Antioxidant Drugs

C H A P TE R   40  Antioxidant Drugs Craig B. Webb

Definitions Oxidative Stress Oxidative stress is an imbalance between prooxidant compounds and antioxidant defenses.1,2 Another term used to describe this summation of pro- and antioxidant molecules is the redox state.

inhibit mitochondrial function, and deplete cellular pyridine nucleotides causing breaks in DNA strands. Nitric oxide can also combine with the superoxide anion free radical to form the RNS peroxynitrite. Peroxynitrite produces cell injury through lipid peroxidation, inhibition of mitochondrial respiration and Na+/K+-adenosine triphosphatase (ATPase) activity, and protein oxidation.

Free Radicals A free radical is any molecular species capable of independent existence and containing one or more unpaired electrons.1,2 Examples include the hydrogen radical (H•), the superoxide free radical (O2•–), and the hydroxyl (OH•) and peroxyl radicals (RO2•). Metabolic processes taking place within the liver constitute a major source of free radical production. The superoxide free radical, for example, is produced by hepatic oxidative reactions and by “uncoupling” of the cytochrome P450 enzyme system. Free radicals are formed during hepatic metabolism of endogenous substances or xenobiotics such as acetaminophen.5

Pathophysiology1,2 DNA Damage ROS cause DNA base-pair modifications, strand breaks, crosslinking, and mutations resulting in uncontrolled growth and malignant transformation. Free radicals are also implicated as initiators of apoptosis, or programmed cell death.

Lipid Peroxidation

The term reactive oxygen species (ROS) is used to describe free radicals containing oxygen.1,2 These are molecules that are formed by the reduction of oxygen and encompass both free radicals and nonfree radicals such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite (ONOO, which is also a reactive nitrogen species). ROS are produced under normal circumstances during normal mitochondrial respiration, and during disease processes such as inflammation, necrosis, and ischemia.

Polyunsaturated fatty acids in cell membranes react with oxygen to produce peroxyl radicals, the primary free radical intermediate of lipid peroxidation. Change in the structure of membrane lipids will change cell membrane fluidity and significantly alter membrane functions such as ion transport, receptor recognition and signaling, and osmotic gradients. Initially a hydroxyl radical removes hydrogen from lipid molecules in the cell membrane, transforming that lipid molecule into a free radical and starting a cycle of reactions whereby a newly formed membrane lipid peroxyl radical extracts a hydrogen molecule from the next lipid molecule and the cycle is repeated.

Reactive Nitrogen Species

Protein Damage

Nitric oxide synthase (NOS) in hepatocytes and Kupffer cells produces nitric oxide (NO•), a reactive nitrogen species (RNS)1,2 in the reaction: l-Arginine + O2 + NADPH → NADP+ + NO• + citrulline where NADPH is nicotinamide adenine dinucleotide phosphate (reduced form) and NADP+ is nicotinamide adenine dinucleotide phosphate. Nitric oxide is also produced by neutrophils as part of the inflammatory process, and from the reaction of glutathione with peroxynitrite. Nitric oxide binds reversibly to free thiol groups, including reduced glutathione (GSH) through the action of glutathione-Stransferase. Nitric oxide is a powerful vasodilator, and acts as an antioxidant through its ability to scavenge lipid peroxyl radicals. Conversely, nitric oxide forms nitrogen-containing reactive intermediates such as nitrotyrosine, which can lead to liver necrosis,

Oxidative modification of endogenous proteins causes unfolding of the tertiary and quaternary structure. Intracellular signaling pathways rely on normal protein structure and function, and ROS can oxidize amino acids within enzymes, rendering them inactive and/ or antigenic.

