Targeting Inflammation Using Novel Nitric Oxide Donors

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Frontiers in Nephrology: Targeting Inflammation Using Novel Nitric Oxide Donors

Gordon Letts* and Joseph Loscalzo†

*NitroMed, Inc., Lexington, Massachusetts; and †Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts

ABSTRACT COX-1, an enzyme that produces a fam- Chimeric molecules are single-chemical entities that possess at least two separate ily of prostaglandins that are considered functions. In the design of new chimeric medicines, the two biologic actions are fundamental to maintaining the health often designed to be synergistic and, thereby, complement each other in activat- and integrity of many organs, including ing a specific target, such as a gene, a receptor, or an enzyme. In most chimeric platelets, blood vessels, and the kid- molecules, one functionality is designed to provide a high affinity to a designated ney.21,22 Inhibiting this enzyme and, site, thereby permitting the targeting of the second functionality, which is usually thereby, eliminating prostanoids that nonspecific. This review focuses on the development of two classes of chimeric promote normal homeostasis (beneficial medicines, anti-inflammatory and diuretic chimeric agents, both of which incorpo- prostanoids) results in a “deficient ” or- rate a nitric oxide moiety into the parent pharmacophore. gan whose ability to function is compro- mised. Reported reductions in various J Am Soc Nephrol 18: 2863–2869, 2007. doi: 10.1681/ASN.2007030321 organ functions (e.g., platelet adhesion and aggregation, renal blood flow, endo- scopic ulceration) all provide support for CHIMERIC NITRIC OXIDE ing enzyme inhibitor is both a direct ni- this mechanistic concept.23–26 DONORS trovasodilator and an effective angioten- The advantage of inhibiting selec- sin-converting enzyme inhibitor whose tively COX-2, an enzyme that is mainly 1 The development of novel chimeric ni- inhibitory activity is equivalent to the induced as a result of an inflammatory tric oxide (NO) donors had its origins in parent captopril. This series of studies set stimulus, is that it will not inadvertently 1,2 studies by Loscalzo’s group showing the stage for the development of many block the beneficial prostanoids that are that N-acetylcysteine potentiates the ac- other chimeric NO-donor compounds, produced by COX-1.10,27,28 The induced tivity of nitroglycerin and of authentic two of which are described next in detail. COX-2 enzyme was thought to generate endothelium-derived relaxing factor, excessive amounts of detrimental prosta- likely by generating S-nitroso-N-acetyl- noids at sites of injury, thereby amplify- cysteine. This S-nitrosothiol prolongs CHIMERIC CYCLOOXYGENASE-2 ing the damage and overwhelming local the bioactivity of NO and maintains the INHIBITORS host defense mechanisms. Although this intracellular thiol redox state of the effec- simple concept is generally true, excep- tor cell (platelet or vascular smooth mus- Selective cyclooxygenase-2 (COX-2) in- tions have become apparent as a result of cle cell). Subsequent studies by this hibitors were developed because they the clinical use of selective COX-2 inhib- group showed that S-nitrosothiols are were conceived to offer an improvement itors.29 In particular, prostacyclin, a ben- naturally occurring species in mammals, over existing nonselective nonsteroidal eficial prostanoid that is generated by the with S-nitroso–serum albumin serving anti-inflammatory drugs (NSAID).9–17 as a bioactive plasma reservoir for NO3,4 The existing NSAID are widely known to to which and from which NO is trans- cause adverse effects, such as gastrointes- Published online ahead of print. Publication date ferred via low molecular weight S-nitro- tinal irritation and/or bleeding; in addi- available at www.jasn.org. sothiols by thiol-nitrosothiol exchange.5 tion, their long-term use produces mild Correspondence: Dr. Joseph Loscalzo, Brigham & In a series of studies of the first true chi- hypertension and a detriment in renal Women’s Hospital, 75 Francis Street, Boston, MA 02115. Phone: 617-525-4833; Fax: 617-525-4830; meric NO-donor molecule, S-nitroso- function, among other abnormali- E-mail: [email protected] captopril, Loscalzo’s group6–8 showed ties.18–20 These unwanted actions have Copyright © 2007 by the American Society of that S-nitrosated angiotensin-convert- been shown to be a result of inhibition of Nephrology

