Cytochrome P450 Enzymes, Drug Transporters and their Role in Pharmacokinetic Drug-Drug Interactions of Xenobiotics: A Comprehensive Review

Drug-Drug interactions (DDI) is a serious clinical issue. An important mechanism underlying of DDI, is induction or inhibition of drug metabolizing enzymes (DMEs) and transporters that mediate metabolism, cellular uptake and effl ux of xenobiotics. DDI cannot be avoided in many cases, as they belong to routine medical practice. Especially DMEs and transporters of small intestine, liver, kidney are the major determinants of the pharmacokinetic profi le of drugs. Enzymes and transporters mediated DDI in these three organs can considerably infl uence the pharmacokinetics and clinical effects of drugs. The purpose this review is to elucidate the effect of cytochrome P-450 (CYP 450) enzymes and transporters mediated DDI on the pharmacokinetics and further its clinical implications. Review Article Cytochrome P450 Enzymes, Drug Transporters and their Role in Pharmacokinetic Drug-Drug Interactions of Xenobiotics: A Comprehensive Review Srinivas Maddi1*, Thirumaleswara Goud2 and Pratima Srivastava1 1GVK Biosciences PVT LTD, Nacharam, Hyderabad, 500076, India 2Creative Educational Society’s College of Pharmacy., Chinnatekur, Kurnool, Andhra Pradesh, India Dates: Received: 23 June, 2017; Accepted: 10 July, 2017; Published: 11 July, 2017 *Corresponding author: Srinivas Maddi, Associate Director, DMPK Biology Division, 28A IDA Nacharam, Hyderabad 500076, Telangana, India, Tel: 919701166299; E-mail:


Introduction
Modifi cation of a patient's clinical response to the administered drug by the co-administration of another drug is defi ned as a Drug-Drug Interaction (DDI). Without affecting the drug kinetics, when a pharmacological response is changed either through agonism or antagonism, those DDIs are termed as pharmacodynamic interactions. When we observe the alterations in drug disposition mainly via induction or inhibition of transporters or metabolic enzymes and drug transporters involved in drug absorption, distribution, metabolism or excretion, those DDIs are termed as pharmacokinetic interactions [1].
Pharmacokinetic DDIs are responsible for approximately 20-30% of the adverse drug reactions in general population, they also account for about 10% of the cases under emergency department and contribute 3-5% of the medication errors in hospitalized patients [2,3]. Since the drug pharmacokinetics can be signifi cantly altered by both transporters and enzyme mediated DDIs, it can potentially affect the therapeutic effi cacy or toxicity of drugs [4]. DDIs based on metabolism are specifi cally due to induction and/or inhibition of cytochrome P450 (CYP 450), they have been considered to be the most dangerous ones [5]. Based on the pharmacological and toxicological effects of both the parent drug and its metabolites, the clinical consequences of CYP induction or inhibition depends and if the victim drug has a narrow therapeutic index this may be particularly signifi cant because DDIs based on metabolism may cause changes in the concentration of drug up to 10 fold whose biotransformation is induced or inhibited [6]. This is particularly signifi cant if the parent is responsible for the pharmacological effect and the affected metabolic pathway constitutes its main route of elimination, induction and inhibition may cause potential toxic effects or complete loss of therapeutic effi cacy, respectively. Vice versa, if the parent compound is a prodrug, enzyme induction likely may result in potential toxic effects whereas, inhibition of its metabolic conversion may cause a reduction in therapeutic effi cacy. When reactive metabolites are generated, induction may be dangerous as they can cause serious idiosyncratic reactions frequently [7].
A recent systematic review revealed that, based on in vitro tests performed at the time of drug development, majority of the new molecular entities have been observed to be perpetrators of metabolic interactions. It has been found that 45% of the new molecular entities are the victims of clinically signifi cant metabolic DDIs [8]. Due to enzyme induction or inhibition it is very diffi cult to manage the wide inter individual variability in the magnitude of drug interactions by dose adjustment [9]. By the FDA regulatory guidance, more complex and refi ned predictive models have been proposed and endorsed subsequently. Recently, it has been pointed out that they do not enhance predictive capacity. The underlying mechanism that alters the drug disposition in liver disease is unknown and hence it is diffi cult to predict the effect of liver insuffi ciency on metabolism based DDIs [10,11]. iii. Sulfo transferases (SULTs) iv. N-acetyl transferases (NATs) [12,13]

Cytochrome P 450
The initial step in the biotransformation of xenobiotic compounds is catalyzed by the CYP450 enzymes in the liver.
These enzymes are members of mixed function oxidase family. They catalyze and introduce an oxygen atom into substrate molecules which often results in dealkylated and hydroxylated metabolites. In humans, more than fi fty CYP450 isoenzymes are known to exist. Based on the similarities of amino acid sequence they are classifi ed into 17 families and 39 sub families. At the amino acid level, proteins from the same family have greater than 40% similarity, whereas in the same sub families have greater than 55% homology [14]. According to the standard nomenclature, the family is designated by a number followed by a letter which designates the sub family and a second number which designates the sub family's individual members. Majorly, the drug metabolism can be carried out by the CYP1, 2 and 3 families and occurs mainly in the liver. In our body, the highest concentration of CYP450 is observed in liver. The signifi cance of extra hepatic metabolism in tissues like lung and intestine however is also recognized.

