional structures. Therefore they can exist in symmetrical and asymmetrical forms. Molecules with an asymmetric centre are chiral. If the molecule and its mirror image are non-superimposable, the relationship between the two molecules is enantiomeric and the two stereoisomers are enantiomers. Since enantiomers have very similar or identical physicochemical properties, it is very difficult to distinguish between them in an achiral environment. However, once in a chiral environment, as in the body, they exhibit clear differences. In fact, most of the physiological processes in nature are stereospecific. Stereospecificity can occur in pharmacokinetic processes, in particular that utilise a carrier protein, receptor or enzyme. In addition, stereoselectivity occurs in pharmacodynamic processes and the differences between enantiomers can be either qualitative and quantitative. 2-arylpropionic acid derivatives (2APAs - profens) are an important subgroup within the class of NSAIDs . These are chiral compounds marketed as racemic mixtures. Some members of the group in an speciesdependent manner undergo a special type of metabolic transformation leading to partial inversion to the optical antipode through a specific conjugation with CoA (coenzyme A) and subsequent epimerization. This metabolic inversion has not only pharmacological consequences (related to clinical effect) but also toxicological consequences such as, formation of hybrid triglycerides and even inhibition of fatty acid ßoxidation. Differences on inversion rate between compounds and species will be discussed as well as its modification by different patho-physiologic processes such as, inflammation.

1.

INTRODUCTION

Symmetry (or lack of it) is a feature of geometric figures with two or more dimensions. As molecules are three dimensional figures, they exist in symmetric or asymmetric forms. The most common basis for molecular asymmetry is a tetravalent carbon atom with four different groups attached to it. This carbon is the *Address correspondence to this author at the Cátedra de Farmacología. Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata. Calle 60 y 118, cc 296 (1900) La Plata. Buenos Aires. Argentina; Fax: +54-221425-7980; Email: [email protected]

1389-2002/01 $28.00+.00

asymmetric centre and the molecule is chiral. If the molecule and its mirror image are nonsuperimposable, the relationship between them is enantiomeric, and the two stereoisomers are enantiomers. Although each member of a pair of enantiomers differs from the other in the spatial arrangement of the groups attached to the chiral centre, most of their physical properties (melting point, boiling point, refractive index, solubility, etc) are identical. The only major difference between the isomers of an enantiomeric pair is the way in which they interact with plane-polarized light.

© 2001 Bentham Science Publishers Ltd.

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Current Drug Metabolism, 2001, Vol. 2, No. 1

Since enantiomers have very similar or identical physicochemical properties, it is very difficult to distinguish between them in an achiral environment [7]. However, in a chiral environment, as in the body, they exhibit clear differences [4]. In fact, most of the physiological processes in nature are stereospecific [4]. A quarter of all therapeutic agents are marketed and administered to humans as racemic mixtures (50:50 of each enantiomer). It is important to highlight that this mixtures are not drug combinations in the sense of two or more co-formulated therapeutic agent, but combinations of isomeric substances whose biological activity may reside predominantly in one particular optical form [8]. The important point of using racemic mixtures is to understand that their use can be thought of as polypharmacy, with the proportions of the various optical forms present being dictated by chemical rather than pharmacological or therapeutic criteria. Moreover, it is now appreciated that pharmacologically less active or inactive isomers may well contribute to the toxicity or adverse effects of the drugs [8]. 2.

ESTEREOISOMERISM

The first reports that some natural products exhibited optical activity were presented by Biot between 1815 and 1818. This author discovered that some natural liquids (oil of lemon, oil of turpentine) and solutions of some natural products (camphor, sugar) were able to rotate the plane of polarized light [9]. The concept of stereochemistry was introduced by Louis Pasteur in 1848, when he noticed that the ammonium sodium salt of tartaric acid crystallised in two forms, differing slightly in the arrangement of the crystal faces, and that a solution of the righthanded crystals deviated plane polarized light to the right (denoted (+)) whereas a solution of the left-handed crystals resulted in an equal deviation, but to the left (denoted (-)). Pasteur also noted that mixing equal weights of the two forms of crystals resulted in an optically inactive solution. As a result, he proposed that the optical activity is