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flammatory mediators. For example, if DYSFUNCTIONAL the endothelium has this mutation in ENDOTHELIUM FUNCTIONAL its NOS-2 gene, then with inflamma- ENDOTHELIUM tion and activation of COX-2 in the vessel wall, oxidative inactivation of NO would occur by virtue of its rapid NSAID or COX-2 = PGI2 reaction with superoxide and the sub- PGI2 sequent generation of the cytotoxic Inflammation = NO – NO – OONO O2 peroxynitrite. In this setting, vascular relaxation would decrease and vascular NORMAL BLOOD PRESSURE ELEVATED BLOOD PRESSURE leukocyte adhesion would increase.35 ––Responsive to drugs and Enhanced response to vasoconstrictors This effect would be further enhanced homeostatic signals – Less responsive to drugs if a selective COX-2 inhibitor were Figure 1. How NSAID- or COX-2–induced inhibition of prostanoids at a site of endothelial given and local prostacyclin produc- inflammation can cause a perturbation in endothelial function, leading to an adverse effect on BP. tion also inhibited. In these cases, local homeostatic responses are virtually nonexistent. This concept is illustrated endothelium and whose actions are im- these cases, the levels of endogenous in Figure 1. portant in maintaining vascular integrity NO are bolstered by NOS-2, and a One way to combat this possible sce- and preventing the activation of circulat- heightened adverse oxidant effect may nario and to improve the clinical useful- ing platelets and the adhesion of neutro- be observed in the context of other in- ness of a COX-2 inhibitor is to transform it phils, is synthesized via both COX-1 and into a chimeric molecule that provides COX-2 pathways.11,30,31 Moreover, the NO. The concept of linking two separate reduction of prostacyclin production in functions into such a single molecule is ex- organs, such as the kidney, can lead to emplified in Figures 2 and 3. In this exam- abnormal organ function that is indistin- ple, the single-chemical entity, NMI-1093, guishable from that caused by nonspe- has two biologic functions: One is the po- cific NSAID.32 tent, selective inhibition of COX-2, and the Another enzyme that is induced along other is the ability to generate NO rapidly with COX-2 (and with similar kinetics) (Schroeder JL, Bandarage RR, Bandarage at the site of inflammation is NO syn- UK, Earl R, Gzawa M, Fang X, Garvey DS, thase-2 (NOS-2). The NO that is gener- Gaston RD, Khanapure SP, Stevenson CA, ated by NOS-2 is part of a local host de- and Wey SJ, NitroMed, Burlington, MA, fense response and has complex 2001, unpublished data).36 In Figure 2, the biology.33 The freshly generated NO is des-NO molecule, NMI-1089, is also capable of supplementing the local site shown, to illustrate the enhanced benefits Figure 2. Chemical structures of levels of constitutive NO as the low-flux NMI-1093 and NMI-1089. of the chimeric derivative. endogenous NO is rapidly oxidized and NMI-1093 has a potency and selectiv- eliminated at the site of an inflammatory ity for COX-2 comparable to the clini- insult.34 The NO reacts with superoxide cally approved drugs, such as rofecoxib. anions that are generated by COX-2 or The des-NO analogue (NMI-1089) also other downstream sources that are acti- retains excellent selectivity for COX-2 vated by inflammatory stimuli to form and is, thus, a good agent with which to an intermediate with powerful cytotoxic examine the benefit of the added NO do- actions, peroxynitrite (ONOOϪ), thereby nor functionality that has been incorpo- helping to eliminate the inflammatory rated into its structure (NMI-1093). The stimulus (if a biologic agent) and creating a relative potencies and selectivity of ac- “noninfective” boundary around the site tion are shown in Figure 4. of injury to localize or prevent further ex- The anti-inflammatory properties pansion of the inflammatory event itself. that are inherent in the chimeric COX-2 Figure 3. Mechanism by which a chimeric 14 Hobbs et al. showed that certain molecule with a high affinity for the COX-2 inhibitor are illustrated in Figure 5. This mutations in the NOS-2 gene result in active site is able to generate NO locally. classic animal model is often used to the generation of high fluxes of NO, The NO is formed by the action of nonspe- compare and rank the efficacies of both which can have severely adverse conse- cific esterases on the nitrate moiety of the selective COX-2 inhibitors and nonselec- quences at an inflammatory site. In chimeric molecule. tive NSAID. As shown, it is evident that