RH + O 2 + NADPH + H +   ROH + H 2 O + NADP +
The isolation of CYP 450 is diffi cult and tedious. The utility of the protein for in vitro metabolism studies have been greatly enhanced by the methods like heterologous expression of recombinant CYP 450 using Baculovirus (BaV) and E.coli [17].

Flavin mono oxygenases (FMOs)
FMOS are associated with the endoplasmic reticulum and catalyze the oxidation of organic compounds using NADPH and molecular oxygen as the source of electrons resulting in the decrease of one of the oxygen atoms. FMOs are similar to CYP450 enzymes but they are mechanistically distinct from the CYP450s, in that they react with NADPH and oxygen in the absence of substrate to form a 4-hydro peroxy fl avin enzyme intermediate. In the cell, FMOs exist in an activated form. Their interaction with a nucleophilic group like phosphate, amine or thiol is necessary for the completion of the catalytic cycle. In human liver, FMO 3 is present in most abundant form and in terms of overall drug metabolism it is believed to be the dominant member [18,19].

UDP glycosyl transferase (UGTs)
By using UDP-glucuronic acid as a donor molecule, UGT catalyzes the glucuronidation of xenobiotics at hydroxyl, sulfhydryl, imino, amino and carboxyl groups. Usually, this generates products which are more hydrophilic. Hence, they are readily excreted in urine or bile.

UDP-glucuronic acid + R  UDP + R-glucuronide
Generally, glucuronidation is classifi ed as Phase-II metabolism. It is the phase that occurs after CYP450 dependent oxidative metabolism. Majority of the compounds need not require prior oxidation because they have functional groups already that can be glucuronidated. Glucuronidation of 5-lipoxygenase inhibitors and UGT2B7 dependent glucuronidation of morphine are the examples of fi rst pass metabolism catalyzed by UGTs. UGTs are located in the lumen of the endoplasmic reticulum. In human two UGT families have been identifi ed that includes UGT1 and UGT2. In human liver, the members of the UGT1 family are expressed in which most of the xenobiotic metabolism takes place which includes UGT1A1, 1A3, 1A4, 1A6 and 1A9 [20][21][22].

Glutathione transferases (GSTs)
The formation of thio ether conjugates between glutathione and reactive xenobiotics by directly adding or displacing an electron withdrawing group can be catalyzed by GSTs.

Sulfo transferase (SULTs)
The conjugation of sulfate groups on to a variety of xenobiotic and endogenous substrates that have the acceptor moieties like hydroxyl and amine groups can be catalyzed by SULT enzymes.

N-acetyl transferase (NATs)
The biotransformation of hydrazine and aromatic amines to the respective hydrazides and amides can be catalyzed by N-acetyl transferases (NAT) by using acetyl co enzyme A as a donor. The O-acetylation of N-hydroxy aromatic amines to acetoxy esters can also be catalyzed by them .

R-NHOH + COA-S-COCH 3  R-NHOCOCH 3 + COA-SH
In humans, there are two NAT isoforms that includes NAT1 and NAT2. NAT1 is expressed in liver and many other tissues, whereas NAT2 is expressed only in the gut and liver [27] In the metabolism of a wide variety of both exogenous and endogenous compounds CYP 450 enzymes are involved that constitute a large family of heme-thiolate protein [28]. In the year 1955, CYP 450 enzymes were fi rst discovered in rat liver microsomes. In the presence of carbon monoxide, they are characterized by an intense absorption band at 450nm [29]. On the smooth endoplasmic reticulum, the CYP 450 mixed function mono oxygenases are located throughout the body; small intestine and liver are the major sites [30]. For a wide range of compounds, these enzymes are responsible for the oxidative (Phase -1) metabolism. Lipophilic drugs can be biotransformed to more polar compounds that can be excreted by the kidneys. In some instances the metabolites can be toxic,

Drug-Drug Interactions Mediates Through CYP Induction
Enzyme induction is a process, in which a drug stimulates a particular isoform of CYP450, and there occurs a gene mediated increase in number of molecules of the DME. Drug that stimulates the enzyme is termed as an "inducer". Induction is a complex, dose related phenomenon requiring the inducer to reach a critical concentration to bind and activate transcription factors at an intranuclear receptor or regulation point, from which up regulation of messenger RNA occurs with subsequent increase in protein production. Induction is a relatively slow process that may start after 3-4 days of exposure to an inducer.
Maximal effect usually occurs after 7-10 days and requires an equal or longer time to dissipate after inducer is stopped.

Drug -Drug Interactions Mediates Through CYP Inhibition
Enzyme inhibition is a phenomenon in which some particular  (Table 1).

