Patient Case

Hypertension is a prevalent disease in the western culture due mostly to poor diet and lack of exercise. Hypertension can occur as an idiopathic disease or a genetic predisposition. According to the Food and Drug Administration (FDA), hypertension is most prevalent in individuals over the age of 55, of African American descent, and those with a past family history of hypertension. Factors like weight, sodium intake, smoking, and alcohol abuse also contribute to an individual's likelihood of developing hypertension.(1) Physicians and pharmacists use the Joint National Committee (JNC) VII Guidelines to classify an individual's blood pressure into four different categories.(2) These categories are: Normal, Prehypertension, Stage I Hypertension, and Stage II Hypertension.(2) If this disease state is untreated, hypertension can lead to life-threatening situations including stroke, blindness, myocardial infarction, kidney failure, and heart failure.(1) To reduce and prevent these life-threatening situations from occurring, patients may be prescribed an anti-hypertensive medication. One first line agent used for treatment of hypertension is an angiotensin-converting enzyme inhibitor (ACE-I).
Nearly two thirds of American adults with hypertension are on an anti-hypertensive managed by their local pharmacist or physician.(3) Enalapril (Vasotec) is an ACE-I, which works to lower the blood pressure in hypertensive patients. Enalapril, a prodrug, works specifically by inhibiting the angiotensin-converting enzyme (ACE) after hydrolysis to the active form, enalaprilat, and suppressing the renin-angiotensin-aldosterone system (RAAS).(4) By preventing the conversion of angiotensin I to angiotensin II, aldosterone secretion is decreased. When an ACE-I is administered, it also inhibits the breakdown of bradykinin, which may lead to a dry cough, often referred to as 'ACE cough'. More seriously, buildup of bradykinin can lead to angioedema. Angioedema is characterized by swelling that occurs under the skin, and it manifests as hives and troublesome breathing. Angioedema is a serious side effect of ACE-Is that is potentially life-threatening.(5)
KT is a 72-year-old overweight African American male who was recently placed on an ACE-I for his stage I hypertension. After failing to lower his blood pressure during nine months of lifestyle modifications, KT was prescribed enalapril 10 mg tablet by mouth once daily for hypertension. Four weeks after initiation of enalapril, KT returned to his local pharmacy to purchase dextromethorphan for a dry cough he had recently developed. While talking to KT, the pharmacist, Paul, realized KT did not have a cold and his cough was non-productive. KT also described some minor swelling and discomfort in his throat. After reviewing KT's profile, Paul noticed KT's enalapril prescription that he had started just four weeks ago. Paul called KT's physician and expressed his concern of ACE cough and angioedema. KT's physician discontinued enalapril, and Paul strongly suggested that KT seek medical attention immediately.