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determined by molecular asymmetry, which produces non-superimposable mirror image structures [10]. The first theory to explain the phenomenon of optical activity was formulated by Van'Hoff and LeBel in 1874. They proposed, independently, that the four valences of a carbon atom are directed toward the corners of a tetrahedron. When a carbon atom has four different groups attached to it, the resulting molecule generally exists as two non-superimposable mirror images (termed enantiomers) and the molecule is dissymmetric or chiral [11]. Carbon is not the only atom that can act as an asymmetric centre; phosphorus, sulphur and nitrogen can also form chiral molecules. The arylpropionic acid derivatives are a group of non steroidal anti-inflammatory drugs (NSAIDs) that possesses an asymmetric centre on their molecules, therefore they are chiral compounds (See Fig. 1)). 3.

NOMENCLATURE

Any material which rotates the plane of polarized light is termed "optically active". An isomer of optically active compound can rotate the plane of polarized light to the left (levorotatory) in which case it will be designated "l or -" or to the right (dextrorotatory) in which case it will be termed "d or +". It is important to bear in mind that this nomenclature refers only to the effects on polarized light and give no information concerning absolute configuration. Indeed, this is a dependent parameter and varies with concentration, temperature, pH and the wavelength of light use to determine rotation. Another system for specifying chirality is the "L/D system" (Fisher convention). This system relies on the chemical correlation of the configuration of the chiral centre to D-glyceraldehyde or L-serine. The compounds which can be correlated to D-glyceraldehyde are named D, those correlated to L-serine are designated as L. It is important to note that although D-glyceraldehyde

Pharmacology of Chiral Compounds

Current Drug Metabolism, 2001, Vol. 2, No. 1 39

CH 3 H3 C

C

H CH 2

COOH

H

CH 3

H H3 C

CH 3

C

C

CH 2

COOH

CH 3

H

R(-) IBUPROFEN

S(-) IBUPROFEN

O C

C

O

H

H CH 3

C

H3 C

COOH

C

C

COOH

R(-) KETOPROFEN

S(+) KETOPROFEN

O

O H

H C

H3 C

CH 3 COOH

C

COOH

R(-) FENOPROFEN

S(+) FENOPROFEN

Fig. (1). Chemical structure of some 2-arylpropionic acids NSAIDs.

is dextrorotatory (rotates the plane polarized light to the right), the compounds correlated with it do not have to be dextrorotatory, i.e could rotate light to the left. Therefore, D-prefix is not correlated with (+) or (-) specific rotation, and the Dcompounds can be l (or -) and vice-versa, Lcompounds can be d or (+). These nomenclature systems has been abandoned in favour of the Cahn-Ingold-Prelog (CIP) nomenclature [12]. This system is based on a set of rules for ordering the substituents attached to the asymmetric atom by ligands precedence rules (1-4). There are different ligands precedence rules, being the simplest : "ligands of the higher atomic number precede those with lower ones". The central atom and the three ligands are viewed from the direction of the vector C→4 , where 4 is a ligand of lowest precedence (See Fig. 2)). If the ligands 1-3 are ordered such that the movement from the ligand of the highest precedence (1) to

the third (3) passing the second (2) in between is in the clockwise direction (sequence 1→2→3) the configuration is designated as R (rectus) on the other hand, if the same requires movement in the anti-clockwise direction the configuration is designated S (sinister). The configurational designation is preceded by the number specifying the location of the chiral centre. The existence of these two systems of nomenclature has led to complications, making recognition of the biological properties of enantiomers difficult. In addition, a third system of signs was proposed in 1989 by Simonyi et al [13], adding further confusions. As emphasised by Brown [14] it is necessary to establish a clear system of nomenclature to convey the fundamental fact that stereoisomers are different compounds with potentially differing biological properties. Stereoisomers are not different forms of the same compound.