Reactive Oxygen Species

Altered Redox State Intracellular changes in ROS cause changes in the redox balance and second messenger signal transduction that may affect cell function, cell proliferation, and gene expression. The upregulation of matrix metalloproteinases, kinases, and transcription factors, such as nuclear factor kappa B (NF-κB), by free radicals may result in the production of mediators, such as tumor necrosis factor (TNF)-α and interleukin-1, in chronic diseases, such as inflammatory bowel disease.2

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SECTION V  Pharmacologic Approach to Gastrointestinal Disease

Drug Classifications and Mechanisms of Action Antioxidant defenses consist of both enzymatic and nonenzymatic processes.3 Antioxidant enzymes include superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. These antioxidant enzymes catalyze chemical reactions that utilize ROS. The end-product of their reactions is often a much less harmful compound such as water, or a metabolite that is subject to further antioxidant reactions, such as hydrogen peroxide. Sulfur-containing glutathione is the most important of the nonenzymatic antioxidants. Thiols exert their antioxidant action through oxidation of the sulfhydryl bond of cysteine. In this way, they scavenge free radical unpaired electrons. The inhibition of lipid peroxidation by α-tocopherol (vitamin E) is another example of a scavenging antioxidant property. These antioxidants are replaceable substrates because they can be returned to their reduced form through simple chemical reactions. Enzymatic and nonenzymatic antioxidant defenses often work synergistically. For example, after the superoxide dismutase enzyme generates hydrogen peroxide from the superoxide anion, the glutathione peroxidase enzyme converts hydrogen peroxide to water by oxidizing GSH to the disulfide form GSSG. The glutathione reductase enzyme completes the process by returning GSSG to GSH: 2 O2•– + 2 H+ → H2O2 + O2 Superoxide dismutase enzyme 2 GSH + 2 H2O2 → 2 H2O + GSSG Glutathione peroxidase enzyme (selenium cofactor) GSSG + NADPH + H+ → 2 GSH + NADP+ Glutathione reductase enzyme (riboflavin cofactor) Much like glutathione, the thioredoxins are ubiquitous thiolcontaining antioxidant polypeptides that are oxidized to a disulfide form while they undergo redox reactions with multiple proteins. Thioredoxins are returned to their reduced form by an NADPHdependent reaction driven by the thioredoxin reductase enzyme, and along with GSH, are critical to cellular redox (oxidative) potential. The Fenton reaction uses metals (copper or iron) as a cofactor during the generation of ROS: Fe2+ + H2O2 → OH• + OH− + Fe3+ The liver serves as the major organ for iron and copper transport and storage. Hepatic copper accumulation is both cause and consequence of chronic hepatitis. Proteins that bind these metals are considered antioxidant. The liver is abundant in metallothioneins or cysteine-rich proteins that bind to various hepatic metals such as copper and zinc. Iron is bound in the hepatocyte to the protein ferritin or hemosiderin. Some of the more important of the metalbinding transport molecules are transferrin and lactoferrin, which bind iron, ceruloplasmin, and albumin, which bind copper, and the exogenous copper-chelating agent penicillamine.

Rational Use of Antioxidants in the Veterinary Patient Therapies of oxidative stress in dogs and cats have been directed primarily at the tripeptide glutathione (γ-glutamylcysteineglycine). During normal hepatic metabolism, glutathione can be directly conjugated to a variety of metabolites and drugs to increase their water solubility and enhance their excretion through the kidney. This process is particularly important in the cat because hepatic glucuronidation is virtually absent in that species. Glutathione is abundant in gastrointestinal mucosa and undoubtedly serves a similar antioxidant role in that tissue.

Reduced GSH concentrations have been reported with hepatic disorders, including inflammation, cholestasis, lipidosis, copper retention, and acetaminophen toxicity.4-6 These studies support the role of glutathione as an antioxidant in dogs and cats and a number of glutathione precursors have been developed.