Background

Enalapril was created via a rational drug design approach based on the inhibition of thermolysin, a zinc protease, and approved for marketing in 1985.(6) Enalapril is a prodrug, and the drug is metabolized into its active form, enalaprilat. Enalaprilat is poorly absorbed orally due to its polarity at the non-proline carboxylic acid.(7) With this polarity, the drug has trouble crossing through the phospholipid membrane of the intestinal tract. However, the carboxylic acid is needed to bind to the target.(6) By adding an ethyl group and changing the carboxylic acid to an ester, enough lipophilicity is gained to increase absorption into the circulation.(7) The inactive enalapril is then metabolized by an esterase to its active form, enalaprilat. Because the prodrug is only needed when administered orally, the drug is given intravenously as enalaprilat.(7)
As stated earlier, enalapril is an inhibitor of ACE. ACE is involved in two signal transduction pathways in the body, one of which is responsible for its clinical action, and the other responsible for both clinical action and off-target effects. The action of enalapril on the ACE enzyme in the RAAS is what is responsible for its antihypertensive effect. Enalapril's off-target effect on the kinin-kallikrein system (KKS) is responsible for the adverse effects some patients experience while taking enalapril or other ACE-Is.(4)
ACE also inactivates bradykinin, a vasodilator, in the KKS.(4) By inhibiting the action of ACE, enalapril reduces the breakdown of bradykinin, which then potentiates the vasodilatory effect of bradykinin on the blood vessels. However, bradykinin also has many other off-target effects. As the breakdown of bradykinin is inhibited, the active form builds up in the body. An excess of active bradykinin in the body can cause a dry, hacking cough that will not go away.(4) As mentioned before, this cough is colloquially referred to as 'ACE cough'. The cough does not respond to anti-tussive therapy and is only ameliorated by discontinuation of ACE inhibition. ACE cough affects between 1.3 and 2.2 percent of patients taking enalapril.(9) A more serious side effect, angioedema, is also associated with a sudden increase in active bradykinin due to ACE inhibition and its vasodilatory effect when initiating treatment. This is rare, but very serious when it occurs.(9)
Angioedema is a localized swelling of the deeper layers of the dermis, affecting the lips, tongue, cheeks, and pharynx. It is divided into three main categories of mechanism, which include a deficiency in C1 inhibitor, allergic angioedema, or medication related angioedema.(10) ACE-I associated angioedema is the most common of medication related incidents, and is thought to be mediated by bradykinin and substance P.(10) In the large-scale OCTAVE trial, which looked at two inhibitors of ACE, omapatrilat and enalapril, angioedema occurred at a rate of 0.68% over 6 months in all patients.(11) Angioedema was also more common with omapatrilat than with enalapril.(11) Risk factors for acquiring ACE-I associated angioedema include the African American race, female gender, previous drug rash, sleep apnea, smoking, age older than 65 years, seasonal allergies, obesity, upper airway disease, and initiation of an ACE-I within one week. (12) In a smaller study of 2,421 patients taking a variety of ACE-Is, nine patients (0.37%) developed angioedema and most of these cases (five) were attributed to the drug enalapril. (12) Although this was an interesting study, ACE-I associated angioedema should be considered a class effect. The best form of treatment for this condition is to simply discontinue the ACE-I and switch medications. Calcium channel blockers (CCBs) and angiotensin receptor blockers (ARBs) are of most use in these patients.(12)
The University of Bath Department of Biology and Biochemistry's 2004 paper, 'Structural Details on the Binding of Antihypertensive Drugs Captopril and Enalaprilat to Human Testicular Angiotensin I-Converting Enzyme', presented two new crystal structures of human testicular angiotensin I-converting enzyme (tACE) in complex with captopril and enalaprilat. Somatic ACE was the isoform utilized in the structure, and it contains two active domains, N and C. The C domain is primarily reviewed in blood pressure regulation, but in recent research, it has been found that inhibiting either one of the two domains prevents hydrolysis of angiotensin I.(6) Of particular importance is the understanding that both domains need to be inhibited to undo the conversion of bradykinin to its inactive product. It can be seen in Table 1, using the concentrations in nM of domain inhibition, that both domains are inhibited with use of enalapril. The accumulation of bradykinin and its binding to the bradykinin 2 (BK2) and neurokinin 1 (NK1) receptors then causes vasodilation and edema in the surrounding tissue.(10) This may confirm why patients taking an ACE-I like enalapril might experience angioedema.

Medicinal Chemistry

Enalaprilat, as seen in the 3D enalaprilat button below is a dicarboxylate-containing ACE-I, meaning it contains two carboxylic acids, both of which are essential to its efficacy.(7) In an article by Patchett titled 'The Chemistry of Enalapril', researchers at a large drug manufacturer used protein assay methods to derive new medications from the already well-studied captopril.(13) While captopril worked well in its antihypertensive effects, it had developed two unfavorable side effects, which were rash and loss of taste. It was believed that captopril's sulfur atom was the root of these adverse side effects. So, the research team removed the sulfur and substituted it with a less toxic bioisostere, a carboxylic acid. The assays also produced a few new surprises, and the researchers had found more parts of the molecule they could rearrange to improve efficacy. With this study, Patchett described the chemistry of enalapril, specifically the parts that improve its affinity for ACE. Enalapril's structure has four distinct parts to it, each of which contribute to the potency and efficacy of the medication in some way.(7,13)

The proline moiety on the bottom right of enalapril increases the drug's potency because it makes the molecule a dicarboxylate, which increases affinity for ACE.(13) Enalapril also has a phenylethyl group on the bottom left, which makes the molecule more hydrophobic. This allows for better absorption of the medication across the gut. Additionally, hydrophobic carbon chains at this site greatly increase potency of the drug when it is in the ACE active site.(13) The ACE active site bound to enalaprilat and without enalaprilat can viewed by clicking the buttons below, which will visually show the binding site with and without enalaprilat bound. The third moiety is a secondary amine, colored blue in the middle of the molecule. This amine's activity greatly depends on the formation of the fourth moiety, which is the ethyl ester.