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NMI-1093 is a potent anti-inflammatory this anesthetized rat model, a short extra- nificant elevation in BP.32,39 This effect drug with a similar efficacy to that of ro- corporeal vascular loop is created, and a can be demonstrated by measurement fecoxib and celecoxib. The compounds silk thread is placed in the loop for 15 of the enhanced sensitivity to the vaso- have similar duration of action and also min. During the time the thread is in constrictor angiotensin II in animals exhibit similar potencies in a second an- contact with circulating blood, the for- after long-term drug administration at imal model of inflammation, the rat paw eign surface of the thread is the site of dosages of drug that do not directly af- edema model (data not shown; Trocha platelet deposition. This “white ” throm- fect BP (7 d).40 As shown in Figure 7, AM, Shumway M, Augustyniak M, bus on the thread can be removed and there is an enhanced constrictor re- Young D, Janero DS, NitroMed, Burling- weighed, reflecting the capacity for plate- sponse to infused angiotensin II in the ton, MA, unpublished data). let activation and adhesion. As illus- presence of a selective COX-2 inhibitor The benefit of the NO donor func- trated, the chimeric molecule can pre- that is not apparent in the presence of tionality that is inherent in NMI-1093 is vent platelet deposition in vivo, whereas an equivalent dosage of a chimeric in- illustrated in Figure 6. Figure 6A demon- both rofecoxib and the des-NO analogue hibitor. This lack of hypertension with strates the ability of NMI-1093 to inhibit cannot.37 chimeric NO donor drugs has also been ADP-induced platelet aggregation in An important role of endothelial reported with naproxcinod.41–43 vitro. The des-NO analogue and rofe- NO is the maintenance of vascular re- Naproxcinod is a chimera of naproxen coxib have no activity in this assay, activity (Cochran E, Trocha M, Young and a mononitrate donor functional- whereas the widely known NO donor D, Marek P, NitroMed, Burlington, ity. This drug, which has recently com- isosorbide dinitrate has similar activity MA, 2001, unpublished data).38 An ad- pleted phase 3 studies, is as effective an to that of NMI-1093. Figure 6B extends verse effect of NSAID and selective NSAID as naproxen itself; however, it this observation to an in vivo setting. In COX-2 inhibitors is a modest but sig-

Figure 4. Inhibition of COX-1 and COX-2 thromboxane production in human whole blood by NMI-1093, NMI-1089, rofecoxib, and celecoxib. PG, prostaglandin (I2 or e2). Data are percentage of inhibition and are the mean of six experiments.

Figure 6. (A) Inhibition of ADP-induced aggregation of human platelet-rich plasma by isosorbide dinitrate (ISDN), NMI-1093, and NMI-1089. Data are percentage of inhibition relative to the aggregation of control platelets that were not treated with a test agent and are means Ϯ SEM (n ϭ 6). (B) Effect of oral administration of rofecoxib, NMI-1093, or NMI-1089 on in vivo thrombus formation in an anesthetized rat Figure 5. Effect of oral administration of equimolar doses (15 ␮mol/kg) of celecoxib, arteriovenous shunt model. Data are ex- rofecoxib, valdecoxib, etoricoxib, NMI-1093, and NMI-1089 in the rat carrageenan air- pressed as thrombus weight (mg) and are pouch model of inflammation. Results are expressed as white blood cell (WBC) content in means Ϯ SEM (n ϭ 5 to 8). *P Ͻ 0.05 the pouch exudates and are means Ϯ SEM (n ϭ 5 to 12). **P Ͻ 0.01 versus vehicle. versus vehicle or rofecoxib.

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HCTZ is to target NO to the kidney, where it can have a multiplicity of actions, all helping to improve the clinical profile of HCTZ itself. These actions include reduced HCTZ-induced electrolyte imbalance, enhanced systemic and renovascular homeostatic control, inhibitory actions on the release of deleterious neurohormones (renin-angiotensin-aldosterone system), reduced metabolic disturbances, and reduced diuretic tachyphylaxis. As shown in Figure 9, the chimeric HCTZ NMI-3377 has a similar potency and duration of action as HCTZ itself. In this rat model, similar urinary volumes and time courses were measured (Kuo JT, Wong AL, Wexler RS, Zifcak BM, Ellis JL, Dube GP, Selog WM, NitroMed, Lexington, MA, 2005, Figure 7. Effect of oral administration of equimolar doses of a COX-2–selective inhibitor or a unpublished data). In Figure 10, the mea- chimeric NO–COX-2 inhibitor (15 ␮mol/kg per d for 7 d) on pressor responses to intravenous surement of elevated NO metabolites is plot- angiotensin II infusions (1, 3, and 10 ng/kg per min) in anesthetized rats. Data are expressed as changes in mean arterial pressure (MAP) and shown as mmHg (n ϭ 4 to 5 per treatment group). does not cause elevations in BP and has macologic agent is illustrated in Figure 8. a reduced gastric irritancy profile.13 In this example, NO has been linked via a In summary, this new class of medi- nonhydrolyzable bond to the diuretic cines, chimeric COX-2 inhibitors, has the hydrochlorothiazide (HCTZ). Linking potential to provide effective anti-inflam- the nitrate to HCTZ targets the release of matory and analgesic activities with a bet- NO to the kidney. ter safety profile than the existing COX-2 Results and guidelines from the Antihy- inhibitors. The benefits of possessing bi- pertensive and Lipid Lowering Treatment to functional activities include minimal gas- Prevent Heart Attack Trial (ALLHAT)44 and trointestinal irritation, improved vascular the joint National Committee on Prevention reactivity (antihypertensive properties), of High Blood Pressure38 suggest that thia- enhanced antiplatelet properties, and im- zide diuretics should be used as first-line ther- proved renal function, among others. apy, either alone or in combination with other antihypertensive agents. There are more than 27 marketed HCTZ combina- CHIMERIC DIURETICS tions, and, although their use is encouraged, these diuretics suffer from a series of adverse The second example of a chimeric phar- effects, particularly when used in higher dosages. The practice of using HCTZ combination products (e.g., Aldactazide) is intended to lower the dosage of HCTZ and to add an agent with a complementary mode of action to assist in adequate BP control, minimizing undesirable effects of either agent used alone in the usual dosages. The adverse effects of Figure 9. Cumulative urine volume in HCTZ include electrolyte imbalance, stimu- conscious rats over 6 h after oral adminis- lation of the renin-angiotensin system, or- tration of equimolar doses of HCTZ (0.3, thostatic hypertension, and renal dysfunc- 1.0, and 3.0 mg/kg; A) or NMI-3377 (0.3, tion, as well as the onset of metabolic 1.0, and 3.0 mg/kg HCTZ equivalent; B). Figure 8. Chemical structures of Ϯ disturbances, such as new-onset diabetes. Data are expressed as ml and are means NMI-3377 (A) and HCTZ (B). SEM (n ϭ 6 to 12 per group). The benefit of creating a chimeric

