Development and Applicability
6.6 DRUG ELIMINATING AGENTS AND MECHANISMS
culture format. They are known as “small wells within large wells,” wells within a well such that cells form organs that are cultured within the small wells, surrounded by the drug-containing large wells described in [32].
This device permitted the maintenance of the human liver cells’ pheno- typic functions for several weeks. It allowed the evaluation of gene ex- pression profiles, Phase I/II metabolism proteins, secretion of liver-specific products, and susceptibility to hepatotoxins. Application of this technique shows prospects in overcoming aspects of the preclinical failures in drug discovery.
6.5.3.2 Integrated Microcell Culture Systems
In a similar arrangement, the microcell culture analog (mCCA) approach was developed to create a cell culture environment that is close to that of humans, to improve the credibility of the drug PK and PD profiles [33].
The device consists of separated chambers connected with microchannels to link the different chambers of cultured cells of the liver, tumor, and mar- row, to simulate the blood flow. The microenvironment provided by the mCCA was intended to simulate more of the in vivo environment in than a conventional monolayer culture.
6.5.3.3 Three-Dimensional Cell Patterning and Culturing
Cell culture systems created to very closely represent the human physi- ological characteristics are interaction prone, as they produce a fertile envi- ronment for cell–cell and cell–matrix interactions [34,35]. This platform is adaptable to variable cell patterning methods.
6.5.3.4 Membrane-Based Multilayer Microfluidic Devices
This technology reproduces the in vivo microenvironment of kidney tubular cells. It was created in an attempt to resolve the issue of the lack of a tissue- like microarchitecture in traditional two-dimensional systems. Using this device, cell polarization and cytoskeletal rearrangement studies have been made more predictive [36].
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of enzymes and the UDP-glucuronosyltransferases (UGTs)r enzymes which limit systemic exposure. The small intestine is regarded as an absorptive organ and may act as a rate-limiting barrier. CYP3A4 is the most abundant P450 present in human hepatocytes as well as the intestinal enterocytes [37–39].
6.6.1 Metabolizing Enzymes: CYPs (P450)
A prominent drug activity in the body is drug metabolism, which is what the body does to the drug. Metabolism refers to the biotransformation of a parent drug by drug-metabolizing enzymes. In addition to bile and re- nal excretion, drug metabolism contributes to determining the fate of the drug in the body. CYPs are the most popular enzyme studied in drug de- velopment. They are the Phase I metabolizing enzymes chiefly involved with oxidation of foreign compounds including the xenobiotics and select medicinal compounds, committing them to the elimination pathway. They could transform nontoxic compounds to toxic reactive intermediates. Drug metabolism is generally regarded in the context of Phase I oxidation. The oxidation activity mostly involves the nonpolar molecules, which are oxi- dized by the addition of oxygen atoms, usually in the form of a hydroxyl moiety (–OH group). Phase II conjugation involves adding a very water- soluble molecule such as glucose (glucuronidation) or sulfate (sulfation) to the organic group, especially at the –OH site. Early determination of the downstream metabolic effect is a key to successful clinical studies. CYPs play a role in drug–drug interactions through drug activation or inhibition [40,41].
Around 75% of approved drugs are metabolized by CYPs, particularly the five major isoforms [42]. Single nucleotide polymorphism has been at- tributed to several species, including CYP2D6 and CYP2C9. As discussed earlier, size and flexibility have been attributed to multiple and simultane- ous ligand binding. CYP3A4 metabolizes the most active of all the CYPs with a reported metabolizing activity of up to 50% of all drugs [43]. In silico methods of modeling ADMET models the protein flexibility, which is a fundamental step in its bioactivity – conformational changes have been reportedly induced by ligand binding. The hepatocytes or liver microsomes are liver cells that are used in clinical trials to model the activity of the me- tabolizing enzymes [44,45].
6.6.1.1 Assays Used in Evaluating Drug Metabolism
Metabolic assay is used to identify metabolic stability in chemi- cal structures in connection with hepatic metabolism. It evaluates the
rate of clearance of a drug using the liver hepatocytes or microsomes.
The disappearance of the parent chemical with time is an example of a change. Metabolic profiling involves incubation of drug compounds with in vitro hepatic metabolic systems or cells of which the major metabolites are identified with mass spectrometry. The metabolites identified enable structure optimization to prevent a choice metabolite from forming. It is also applicable in the selection of animal species with similar physiology that produce identical metabolites to humans for assigning the “relevant animal species.”
6.6.2 Drug–Drug Interactions
The effects of a drug depends on the concentration levels at various sites of drug action (relates to the PD), or the blood or tissue concentration of the drug. Elimination of a drug or its active metabolites therefore affects the concentration at the sites of drug action. This occurs either by metabo- lism to an inactive metabolite that is excreted, or by direct excretion of the drug or active metabolites. Identifying metabolic drug–drug interactions that elicit these changes enables the determination of the extent to which toxic or active metabolites are formed [46].
One example is the inhibition of the metabolism of a nondrowsy an- tihistamine, terfenadine, by the antifungal drug etoconazole. Concomitant administration of both terfenadine and ketoconazole lead to fatal cardiac ar- rhythmia because of the elevated level of terfenadine. Terfenadine has been recalled owing to its drug–drug interaction potential. Ketoconazole is a well-known potent inhibitor of CYP3A4, the P450 isoform responsible for metabolism of terfenadine.