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2

3

C 1

C

4 1

3

CLOCKWISE R CONFIGURATION

4 2

COUNTERCLOCKWISE S CONFIGURATION

Fig. (2). Schematic representation of the Cahn-Ingold-Prelog (CIP) nomenclature. The central atom and the three ligands are viewed from the direction of the vector C→4 , where 4 is a ligand of lowest precedence . If the ligands 1-3 are ordered such that the movement from the ligand of the highest precedence (1) to the third (3) passing the second (2) in between is in the clockwise direction (sequence 1→2→3) the configuration is designated as R (rectus) on the other hand, if the same requires movement in the anti-clockwise direction the configuration is designated S (sinister).

4. ARYLPROPIONIC TIVES

ACID

DERIVA-

While there is confusion over the nomenclature on this area, the convention has been to categorise the α-methyl-substituted phenylacetic acids (eg. Ibuprofen, fenoprofen, ketoprofen, naproxen) as arylpropionic acid derivatives (APAs) [15]. 4.1. Stereoselective Pharmacodynamics The primary anti-inflammatory action of the NSAIDs is mediated by inhibition of the prostaglandin cascade. There are two isozymes of cyclooxygenase (endoperoxide synthase), that catalyse the first step in the prostaglandin synthesis: the conversion of arachidonic acid to prostaglandin H2 (PGH2). Cyclooxygenase 1 (COX-1) was initially purified from sheep vesicular glands [16 - 17] and is constitutively expressed in most tissues [18] and in blood platelets [19]. COX-1 is probably involved in the production of prostaglandins involved in cellular "housekeeping" functions, such as coordinating the actions of circulating hormones [20

- 21 -22] and regulating vascular homeostasis. COX-2, which shares about 62% aminoacid identity with COX-1, is expressed only following cell activation [23 - 24]. The biosynthesis of COX2 is stimulated by serum, growth factors and phorbol esters in fibroblasts [25] and by lipopolysacharide in monocytes/macrophages [24]. The observations that expression of COX-2 is stimulated by mediators of inflammation and that expression is inhibited by glucocorticoids [26] suggest that COX-2 may produce prostanoids involved in inflammation (More extensive reviews on this subject [22 - 27] ). For the 2 arylpropionates, the most important structural feature for antiprostaglandin activity is the stereochemistry at the α carbon. All studies have shown that the S(+) enantiomers are many times more potent COX inhibitors agents than the R(-) enantiomers [28]. There are few reports concerning the relative ability to inhibit COX-1 and COX-2 enzymes. Meade et al. [29] reported a 2 fold preference for COX-2 inhibition for S(+)ibuprofen. Naproxen (a pure S(+)compound) shown a COX-1/COX-2


potency ratio of 6.3 [30]. Assays in canine cells have shown a COX-1/COX2 ratio for carprofen of 129, 181 and 4.9 for the racemic mixture, S(+)enantiomer and R(-)enantiomer, respectively [31]. Although inhibition of cyclooxygenase is considered the major mechanism of action of NSAIDs, there have been studies reporting antiinflammatory effects independent of cyclooxygenase inhibition. For example, they have been shown to modify the oxidative burst induced by a range of stimuli in human neutrophils [32] and bovine neutrophils [33]. Also inhibition of ßglucuronidase release from the same type of cells in man [34] and from horse synoviocytes [35] have been reported. It is interesting the fact that for some of these effects enantiomers are equipotent [34 - 36]. On this context, Brune et al., [37] have observed on clinical trials that pure S(+)enantiomers are superior antiinflammatory drugs but only equal to racemic mixtures as analgesics, suggesting that this is because the R(-) enantiomer contributes to this effect. This suggestion is supported by an study on flurbiprofen, which applied an experimental pain model based on both chemo-somatosensory event related potentials (CSSERP) and subjective pain rating [38]. This authors shown that both flurbiprofen enantiomers produce equipotent analgesic effect. An important fact that makes difficult to analyse the pharmacodynamics of APAs is the characteristic metabolic chiral inversion these drugs can undergo. Therefore it is very important to avoid conclusions based exclusively on in vitro studies.