Enalapril's ethyl ester is the part of the molecule that makes it a prodrug. When enalapril is given orally, there are only two moieties on the molecule that may ionize. The first is the carboxylic acid on the proline, which has a pKa of 2.7 At physiological pH of 7.4, this carboxylic acid is predominately in its ionized form, COO-.
The second moiety that can be charged on enalapril is the secondary amine. This amine has a pKa of 5.49, which makes it predominately neutral at physiological pH. This is important for the bioavailability of the drug. If enalaprilat were to be given orally as it is shown above, there would be a carboxylic acid in place of the ethyl ester. Because this carboxylic acid is so close to the secondary amine, it would ionize the amine and thus change its pKa to 8.02.(7) This would make the amine predominately ionized at physiological pH, greatly decreasing its oral bioavailability.(7) Because of this, enalapril is given as a prodrug so that it may first cross the intestinal lumen before bioactivation.

Once through the intestinal lumen, enalapril is hydrolyzed by carboxylesterase-1 (hCE-1), shown. Though hCE-1 is not a CYP450 enzyme, it is found predominately in the liver and is a bioactivator of many drugs. (14) With hydrolysis, enalapril becomes enalaprilat, and the non-proline carboxylic acid is exposed, allowing tight binding of the drug to ACE.

As seen by clicking the button below labeled, Zinc atom in the ACE, the ACE is a zinc metallopeptidase and is found throughout the body.(8) As seen in the image, enalaprilat is buried in the enzyme, so there are many interactions that hold the drug in its active site. Enalaprilat's ability to mimic the transition state of angiotensin I hydrolysis is thought to be the reason for its strong binding capacity. Enalaprilat possesses a tetrahedral carbon in place of the labile peptide bond in angiotensin I. The secondary amine, the carboxylic acid, and phenylethyl groups all contribute to the overall binding of the compound to ACE. The secondary amine on enalaprilat is located at the same position as the labile amide nitrogen on angiotensin I. Additionally, the ionized non-proline carboxylic acid can form an ionic bond with the zinc atom of ACE, and the phenylethyl group mimics the hydrophobic side chain of the Phe amino acid, which is present in angiotensin I.

Click on the buttons below to view the secondary structures of ACE.

Along with a zinc ion, there are amino acids that help with the binding of enalaprilat. These amino acids create electrostatic interactions with the drug to help stabilize it in the site.(6) There are six amino acids, two chloride ions, and one zinc molecule that help with this interaction. A water molecule binds to the nearby Glu384, resulting in a polarization between the negative glutamate carboxylate group and the positive zinc ion. During this reaction, the nucleophile (water) attacks the carbonyl on the enalaprilat, which pushes electrons out on the oxygen to form a stable tetrahedral intermediate. Zinc then interacts with the oxygen on the enalaprilat, which stabilizes the transition state and makes the reaction happen more quickly, because the zinc acts as a catalyst. The image shows the zinc molecule of ACE as it relates to the amino acids in the binding site.(6) The drug enalaprilat is then stabilized by hydrogen bonding interactions between Ala354 and an amide on His353. In the C domain active site, there are two buried chloride ions. These chloride ions are also used to help stabilize the active site by creating electrostatic interactions with the amino acids surrounding them. The main amino acids that these chlorides react with are the Tyr and Arg residues. (6) As stated earlier, the most important interaction in the drug binding to ACE is with the zinc ion.

