Montelukast: A Patient Case Related to Medicinal Chemistry and Drug Design
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A 54 year old woman, KT, with a history of asthma, developed pruritus and jaundice one month after starting therapy with montelukast (Singulair) 10mg by mouth daily. KT was admitted to the hospital with a newly presenting combination headache and dizziness that had been increasing over the past two to three weeks. On examination, KT was jaundiced but had no fever or enlargement of the liver or spleen. KT's liver tests showed an increase in total serum bilirubin, modest elevation of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Upon investigation of a current medication list, it was discovered that she was concurrently taking gemfibrozil 600mg by mouth twice daily and her dose of montelukast. Since gemfibrozil, a CYP2C8 inhibitor, causes montelukast, a CYP2C8 substrate, to increase in plasma concentration by multiplicative amounts and lead to a possible toxicity, the pharmacist mentioned the interaction to the patient. He also mentioned that montelukast has been linked to rare cases of liver injury.1 After a brief conversation with KT, it was discovered that she had recently changed primary care providers and had only started the montelukast therapy about a month prior. The pharmacist said they were going to contact the patient's current primary care provider to determine an alternate therapy for either the patient's hyperlipidemia or for the asthma.
Based on KT's reaction circumstances, it is important to recognize how montelukast can react with both medications and a patient's own metabolism. One may not initially realize just how intricate a body's metabolic enzymes are, to what extent they interact with a medication like montelukast, and what kind of effect enzyme inhibitors can have on the montelukast in the patient's body.
Asthma has been a concern for decades, which has led numerous medical personnel to actively discover or simply try various methods of prevention. After continuing research and exploration of the structure and function of those medicinal preventions like montelukast were health care providers able to determine why episodes like KT's happened.
In 1938, two physiologists, W. Feldberg and C. H. Kellaway, published a paper characterizing a 'slow reacting substance' they observed in dog and monkey lungs after being treated with cobra venom. They discovered that this substance somewhat resembled the action of histamine.2 Similarly, in 1960, Brockelhurst isolated a substance from guinea pig lungs and named it 'slow reacting substance of anaphylaxis (SRS-A).' He discovered that the substance caused the slow contraction of smooth muscles in the lung and other tissues and is more potent than the contraction induced by histamine.3 In 1979, Samuelsson proposed a structure for SRS-A and coined the name 'leukotriene (LT).' This structure was found to consist of a variable mixture of compounds (LTC4, LTD4, LTE4, and LTB4) and led researchers to hypothesize that leukotrienes are important mediators of asthma.4 This collection of observations and discoveries eventually led to the development of a safe and effective orally absorbed drug to treat asthma.
Initially, a variety of drug targets were proposed, such as the blocking of 5-Lipoxogenase and LTD4 receptors. The search for an SRS-A receptor antagonist has led to the discovery of FPL-55712, a possible LTD4 antagonist. FPL-55712 enables scientists to accelerate their medicinal chemistry efforts and led to the development of montelukast (Singulair).5 Montelukast was developed from other weakly antagonistic quinolone derivatives (e.g. Verlukast, MK-0571 and MK-0679).
A number of changes were made to these derivatives to increase potency and improved pharmacokinetics. The dithioacetal moiety was replaced, as this had been shown to be a site of metabolism for previous candidates. It was noted that the vinyl group exhibited excellent pharmacokinetics with extended half-lives. Replacement of one sulfur atom with a methylene group in the dithioacettal moiety, as well as an addition of alkyl substitution into the carbon chain, was found to increase potency. Additionally, an alkyl substitution on the thioether chain also led to an increase in potency, and a phenyl group inserted into the carbon chain was shown to boost activity. Most importantly, insertion of a cyclopropylmethyl moiety enhanced the potency by at least 10-fold. The compound selectively inhibits LTD4 at the cysteinyl leukotriene receptor cysLT1. With these findings, the new compound was called MK-0476 and selected for development in 1991.4 This is the drug we know now as montelukast.