Cytochrome P450 Mediated Drug-Drug Interactions In Hepatic Dysfunction
In the pathogenesis of various liver diseases, the hepatic CYPs are involved. Hepatotoxicity can be induced by CYP mediated activation of drugs to toxic metabolites (e.g.: Halothane and Acetaminophen). In some cases, covalent binding of the toxic metabolite to CYP leads to immune mediated hepatotoxicity and anti-CYP antibodies. In the serum of patients with type II auto immune hepatitis, anti-CYP 2D6 antibodies are present.
The mechanism and the pathogenic signifi cance remain unclear. Various studies revealed the role of CYP2E1 in the pathogenesis of non-alcoholic steatohepatitis and alcoholic liver disease. Increased CYP2E1 activity is associated with lipid peroxidation and the production of reactive oxygen species with secondary damage to mitochondria and cellular membranes can be seen in these conditions. It has also been postulated that CYP2E1 has a role as a cofactor for hepatocellular carcinoma due to its ability to activate carcinogens. Particularly for the drugs mediated by CYPs, drug metabolism can be impaired in patients with liver disease.
The activity and content of CYP1A, 2C19 and 3A seems to be more vulnerable to the effect of liver disease whereas CYP2C9, 2D6 and 2E1 are less affected. Based on the etiology of liver disease, the pattern of CYPs isoenzymes alternations also varies. It has been demonstrated that a strong relationship was observed between the activity of CYPs and the cirrhosis severity. To assess the hepatic functional reserve, the usefulness of measuring CYP activity remains uncertain [35].

Effect of hepatic dysfunction on enzyme Inhibition
In hepatic dysfunction, fi ve types of factors can affect the extent of inhibitory DDIs.
2. Hepatic extraction ratio of the drug with inhibited metabolism.
3. Reduced liver uptake of the inhibitor.
4. Nature of the inhibitory interaction (reversible or irreversible).

Reduced enzyme content
In hepatic dysfunction, the expression of various CYP enzymes has been shown to be markedly decreased especially, CYP1A2 and CYP3A4. Decreased enzyme content results in reduced inhibitory effect [36,37] .

Hepatic extraction ratio of the drug with inhibited metabolism
The hepatic clearance of drugs with a low extraction ratio is decreased in proportion to the degree of enzyme inhibition, as to the clearance of these drugs depends on the metabolic capacity of the liver. The clearance of drugs with a high extraction ratio can be determined by liver perfusion and should be unaffected by a reduction in intrinsic clearance caused by enzyme inhibition [38].

Reduced liver uptake of the inhibitor
For various basic drugs which are structurally unrelated, decreased drug uptake by the cirrhotic liver has been observed by the in vitro studies [39].

Nature of the inhibitory interaction (reversible or irreversible)
Liver dysfunction may differentially affect the accumulation kinetics in the liver cell of reversible and irreversible inhibitors.
In between the intra and extra cellular spaces, reversibly binding molecules rapidly equilibrate whereas the binding of irreversible inhibitors is time-dependent and if the inhibitor concentration exceeds that of the enzymatic protein it can precede up to total enzyme inhibition.

Drug-Drug Interactions Via Non Microsomal Enzyme Inhibition (Other than CYP 450)
Non-microsomal enzymes may be inhibited by certain agents to produce signifi cant drug interactions. Allopurinol

Drug-Drug Interactions Via First Pass Metabolism
Drug interactions sometimes depend on extent of fi rst pass metabolism of drugs. Oral bioavailability of a drug is likely to be increased if its fi rst pass metabolism is inhibited by another drug given concurrently that competes with it for fi rst pass metabolism. Propranolol increases bioavailability of chlorpromazine by decreasing its fi rst pass metabolism.
Propranolol decreases breakdown of lidocaine by decreasing hepatic blood fl ow, because Lidocaine metabolism depends on the hepatic blood fl ow. For ABC transporter gene, around seven sub families were identifi ed, encoding for 49 different proteins [45,46]. In particular, transporters belonging to the ABCB, ABCC and ABCG sub families, have specifi cities of drugs [47]. In the transport of a wide range of substrates, SLC and ABC transporters are involved and they share a wide distribution in the body [48].   [55]. Crystal structures of mammalian P-gp were reported very recently, exhibiting distinct incomplete over lapping drug-binding sites in the internal cavity of the protein that provides the fi rst molecular basis for its multispecifi city [56]. P-gp expressed in various tissues is located on the apical side of intestine, kidney epithelia and liver where it decreases systemic drug exposure by limiting oral absorption and enhancing biliary and urinary excretion [ 57].

Transporters for hepatic drug elimination
With high protein binding from the circulation, the liver has the capability to extract the drugs effi ciently. Frequently, the hepatic uptake of drugs is followed by phase-I and A strong association of SLCO1B1 variants with an increased risk of myopathy due to simvastatin was emphasized by a recent genome wide study. With high statin blood concentrations, these genotypes are known to be associated [64].
In the hepatic uptake of type I organic anions like indomethacin and salicylate, OAT2 could be involved and is expressed moderately [65].

Transporters involved in tubular secretion of drugs
Transporters involved in tubular secretion of drugs include P-gp, OATP and OCT, their inhibition decreases the renal elimination, and leads to increased serum drug concentrations.
Both probenecid and penicillin are transported through OATP.
Probenecid competitively binds to OATP and gets excreted,