Levy & Boddy [3] have proposed that pharmacokinetic parameters for chiral compounds may be classified according to three levels of organisation in the body (macromolecules, whole organ and whole body), being the hybrid character of parameters inversely proportional to the degree of stereoselectivity expected in those parameters. The pharmacokinetic characteristics of the enantiomers usually observed after administration of a racemic mixture differ from some of this drugs. These differences are also specie-specific (Table 1). Table I. Differences on AUC of Enantiomers of Some APAs on Different Species after Administration of Racemic Mixtures Compound IBUPROFEN

Reference

-

Man Horse Sheep Dog Rat

S(+) > R(-) ? ? ? S(+) = R(-)

[39]

FENOPROFEN

-


S(+) > R(-) S(+) > R(-) S(+) > R(-) S(+) > R(-) S(+) > R(-)

[39] [40] [41] [40] [86]

KETOPROFEN

-


S(+) = R(-) S(+) > R(-) S(+) < R(-) S(+) > R(-) S(+) > R(-)

[42] [43 - 44] [45] [42] [86]

CARPROFEN

-


S(+) = R(-) S(+) < R(-) S(+) < R(-) S(+) < R(-) S(+) > R(-)

[42] [46] [42 - 47] [47 - 95] [72]

4.2. Stereoselective Pharmacokinetics Enantiospecificity in pharmacokinetics arises because of enantioselectivity in one or more of the processes of drug absorption, distribution, metabolism and excretion. Enantioselectivity results from the interaction of chiral drugs with a chiral system, the body.

Species

4.2.1.

[68]

Absorption

Arylpropionic acid derivatives as well as the majority of drugs are absorbed by diffusing passively through membrane matrices, therefore, stereoselectivity on this process is not important. This is due to the fact that enantiomers do not differ in their lipid and aqueous solubility.

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However, differences can arise on dissolution rates since, the aqueous solubility and crystal forms of racemates can differ from those of individual isomers. In fact , using DSC and x-ray diffractometry has been demonstrated that aqueous solubility of racemic, S(+) and R(-)ketoprofen differ (182, 259 and 304 µg/ml, respectively) [48]. Also, when matrix tablets are made with racemic and physical mixtures of ketoprofen, stereoselective drug release is observed, being faster the release of the S(+) enantiomer compare to that of its optical antipode. This effect is more marked when chiral excipients such as, hydroxypropylmethylcellulose are used [48]. 4.2.2. Distribution 4.2.2.1. Plasma Protein Binding NSAIDs are strongly bound to plasma protein. These drugs are administered for treating inflammatory processes, where there is a major increase in acute phase proteins and a decrease in albumin level, which can have a significant effect on binding that can be reflected on clinical efficacy [49]. Most NSAIDs are bound to human serum albumin specially at two sites, site I and site II [50]. In human beings the specific binding site for APAs has been identified as site II (indole- and benzodiazepine-binding areas) [51]. Most studies have shown that interactions with this site are enantioselective [52 - 53]. Binding is favourable for S(+)enantiomers and dependent of albumin concentration [54 - 5]. This is not true for horses, at least for ketoprofen. Extent of protein binding of ketoprofen enantiomers in this species is similar and appears not to be concentration dependent [55]. In vitro studies on plasma protein binding of ibuprofen, ketoprofen and 2-phenylpropionic acid have shown that the presence of one enantiomer affects the protein binding of the other in a racemic mixture.

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mentioned, NSAIDs are highly bound to plasma protein, being this feature a disadvantage for penetration into joints. However, due to the increased irrigation in inflamed sites, leaking of plasma proteins carrying NSAIDs is observed. This fact explains the lack of relationship between plasma half-life and observed effect [43 - 56]. This has been demonstrated for ketoprofen in a range of species (bovine, ovine, horse) where the AUC for exudate is almost twice that for transudate (56 -57 - 58]. An important point to bear in mind when analysing penetration of APAs into inflamed sites is the pharmacodynamic differences between enantiomers. As previously mentioned, S(+)enantiomers are the pharmacologically active inhibitors of cyclooxygenase (eutomers). Therefore, it is expected that S(+)enantiomers modulate their own distribution by inhibiting inflammation and thus decreasing permeability and access to the "inflamed sites" [58 - 59]. Since collection of synovial fluid is an invasive procedure, studies on time course of synovial fluid concentrations in humans are scarce. However, there are some experimental models for studying the disposition of NSAIDs on extravascular compartments. Oelkers et al., [59] have developed a model consisting on skin suction blister, on which inflammation can be induced by u.v exposure of skin. Studies on flurbiprofen applying this model have shown no differences on the rate constants of transfer into and out of the blister between enantiomers. Trans-synovial transport is a diffusioncontrolled process, regulated by physico-chemical properties of the drug and the pathophysiological status of the synovial membrane [60]. Therefore, differences between enantiomers are not expected. However, this could be overshadowed by the enantioselectivity on plasma protein binding as well as differences on S:R ratios on plasma.