Discussion

Aforementioned in sections above, enalaprilat binds to ACE by mimicking the transition state of angiotensin I, with the aid of a zinc ion for stability. Enalaprilat inhibits the enzyme by virtue of interactions with its phenylethyl group, carboxylic acid, and secondary amine at ACE's Ala354, Glu384, His353, and Tyr and Arg residues.(6) Enalaprilat can cause adverse effects like cough and angioedema through the inhibition of bradykinin catabolism mediated by the KKS.(4) The exact process of catabolism of bradykinin, an oligopeptide, is not fully known, but it is postulated that it is cleaved via two active sites - N and C domains - on ACE, which are blocked with the use of inhibitors like enalapril.(15) As stated in a previous section, the proline moiety enhances the binding affinity of enalaprilat for ACE.(7)
The vasodilatory effect of bradykinin is manifested mainly through binding of the B2 receptor.(16) B2 is a G-protein coupled receptor that has been associated with essential hypertension, as well as other cardiovascular disorders.(16) It has been speculated that polymorphisms in the genes coding for B2 receptors could explain why certain people are more susceptible to adverse effects like angioedema when taking ACE-Is or non-steroidal anti-inflammatory drugs (NSAIDs).(6) It is possible that KT has one such polymorphism, which may have potentiated bradykinin vasodilatory effects. An exact mechanism for ACE-I-attributed cough has yet to be determined, but Mukae et al. theorize that it is due to a local accumulation of bradykinin. This accumulation spurs the release of histamine as well as other pro-inflammatory compounds, which work to exacerbate the normal cough reflex.(16)
Upon further review, it was found that KT was also regularly taking ibuprofen, an NSAID, concomitantly with enalapril. This potentially augmented the adverse effects of the ACE-I, leading to his angioedema and cough. KT has a variety of risk factors, including advanced age, African American descent, and being overweight, which increases his likelihood for angioedema.(12) Substituting an ARB, such as losartan, for the offending ACE-I is the standard method for relieving adverse effects. A second option would be to screen KT for relevant B2 polymorphisms that could be contributing genetic factors for his angioedema. Those individuals who are positive for specific B2 polymorphisms that have demonstrated increased incidence of adverse effects like angioedema could then be considered for alternative therapies. Presently, genetic testing for B2 receptor polymorphisms is not commonplace, but could be effective in the future for preventing such adverse effects from occurring. Another option could be the introduction of bradykinin inhibitors, such as icatibant, to treat acute angioedema attacks. In comparison to choosing an ARB, icatibant is not particularly feasible at this time due to its high cost and route of administration, but further studies may warrant its use in the future.(17) As a final note, the pharmacist consulted with KT's primary care physician, and recommended the switch of enalapril to losartan.

References

1. U.S. Food and Drug Administration. High Blood Pressure (Hypertension). Available at: http://www.fda.gov/forconsumers/byaudience/forwomen/ucm118529.htm. Accessed November 19, 2013.

2. U.S. Department of Health and Human Services. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure, NIH 04-5230. Washington, DC: National Heart, Lung, and Blood Institute. http://www.nhlbi.nih.gov/guidelines/hypertension/jnc7full.pdf. Published August, 2004. Accessed November 20, 2013.

3. Centers for Disease Control and Prevention. High Blood Pressure Facts. Available at: http://www.cdc.gov/bloodpressure/facts.htm. Accessed December 5, 2013.
4. Sayed-Tabatabaei FA, Oostra BA, Isaacs A, van Duijn CM, Witteman, JCM. ACE polymorphisms. Circ Res [serial online]. May 12 2006;98:1123-1133. Available from: American Heart Association, Dallas, TX. Accessed November 19, 2013. 5. Winters ME, Rosenbaum S, Vilke GM, Almazroua FY. Emergency department management of patients with ACE-inhibitor angioedema. J Emerg Med [serial online]. November 2013;45(5):775-780. Available from: Elsevier, Amsterdam, North Holland, Netherlands. Accessed November 19, 2013. 6. Natesh R, Schwager SLU, Evans HR, Sturrock ED, Acharya KR. Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme. Biochemistry [serial online]. July 13, 2004;43(27):8718-8724. Available from: ACS Publications, Washington, DC. Accessed October 10, 2013. 7. Harrold M. Angiotensin-converting enzyme inhibitors, antagonists and calcium blockers. In: Lemke TL, Williams DA, eds., Foye's Principles of Medicinal Chemistry: Sixth Edition. Baltimore, MD: Lippincott Williams & Wilkins, a Wolters Kluwer business; 2008: 743-746. 8. Turner AJ, Tipnis SR, Guy JL, Rice GI, Hooper NM. ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors. Can J Physiol Pharmacol [serial online]. April 2002;80(4):346-353. Available from: NRC Research Press, Ottawa, Ontario, Canada. Accessed November 25, 2013.

Acknowledgements

We would like to thank the following preceptors for participating in the discussions about enalapril and contributing to our knowledge of pharmacy practice: Paul Klubertanz RPh, Target, Waukesha, WI; Daniel Sem, PhD, Mequon, WI; and Christopher Cunningham, PhD, Mequon, WI.'

Author Contributions

Gratitude is owed to Benjamin Knapp and Bridget Ellerman for their work on the case synopsis. Thank you to Erin McGurty and Nicole Savatski for their contributions to the development of the background. All medicinal chemistry analyses and images are credited to the hard work of Kaitlin Cooper-Johnson, Chad Seubert, and Krista Thuening. Discussion of the enalapril patient case is credited to Kimberly Maerz and Aimee Andrewjeski. Lastly, many thanks to Kaitlin Cooper-Johnson for compiling and editing the paper.'