Montelukast has 60-70% oral bioavailability, over 99% plasma protein binding and exclusively eliminated into the bile. It is the substrate for CYP2C8, CYP2C9, and CYP3A4. According to its product information, the elimination of montelukast occurs mainly via metabolism by CYP3A4 and 2C9 in vitro. However, recent studies show that montelukast is extensively and primarily metabolized via CYP2C8. Six types of major metabolites present themselves after analysis of the HPLC-radioactivity profiles for human bile after an oral dose of montelukast.6 Five of them are primary metabolites of montelukast: acyl glucuronide (M1), a sulfoxide (M2), a phenol (M3), both diastereomers of the 21-hydroxy analog (21 S- and 21 R-, M5a and M5b), and the 36-hydroxy analog (M6a and M6b).6 Although M4 has the highest concentration among all of the metabolites, it is actually a dicarbosylicacid, further oxidized from M6.7
Through in vitro study, montelukast is metabolized into M5 by CYP3A4 through hydroxylation process and metabolized into M6 by CYP2C9. However, in vivo study, montelukast is demonstrated to be metabolized by CYP2C8 into M6, and M6 is further metabolized into M4 as well as through 2C8.7 After comparing the use of a combination of montelukast, a 3A4 inhibitor, and 2C9 inhibitor with a combination of montelukast and 2C8 inhibitor, it was concluded that CYP2C8 is the dominant enzyme in the elimination of montelukast in humans, accounting for about 80% of its metabolism.7
'Cytochrome P450 2C8 is one of the principle drug metabolizing enzymes in the liver.'8 However, no general pharmacophore model has been proposed for CYP 2C8 due to the large diversity of the enzyme's preferred substrates. The R-enantiomer of montelukast, a large potent competitive inhibitor, is one of the enzyme's preferred substrates. Therefore, due to the increased binding property of montelukast and the resulting decreased ability to quickly leave the liver, a conclusion can be made as to the buildup of the drug and metabolites. This buildup can reasonably lead to hepatotoxicity.8 However, this hepatotoxicity is rare, as evidenced in a meta-analysis in which only 4 of the 97 studies on liver toxicity were caused by montelukast.1 The metabolism is heavily dependent on the functions of the enzymes, as well as how it fits into the various binding pockets and is changed into metabolites.
Backbone structure of CYP2C8CYP2C8 is one of the major drug metabolizing enzymes expressed in the human liver.9 A study that looked at the inhibition of CYP 2C8 by several drugs identified montelukast as being a potent inhibitor of clinical relevance of the CYP 2C8 enzyme.10 The structural determinants needed for substrate specificity of CYP 2C8 were investigated in a separate study using site-directed mutants that were chosen based on pharmacophores and 3-D models. The analysis of the structural features common to CYP 2C8 substrates included a substrate pharmacophore where the site of oxidation is 12.9, 8.6, 4.4, and 3.9 angstroms from features that could create ionic or hydrogen bonds.11 CYP 2C8 has a relatively large active site binding cavity, which would be consistent with the large size of montelukast. Due to the active site being so large, it may be able to accommodate large substrates like montelukast in different binding positions.9
Using crystal structures of CYP 2C8 and the R-enantiomer of montelukast, it was demonstrated that montelukast has a high affinity for the inhibitor of the CYP 2C8 enzyme, with a KI of 9 nM. It has a tripartite structure that fits into the three branches of the binding site cavity. It was the largest of the four compounds that was studied, having three branches that were connected to an asymmetric carbon atom. The crystal structure showed that montelukast fit the size and shape of the active binding site cavity well, and it did not require any changes in the tertiary structure to do so. 9
Hydrophobic binding pocketThe longest of the three branches consists of the terminal choloroquinoline ring of montelukast, which sits in the distal hydrophobic pocket of the binding site. This branch exhibits 32% lower B-factors than the average B-factor that was observed for montelukast and gives this branch a planar conformation that maintains conjugation of the ethenyl group with flanking aromatic rings. This indicates that it is the most sterically strained part of the ligand substrate complex.