4.2.2.2. Passage to Synovial Fluid

4.2.3.

Stereoselective metabolism

NSAIDs are drugs extensively used in the treatment of rheumatic diseases, being on this situation, the synovium the major site of action. As

It is well recognised that stereochemical factors play an important role in xenobiotic metabolism. Enantioselectivity may be reflected either as a



difference in the biotransformation of chiral compounds (substrate enantioselectivity) or in production of chiral metabolites (product enantioselectivity).

severity of the inflammatory reaction and the reduction on the inversion rate of ketoprofen. The same is true for ibuprofen in humans as a consequence of stress [61].

2-Arylpropionic acids or profens show particular kinetic properties associated with the chiral inversion metabolic phenomenon. This metabolic way allows the R enantiomer, generally inactive, to transform into the enantiomeric form S, responsible for the therapeutic effects [4]. Thus, the stereoconversion is responsible for the disparity of the results obtained in vivo-in vitro on the prostaglandin inhibitory action of enantiomers. Besides, there is an important inter-compound and inter-species variation.

Inversion Mechanism

The degree of chiral inversion can be modified by the presence of inflammation. Verde et al., [58] have shown in horses a correlation between the

The inversion mechanism is carried out in three stages [62 - 63]. 1).

In a first step, the carboxylic substrate is transformed by a microsomal or mitochondrial acyl-CoA ligase into an intermediary thioester. The involved enzyme would be the long chain fatty acids ligase (E.C.6.2.1.2.3). The thioesterification process involves the incorporation of CoA-SH on the carboxylic function of the enantiomer when a stereospecific enzyme is present [64 -65- 66] (Fig. 3)). The enantiospecificity of the substrate of this enzyme determines the unidirectional

Long-chain fatty acid CoA synthetase CoA ATP Mg ++

COOH

Hydrolysis R-(-)-Profen-CoA

R-(-)-Profen

C H

CH 3

Enzymatic epimerization

R-(-)-Profen

S-(+)-Profen-CoA

Hydrolysis

COOH C H

CH 3

S-(-)-Profen

Fig. (3). Chiral inversion process of aryl-2-propionic acids or profens. A three-step process which begins with the enantiospecific enzymatic formation of a thioester between the R-enantiomer of the 2-APA and CoA. This thioester may be hydrolysed to regenerate the R-enantiomer or may undergo epimerization to yield the thioester in which the 2-arylpropionyl moiety has the S configuration. Subsequent hydrolysis of this S(+)profen-CoA completes the inversion process.

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Landoni and Soraci

character of the inversion process of the profens. 2).

3).

The thioester formed is later racemized by a reversible enzymatic system: racemase or epimerase of the 2-arylpropioniates to a new thioester presenting an opposite configuration [67]. Finally, a hydrolase releases the enantiomer completing the chiral inversion process.

Inversion Direction The inversion direction can be studied by in vivo or in vitro methods. In vivo, the intravenous administration of each of the enantiomers (R and S) individually to an animal species shows in plasma the decrease of one of them and the simultaneous appearance of its optic antipode. In vitro, the initial reaction of the thioesterification with CoA is the limiting and specific stage. The inversion exploration may be the result of the correlation determined in vivo and of the intensity of the thioesterification reaction. The intensity and the direction of chiral inversion of profens depend on the eventual substrate stereospecificity of the thioesterification. Experimental studies show many different situations according to the compound and the animal species involved. -

Non-inversion compounds: certain profens are not inverted in vivo neither do they play the role of substrate of the acyl-CoA ligase in vitro. An example of that is carprofen in humans and rats [68].