6.6.2.1 Types of Drug–Drug Interactions
There are two types of drug–drug interactions: metabolism driven and ab- sorption and excretion driven. Metabolism-driven drug–drug interaction has been widely characterized as it has the most impact and is the reason why it is the subject of most biological investigations in drug discovery.
Metabolism-driven drug–drug interactions can differ among individuals based on genetic variation of a polymorphic enzyme. For example, a strong CYP2D6 inhibitor results in the increase of the plasma levels of a CYP2D6 substrate in subjects who are extensive metabolizers of CYP2D6, but which is reduced in subjects who are poor metabolizers of CYP2D6. This is be- cause there are no available active enzymes to be inhibited. Inhibition of
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drug interactions for a specific drug may occur based on a combination of drug–drug interactions, which follow multiple interaction mechanisms:
• Simultaneous inhibition and induction of one enzyme transporter by a drug
• Inhibition of drug elimination by inhibitor of the same enzyme that metabolizes the drug
• Inhibition of enzyme/transporter in subjects with varying degrees of impairment of drug eliminating organs (e.g., liver or kidney)
Drug-metabolizing enzyme induction strategies have been used in in vitro human liver-based experimental systems to evaluate drug–drug interactions. A drug that can induce any of these enzymes, for example, CYP1A2, CYP2A6, CYP2B6, CYP2C9, and CYP3A4, could cause in- ductive drug–drug interactions [47]. Drug–drug interaction assays are in general focused on P450 isoforms, with CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 as the major isoforms studied. The same experimental systems are applicable for evaluating drug–drug interactions involving Phase II [46] and transporters [48,49].
Absorption and excretion-driven drug metabolism involves biliary and renal drug transporters and small plasma protein binding (the risk is low; may be important for highly bound drugs with narrow therapeutic windows).
6.6.2.2 Plasma Binding Proteins as Drug Eliminating Compounds Model plasma binding proteins are human serum albumin (HSA) and alpha 1-acid glycoprotein, known to bind a high number of drugs. Interaction of these types of proteins with drugs leads to elimination of a fraction of the drug in a protein-bound state while the free and unbound portion ex- erts the therapeutic action. They have important biological roles, mostly in the transport of important biomolecules that are fairly insoluble. Some ex- amples are hormones, vitamins, and fatty acids. These proteins have several binding sites for drugs located in their IIA and IIA domains [50].
The drug’s ligands that bind to negatively charged sites are warfarin, azapropazone, and dansylamide [51]. The aromatic carboxylic acids like ibupro- fen, diazepam, and arylpropionic acids bind to the smaller site II (the diazepam site), while propofol and oxyphenbutazone, among others, bind within the five other sites identified in the three domains. The drugs that can bind to at least two sites are azapropazone, indomethacin, and fatty acids. The in silico 3D HSA paradigm was utilized to terminate binding of methionine aminopeptidase-2 inhibitors plasmaproteins [52,53]. Lipophilicity and the charge of a molecule at physiologic pH affect the ability of a drug molecule to bind to the plasma
proteins, but increases with lipophilicity or cationic charge and decreases with increasing anionic charge at physiologic pH [54].
6.6.3 Transporters
Transporters are present with varying abundance in all tissues in the body and play important roles in drug targeting, drug absorption, drug distribution, and elimination. The safety profile of a drug is altered when its concentration or that of its metabolites is affected in various tissues.
Transporters are expressed in the renal tubule cells. They play a role in the active renal secretion and reabsorption of endogenous compounds and xenobiotics. A large superfamily of proteins transport molecules across the membrane barriers within the human body and about 48 members have been identified [55]. P-glycoprotein (Pgp) is the most important efflux transporter and very active across the intestinal epithelium and the blood–
brain barrier. Verapamil and cyclosporin are examples of drugs transported through the Pgp–efflux pathway. The multidrug resistance protein MRP1 and the MRP2 transporter, as well as multidrug and toxin extrusion pro- teins, are major transporters on the apical membrane. The synchronous ac- tivities of these transporters promote active renal secretion of compounds.
Meanwhile, peptide transporter 2 and system L amino acid transporters, etc., are more associated with active reabsorption.
There are marked species differences in transport function and trans- porter expression of these transporters. Along the proximal tubule is re- markable heterogeneity and this has posed difficulty in prediction of renal elimination of actively secreted compounds from in vitro data [56]. A recent study indicated that monkeys are superior to rats and dogs, a notion that is supported by the fact that monkeys are evolutionarily closest to humans, and which is the reason why both uptake and efflux transporters in mon- keys have been shown to be more predictive of humans than the other species mentioned [57,58]. They are actively involved in the multidrug re- sistance effect, an activity that reduces drug potency.
Multidrug resistance protein 1 is a Pgp, the first ABC transporter and the most characterized transport protein. Its role in expelling molecules out of the cell results in reducing the fraction of drug available for the thera- peutic function. This is further exacerbated by its broad substrate specificity as it can recognize a wide range of drug compounds regardless of charge distribution or molecular structure. The transporter substrate binds to its high-affinity binding site on the membrane domains resulting in multiple simultaneous binding to ligands in up to seven binding sites [59].
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6.6.3.1 UDP-Glucuronosyltransferases
The UDP-glucuronosyltransferases (UGTs) are Phase II enzymes that induce drug metabolism through covalent addition of a glucuronic moi- ety to a drug or endogenous compound. The three major ones are UGT1A1, UGT1A4, and UGT2B7, which account for 15, 20, and 35% of the UGT-metabolized drugs, respectively [60].
6.7 APPLICATION OF ZEBRAFISH AS A MODEL