Chloroquinolone Ring Interacting With Hydrophobic PocketThe shortest branch interacts with the side chain of serine-100 and amide hydrogen of serine-103 in the CYP 2C8 protein, which donates hydrogen bonds to the carboxylate moiety of montelukast.9
Hydrogen Bonds between Carboxylate moiety and SerinesThe third branch, terminating with a tertiary alcohol group, is positioned close to the heme iron. Carbon 4 of the phenyl ring is positioned 4.2 angstroms from the heme iron.9 The tertiary alcohol group also has the potential for hydrogen bonding with the hydroxyl group with the carbonyl of Val-296.9
Heme InteractionsThe carboxylate residue is positioned near Ser-100, which donates a hydrogen bond to the carboxylate moiety. The last branch with a terminal tertiary alcohol group is close to the heme iron of the CYP 2C8. This is where the 'reactive iron-oxo intermediate is generated during catalysis.'8 Many of the interactions between montelukast and CYP 2C8 that determine the binding orientation are hydrophobic in nature.8 When looking at the structure of montelukast, we find it has greater hydrophobic properties than hydrophilic. Therefore, if montelukast is the only medication involved with CYP 2C8, it will easily occupy all of the CYP 2C8 enzyme in the patient's liver. Also, the high protein binding property of montelukast results in difficulty leaving the liver. Since montelukast has a twenty-four hour duration in the human body, long term usage will cause a large accumulation of the drug and its metabolites remaining in the liver. We can conclude that montelukast has a potential hepatic toxicity reaction because of its chemical structure.
As previously mentioned, montelukast is a leukotriene receptor antagonist that is used to treat asthma and seasonal allergies. Its main target protein is the CysLTR1 receptor in the lungs and bronchial tubes. By binding to this receptor, it blocks the action of leukotriene D4. However, the action of montelukast can be inhibited by binding to a class of CYP enzymes, specifically the CYP 2C8 protein. Montelukast is a large anionic inhibitor that exhibits a tripartite structure and fits relatively well into the active site of the CYP 2C8 enzyme.12
The other medication on the patient's medication list, gemfibrozil, is indicated for patients with hyperlipidemia. It reduces plasma triglycerides and very low-density lipoproteins as well as increases high-density lipoprotein concentrations. The mechanism of action of gemfibrozil is not completely understood, but it is mainly used in patients with elevated triglycerides associated with type IV hyperlipidemia which often times results in a significant increase in LDL.12 Gemfibrozil is systemically well absorbed and 70% is renally excreted. As it relates to KT's patient case, gemfibrozil is a strong inhibitor of cytochrome P450 CYP 2C8 in vivo. It was recently found to markedly increase the plasma concentrations of montelukast in humans.13 A major metabolite of gemfibrozil is 1-O- glucuronide. It is a potent and selectively mechanism-based inhibitor of CYP 2C8. It increases the concentration of montelukast, increasing the AUC greatly.
The closest contact with montelukast in the binding site occurs in the upper and distal portions of the substrate-binding cavity, and the interactions of CYP 2C8 with the largest chemical groups of montelukast are the major determinants of the binding orientation. The study concludes that overall, the size, shape, hydrophobicity and polarity of montelukast, as well as the binding site of CYP 2C8 all contribute to the KI value of 9nM and the high binding affinity of montelukast with CYP 2C8.9 The size of each branch of the cavity determines the binding orientation of the largest component of montelukast in the active site; however the polarity determines the orientation of the two smaller components of the montelukast structure.9 This positions the carboxylate moiety in the solvent access channel and the hydrophobic moiety near the heme iron. The benzyl ring of montelukast is also positioned close to the heme iron, and this suggests that it could be oxidized by the CYP 2C8 enzyme. 9
Although the study determined that montelukast is a potent inhibitor of the CYP 2C8 enzyme in vitro, it did not generally conclude the same in vivo. Though, a later study demonstrated the catalyzed metabolism of montelukast by CYP2C8 over CYP 2C9 in vivo. The study noted that CYP 2C8 'catalyzed the depletion of montelukast and mediated the further metabolism of M6 more actively than did any of the other P450 forms.'14 Therefore, one can safely conclude that CYP 2C8 is a major contributor to the metabolism of montelukast, and inhibiting it will cause a significant rise in serum concentration.