-

Unidirectional inversion: this situation occurs when the acyl-CoA ligase has high enantioselectivity. Normally the inversion process is carried out from the R to the S enantiomer, being this process the most frequently found in relation to the profens.

-

Reciprocal inversion: the lack of stereoselectivity of the acylCoA ligase may lead to the formation of two possible thioesters: R-profen-CoA and S-profen-CoA, easily racemized to the forms S-profen-CoA and R-profen-CoA. An example of it is the acid phenyl-2-propionic and the hydratropic

acid, in rat and dog, ketoprofen in mouse and ibuprofen in guinea pig, rat and rabbit [69 70 - 71 - 72]. However, a pitfall of some in vivo studies is the high dose of racemate administered, which may cause the lost in the stereoselectivity of the substrate. Indeed, Knights & Jones [73], pointed out that at high concentrations (1mM) the S ibuprofen enantiomer would be metabolised to the thioester intermediary state. A similar situation has been described with rat microsomes for the S enantiomer of fenoprofen [74]. Cellular Location of Inversion Enzymes a)

AcylCoA ligase

In vitro studies in different animal species have determined that the acylCoA ligase of long chain fatty acids (E.C.6.2.1.2.3) would be the enzyme responsible for ibuprofen and fenoprofen thioesterification [73]. These enzymes are distributed at microsomal, peroxisomal and mitochondrial level [75]. Tanaka et al. [76] purified an acylCoA long chain synthase from rat liver as the responsible for the activation of saturated fatty acids between 10 and 18 C. Knights & Jones [73] reported a biphasic kinetics on the formation of palmytoil CoA, implying in consequence two isoenzimes in that process: one of high affinity and low capacity and another one of low affinity and high capacity. Besides, fenoprofen and ibuprofen competitively inhibit the isoform of high affinity, while ketoprofen and naproxen would not have this inhibiting effect. However, Tracy & Hall [77], Benoit et al. [78] and Soraci & Benoit [79] have demonstrated a monophasic process not being able to determine the implications of the isoenzime of high affinity. The thioesterification of R(-)-fenoprofen is inhibited by palmitate at 20 µM in all animal species studied. b)

Racemase

Schieh & Chen [67], isolated a non-enantioselective epimerase of 2-arylpropionic from homogenate of rat liver. This enzyme is primarily located on mitochondria and cytosol.


c)

Hydrolase

Hutt & Caldwell [68] have studied the activity of hydrolases using homogenates of rat liver. These studies showed that the enzyme is deprived of enantioselectivity and has a maximum activity at cytoplasmic and mitochondrial level. Sites of Inversion Several in vivo studies with APAs have shown that liver, kidney, intestine and lungs participate in the enantioselective metabolism. The R enantiomers of benoxaprofen [80 - 81] and ketoprofen [82] orally administered are pre-systemically inverted. Berry & Jamali [83] studied the participation of liver, stomach and different intestinal segments (duodenum, jejunum, ileum, cecum and colon) in the presystemic and systemic inversion of fenoprofen. They have also shown that the liver and all the intestinal segments take a significant part in the chiral inversion process, while the stomach does not. The observed differences on chiral inversion at presystemic level could explain the differences on bioavailavility between enantiomers. Hall et al. [81] have demonstrated the participation of the lungs on the chiral inversion of fenoprofen and ibuprofen. They have concluded that due to the anatomical location of the lungs the pulmonary metabolism could account for a first pass effect after the intravenous administration of these compounds. Recent studies have involved the brain as a possible site of inversion for ketoprofen. Biological Significance of the Inversion Process From a pharmacological point of view it is easy to conceive the consequences of this phenomenon due to the fact that a generally inactive molecule, under the R configuration becomes an S molecule provided with anti-inflammatory effect. However, the deepest biological significance is linked to the most diverse fates of the intermediary thioester, since this newly formed thioester could play a pivot role in numerous metabolic pathways acting as substrate [84 - 4], such as:


•

Conjugation with Aminoacids

The newly formed thioester group can interact with aminoacids, in particular glycine, taurine and glutamine, [85]. This is important in cats and dogs since these species conjugate mainly with aminoacids [85 - 86]. Tanaka et al [70] observed in dogs that after oral administration 2phenylpropionic acid conjugates with glycine. This conjugation process is not stereoselective since these species can conjugate the two thioesters of the two enantiomers in similar extent . The main places for conjugation are the kidney and the liver, being the responsible enzymes of mitochondrial origin [85]. •