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the kidney are seen in reduced circulating levels of the renin and aldosterone (Figure 11). This effect has been demonstrated in rats in which minipumps that administered equimolar doses of either NMI-3377 or HCTZcontinuouslyfor4wkwereimplanted (Melim TL, Gordon LJ, Wong HL, Wexler RS, Ellis JL, NitroMed, Lexington, MA, 2005, unpublished data). The HCTZ-induced increases in these vasoconstrictor hormones were significantly attenuated in the NMI- 3377–treated animals, and these actions would be expected to translate into improved endothelial function, reduced chronic fi- brotic changes in the cardiovascular system and kidney, and better BP control. In SHR-SP–treated rats that received an NO synthesis inhibitor (to suppress endogenous NO and, thereby, better as- Figure 10. Levels of nitrite (␮M) (upper graph), NO-HEME (nM) (lower left graph), and sess the benefit of the chimeric mole- S-nitrosothiols (RSNO; nM) (lower right graph) in the kidneys of rats after oral administra- cule), NMI-3377 produced a significant tion of equimolar doses of HCTZ and NMI-3377 at 0, 15, 30, 60, 180, and 360 min. Data reduction in the excretion of urinary al- Ϯ ϭ are means SEM (n 6 per group). bumin when compared with HCTZ. This model involves treatment of groups of ted. As shown, increases in renal levels of the animals with vehicle or equimolar doses derivative metabolites of NO, nitrite, S-nitro- of HCTZ or NMI-3377. After 7 d, L-ni- sothiols, and NO hemes are apparent for ap- troarginine methylester (NOS inhibitor) proximately 3 h after dosing (Dhawan V, was introduced, and all animals were Dube GP, Ellis JL, Schwalb DS, Shumway then monitored for an additional 25 d MJ, Warren MC, Zemtseva IS, Janero DR, before being killed and measurement of NitroMed, Lexington, MA, 2005, unpub- renal function. Figure 12 shows that the lished data). improvement in the excretion of uri- The results of enhanced NO delivery to naryalbumin in animals that were

Figure 11. Plasma renin (A) and aldosterone (B) levels before (day 0) and after 29 d of oral administration of vehicle, HCTZ (10 mg/kg per d), or NMI-3377 (10 mg/kg per Figure 12. Cumulative urinary albumin in rats after oral administration of vehicle alone (black), d HCTZ equivalent). Data are expressed as vehicle plus L-nitroarginine methylester (L-NAME; green), HCTZ plus L-NAME (blue), NMI-3377 relative units (renin) or pg/ml (aldosterone) plus L-NAME (yellow), or valsartan plus L-NAME (red). Rats were treated for 35 d (10 mg/kg per and are means Ϯ SEM (n ϭ 8 per group). d, HCTZ or HCTZ equivalent). Data are expressed as mg and are means Ϯ SEM (n ϭ 6to10per *P Ͻ 0.05; **P Ͻ 0.001. group). *P Ͻ 0.05 versus L-NAME or vehicle; ϩP Ͻ 0.01 versus L-NAME or vehicle.

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