Based on available research and the specific situation presented by KT, her primary care provider decided to discontinue the montelukast and keep patient on gemfibrozil. KT had been on gemfibrozil for approximately two years, and she tolerates it well without any complaints, signs or symptoms of end organ damage, or any adverse reactions. Since there are so many drugs available for treating asthma, and KT's lipid panel is best treated with her gemfibrozil, the pharmacist suggested the doctor use an Advair inhaler instead.
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5. Zwaagstra ME, Schoenmakers S, Nederkoorn P, et al. Development of a three-demensionalCysLT1(LTD4) antagonist model with an incorporated amino acid residue from the receptor. Journal of Medicinal Chemistry. 1998; 41 (9): 1439-1445.
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8. PDB ID: 2NNI
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10. Walsky RL, Gaman EA, Obach RS. Examination of 209 drugs for inhibition of cytochrome P450 2C8. J Clin Pharmacol. 2005;45(1):68-78.
11. Melet A, Marques-soares C, Schoch GA, et al. Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants. Biochemistry. 2004;43(49):15379-92.
12. Gemfibrozil. In: Micromedex [database online]. Truven Health Analytics. http://0-www.micromedexsolutions.com.topcat.switchinc.org/micromedex2/librarian/ND_T/evidencexpert/ND_PR/evidencexpert/CS/654757/ND_AppProduct/evidencexpert/DUPLICATIONSHIELDSYNC/2E12E1/ND_PG/evidencexpert/ND_B/evidencexpert/ND_P/evidencexpert/PFActionId/evidencexpert.DisplayDrugpointDocument?docId=249900&contentSetId=100&title=Gemfibrozil&servicesTitle=Gemfibrozil&topicId=dosingAndIndicationsSection&subtopicId=fdaSection. Accessed November 26, 2014.
13. Karnonen, Tiina, Neuvoene J, Pertii, Backman T, Janne. The CYP2C8 inhibitor gemfibrozil does not affect the pharmacokinetics of zafirlukast. Europen Journal of Clinical Pharmacology. 2011; 67:151-155. Doi: 10.1007/s00228-010-0908-0.
14. Filppula AM, Laitila J, Neuvonen PJ, and Backman JT. Reevaluation of the microsomal metabolism of montelukast: major contribution by CYP2C8 at clinically relevant concentrations. Drug Metabolism Disposition. 2011; 39(5): 904911.
We would like to thank the following academic advisors from Concordia University Wisconsin-School of Pharmacy for participating in discussions about this project: Christopher Cunningham, Ph.D., Daniel Sem, Ph.D., and Ernest Stremski, MD, MBA. We would also like to thank preceptor Dr. Lee Sun from Waukesha Memorial Hospital in Waukesha, WI, for participating in discussions about this project and contributing to the development of our knowledge of pharmacy practice.
Thank you Christina Cash and Eleni Toumplis, for writing the discussion and helping with editing, John Gerovac, Elizabeth Stubenvoll, and Soutsada Thongvathsa for producing Jmol images and writing the medicinal chemistry section, Colleen Kom for writing the case synopsis, portions of the discussion piece, and compiling the individual contributions, Kayla Phillips for writing the background section, Mai Yang for writing about drug development and help with editing, and Nan Yang for writing the background section and helping with editing.