Oxidation of Fatty Acids

Numerous profens have been identified as responsible for the inhibition of the mitochondrial oxidation of fatty acids. This characteristic could explain the observed accumulation of fatty acids at hepatic level, leading to the formation of a mild microvesicular esteatoses [87]. Pirprofen, ibuprofen and flurbiprofen have been identified as inducers of this phenomenon. There are several mechanisms explaining this observation: - A competition between the R enantiomers of the compounds and the fatty acids for the CoA pool [88]. - A competition between the R enantiomers and the fatty acids for the palmitoil CoA ligase. This competition is not observed when naproxen (a pure S compound) is administered [65]. - An inhibition of the β oxidation of palmitate, via a non-stereoselective mechanism independent of CoA such as the disconnection of the oxidative phosphorilation [87]. - Acylcarnitine The thioester of CoA formed could serve as substrate for the formation of acylcarnitine. Acylcarnitine formation is a necessary biochemical step for allowing the fatty acid passage

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through the mitochrondrial membranes. Zhao et al [87] have proposed that the thioester Ribuprofenyl CoA and R-flurbiprofenyl CoA could be responsible for the formation of a small quantity of R-acylcarnitine. This mechanism could so indirectly opposed the mitochrondial fatty acids ß-oxidation. - Incorporation to the Acylglycerols Since 1978 [89] it is known that a range of xenobiotics containing carboxilic functional groups (e.g. 2-arylpropionic acids) could replace the endogenous fatty acids in triacylglycerols to form unnatural glycerolipids. In vitro studies on rat hepatocytes and adipocytes have shown that incorporation into triacylglycerols is specific for the R enantiomers. Moreover, it was demonstrated that fenoprofen inhibited endogenous triacylglycerol synthesis in vitro at concentrations reached in vivo. [74 - 89]. 4.2.4

Stereoselective Excretion

The major urinary and biliary metabolites of APAs are their acylglucuronides; stereoselectivity in this reaction has been reported [90 - 91 - 92]. Glucuronic acid is a chiral molecule and has the ß-D-configuration. Selectivity on the excretion of glucuronides seems to be dependent on either the compound or the species. For benoxaprofen and ibuprofen in man, as well as 2-phenylpropionic acid in mouse, excretion of the S (+)-conjugates is greater than that of the R (-). The opposite is true for fenoprofen and ketoprofen in rabbit. In rats important differences has been observed on the pharmacokinetics of ketoprofen and ibuprofen enantiomers and this is due to the high degree of inversion of the R (-) enantiomers as well as to the preferential biliary excretion of the S (+) enantiomers. In fact, in rats enterohepatic circulation is suggested to occur particularly for S (+) ketoprofen. The fraction of enterohepatic circulation being estimated on 75% for S (+) ketoprofen versus 8.2% for its antipode [93]. Of the S (+) ketoprofen excreted in bile 84% is reabsorbed, while only 26% of the R (-) suffers the same process [93].

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Carprofen is not inverted in rats, dogs, horses and man, being excreted almost exclusively on bile. In horses and dogs, enantioselectivity in liver glucuronidation and subsequent biliary excretion of S(+) enantiomer has been reported [94 -95]. This fact could explain the predominance of R (-) carprofen in plasma after administration of the racemate in the species. The opposite is observed for this drug in rats, on which S (+) carprofen is the predominant enantiomer in plasma, glucuronide conjugation being stereoselective for the R (-) enantiomer [72]. Disposition of carprofen in man is unique since both enantiomers show similar bioavailabilities and steroselectivity in plasma glucuronides is negligible [96 - 97]. Acylglucuronides formed from carboxilic acid can undergo hydrolysis, acylmigration and covalent binding to proteins. Intestinal pH might alter hydrolysis of these compounds and consequently the extension of enterohepatic circulation. In fact, for carprofen has been reported that acidic pH reduced hydrolysis and acylmigration leading to stabilisation of the acylglucuronides [98]. 5. REGULATION OF CHIRAL MEDICINAL PRODUCTS In spite of the number of years elapsed since the first description of optical activity of organic molecules in solution by Pasteur, the importance of chirality is not fully appreciated. The lack of appreciation of the fact that stereoisomers are different compounds and that enantiomers often exert different biological effects is a factor leading to failure to recognise the significance of chirality in assuring safety, quality and efficacy of medicinal products. One of the most commonly used argument against pure enantiomeric products has been the high cost of producing compounds in enantiomerically pure forms. Nowadays, this argument is not valid anymore, since there are commercially available HPLC and gas chromatographic chiral stationary phases, which allow separation of enantiomers [99].