S-adenosyl-L-methionine (S-adenosylmethionine) S-adenosyl-l-methionine (SAMe) is a key component of several metabolic pathways, including methylation, sulfuration, and aminopropylation reactions, but it is perhaps most important clinically as a glutathione precursor. Oral administration of SAMe (48 mg/kg q24h) increases plasma SAMe concentrations, hepatic GSH concentration, and hepatic GSH-to-GSSG ratio in healthy cats.7 These same doses have been shown to diminish erythrocyte peroxidation and osmotic fragility. SAMe conserves red blood cell and hepatic GSH concentrations in dogs with experimentally induced steroid hepatopathy,8 and limit Heinz body formation and erythrocyte destruction in cats and dogs exposed to acetaminophen.9,10 Although N-acetylcysteine (NAC) is traditionally considered the antioxidant of choice, comparative efficacy studies show that SAMe may be a more effective treatment of acetaminophen toxicity in mice.9 SAMe also may be useful in the treatment of pancreatitis and gastric ulceration.11,12

N-acetylcysteine NAC is the thiol donor most frequently used in emergency cases. The oral bioavailability of NAC is limited, but when given intravenously it results in a rapid increase in GSH synthesis and has direct antioxidant activity. NAC is still considered the standard of care in cases of acetaminophen toxicity, and is useful in the detoxification of hepatotoxins, as well as in cases of ischemia–reperfusion injury.

Vitamin E (α-Tocopherol) Vitamin E inhibits lipid peroxidation, thereby stabilizing cell membranes, and preventing changes in membrane fluidity and function. Supplementation with vitamin E reduces the production of the proinflammatory mediators NF-κB and TNF-α, and decreases collagen production associated with hepatic inflammation. Vitamin E concentrations are depleted in several forms of liver injury, including hypoxia.13 Vitamin E and cysteine supplementation ameliorates acetaminophen, but not onion powder, toxicity in cats.14 Vitamin E plus selenium combinations also have been used for tetracyclineinduced hepatotoxicity in the cat.15 Dogs treated with vitamin E for 3 months demonstrated an increase in serum and hepatic vitamin E concentrations and an improved hepatic GSH-to-GSSG ratio.16 Vitamin C may help to recycle vitamin E to a useful anti­ oxidant form, but because vitamin C can act as a prooxidant and promote hepatic iron storage, supplementation is generally not recommended.

Milk Thistle (Silymarin, Silibinin) Silymarin is an extract of the milk thistle plant that contains a mixture of bioactive flavonolignans, the most powerful of which is silibinin. Silymarin reduces hepatic iron accumulation, collagen deposition, and fibrosis following severe hepatic injury, and has been reported to improve survival in cirrhosis,17 but not in hepatitis C or alcohol-induced hepatitis.18 Silibinin administered at a dosage of 50 mg/kg reduces elevations in alanine aminotransferase, alkaline phosphatase, and total bilirubin, as well as the hepatic hemorrhagic necrosis following amanita mushroom or CCl4 poisoning in the dog.19,20

CHAPTER 40  Antioxidant Drugs



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Table 40-1 Antioxidant Agents Used in Dogs and Cats Drug Classification

Dose

S-adenosyl-L-methionine 20 mg/kg PO q24h xenobiotics, hepatotoxins 140 mg/kg initial dose IV, N-acetylcysteine 70 mg/kg BID-QID Vitamin E 10 to 15 units/kg PO, BID, q24h (for liver disease) Silibinin

5 to 15 mg/kg q24h, upto 30 mg/kg q24h

Ursodeoxycholate

10 to 15 mg/kg PO q24h

Indications

Side Effects

Trade Names

Acetaminophen toxicity, Zentonil (Vetoquinol) Acetaminophen toxicity, xenobiotics, hepatotoxins Cholestatic liver disease, hepatitis, cirrhosis, copper-associated hepatopathy, hepatotoxins Amanita mushroom poisoning, other hepatotoxins, hepatitis, cirrhosis, cholestatic disorders Cholestatic disorders, cholangitis, hepatitis, copper-associated hepatopathy

Nausea, emesis

Denosyl (Nutrimax)

Hypotension, allergic reactions Platelet aggregation

Mucomyst (Apothecon) Mucosil-10 (Day Labs)

Suppression of cytochrome P450

Marin (Nutramax)

Anorexia

Actigall (Watson)

A recent study evaluated the therapeutic efficacy of combining silymarin with metronidazole for treating canine giardiasis.21 Although the silymarin dose (3.5 mg/kg/day combined with 50 mg/ kg/day of metronidazole) was quite small, a positive response was noted earlier in the silymarin-supplemented dogs.21