The Food and Drug Administration was the first Authority to quote the subject of chirality in a regulatory statement [100], which said "the agency is impressed by the possibility that the use of single enantiomers may be advantageous (1) by permitting better patient control, simplifying dose response relationship (2) by reducing the extent of inter-patient variation in drug response". The FDA and the European Union on Proprietary Medicinal Products (CPMP) [101] have both issued formal guidelines. The regulatory authorities of Switzerland, Australia and Nordic Countries have also promulgated formal guidance. When analysing the regulatory guidelines for chiral compounds it is necessary to differentiate between new drugs from purified enantiomers of approved drugs. For new drugs, although each authority has different ways to deal with this matter, there are some points in common. In all cases the first requirement is to report the chiral character of the molecule and to identify the stereoisomer(s) responsible for the pharmacological activity. Moreover, stereochemical features of the molecule such as, assignment of absolute configuration, an enantioselective analytical method and the full description of the synthesis (with special attention to the formation of the chiral centre) must be informed. Probably the most important addition to the common requirement for drug approval is the chemical, preclinical and clinical justification of the stereoisomeric form chosen for marketing. When a racemic mixture is chosen, the requirements are similar to that for any new chemical compound. However, there are additional requirements such as, a). - The justification for the election of the racemate over the single enantiomer; b). - The pharmacokinetic evaluation of individual enantiomers and c). - The description of any metabolic chiral inversion occurring in vivo. The use of the racemic mixture instead of the single enantiomer is justified when: there is little


difference between enantiomers in activity, pharmacokinetics and toxicity; optical instability of enantiomers in vitro or in vivo and an inability to obtain the desired enantiomer in sufficient quantity at acceptable and reproducible optical purity [8]. If a single isomer is chosen, the only additional requirements are, the justification for the selection, the documentation of its synthesis (specially the formation of the chiral centre) and the confirmation of optical stability in formulation, on storage and in vivo [8]. Regulations concerning purified enantiomers of approved drugs are a controversial issue. So far, FDA does not recognise purified enantiomers of approved drugs as "new drugs". Therefore, it does not grant them a period of market exclusivity. However, since 1997 FDA has opened a formal comment period for considering whether to grant new drug status to these purified compounds and to afford them up to five years of market exclusivity (FDA). [102 - 103]. 6.

CONCLUSIONS

Chirality is a very important issue in pharmacology due to the clinical consequences of administering a racemic mixture not knowing the pharmacokinetics and pharmacodynamics of each enantiomer. In the special case of APAs, it has been shown that the pharmacological activity resides on the S (+) enantiomers, being the R (-) enantiomers not only pharmacologically inactive but also toxicologically important due to its capacity to replace one of the three fatty acids constituting the formation of a hybrid triacylglycerols. Important differences on the disposition of APAs have been reported not only between species but also between drugs. These differences could be on distribution, metabolism and excretion, to a lesser degree on absorption. The members of this group of drugs can suffer metabolic chiral inversion from the R (-) enantiomer to the active S (+) enantiomer.

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However, it is important to bear in mind that metabolic inversion is species and drug dependent and also can be modified by the presence of inflammation or stress and this modifications can be reflected clinically.

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The significance of stereochemical considerations has become an issue for researchers, pharmaceutical industry and regulatory authorities. However, this has not taken place on academia. The most commonly used textbooks for undergraduate students on biomedical sciences do not have a chapter devoted to chirality. It is desirable to change this in the near future.

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