Ursodeoxycholic Acid (Ursodiol) Ursodiol differs from other antioxidants in that it is a prescription medication and it is normally prescribed for its choleretic, not antioxidant, properties. In a rat model of alcoholic steatohepatitis, ursodeoxycholic acid (UDCA) treatment improved liver morphology; decreased aspartate aminotransferase, γ-glutamyltransferase, and liver triglyceride content; and normalized cytochrome P450 enzyme content and markers of oxidative stress.22 UDCA administration (10 mg/kg) is safe, and is currently being used in a number of canine hepatopathies, including copper retention and chronic hepatitis.23 Ursodiol appears to have no prokinetic effect on the biliary system.

Other Diseases Feline intestinal ischemia–reperfusion injury is classically associated with oxygen free radical generation, and can be prevented by treatment with superoxide dismutase.24 Canine gastric dilation-volvulus is another example of an ischemia–reperfusion injury in which the iron-chelator deferoxamine reduces the severity of injury during the period of reperfusion.25 The free radical scavenger, fullerenol, was found to ameliorate changes in GSH in a canine experimental model of ischemic bowel syndrome.26 None of these antioxidant strategies have become best practice standards in cases of gastro­ intestinal ischemia–reperfusion injury.

Combination Therapy The first step in designing an effective antioxidant treatment plan is to define the oxidative damage that is most important in disease progression. Lipid peroxidation, DNA damage, or the signaling of inflammatory cytokines are just a few of the consequences of free radical production. A combination of these effects is likely to require a combination of antioxidants to return to homeostasis.

Combination antioxidant therapy has been used to beneficial effect in racing sled dogs and in cats with renal insufficiency.27-29 In the meantime, logical choices for antioxidant treatments in dogs and cats can be made based on the species, the disease, evidence from studies in other animal species, the small number of veterinary studies currently available, theory, lack of toxicity, and best practice standards. Table 40-1 lists antioxidant agents used in dogs and cats.

References 1. Parks DA, Bulkley GB, Granger DN: Role of oxygen-derived free radicals in digestive tract diseases. Surgery 94:415, 1983. 2. Kruidenier L, Verspaget HW: Review article: oxidative stress as a pathogenic factor in inflammatory bowel disease—radicals or ridiculous? Aliment Pharmacol Ther 16:1997, 2002. 3. Jones DP: Redefining oxidative stress. Antioxid Redox Signal 8:1865, 2006. 4. Center SA, Warner KL, Erb HN: Liver glutathione concentrations in dogs and cats with naturally occurring liver disease. Am J Vet Res 63:1187, 2002. 5. Spee B, Arends B, van den Ingh TS, et al: Copper metabolism and oxidative stress in chronic inflammatory and cholestatic liver diseases in dogs. J Vet Intern Med 20:1085, 2006. 6. Webb CB, Twedt DC, Fettman MJ, et al: S-adenosylmethionine (SAMe) in a feline acetaminophen model of oxidative injury. J Feline Med Surg 5:69, 2003 7. Center SA, Randolph JF, Warner KL, et al: The effects of S-adenosylmethionine on clinical pathology and redox potential in the red blood cell, liver, and bile of clinically normal cats. J Vet Intern Med 19:303, 2005. 8. Center SA, Warner KL, McCabe J, et al: Evaluation of the influence of S-adenosylmethionine on systemic and hepatic effects of prednisolone in dogs. Am J Vet Res 66:330, 2005. 9. Temeus MV, Kiningham KK, Carpenter AB, et al: Comparison of S-Adenosyl-L-methionine and N-acetylcysteine protective effects on acetaminophen hepatic toxicity. J Pharmacol Exp Ther 320:99, 2007. 10. Wallace KP, Center SA, Hickford FH, et al: S-adenosyl-Lmethionine (SAMe) for the treatment of acetaminophen toxicity in a dog. J Am Anim Hosp Assoc 38:246, 2002.

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11. Lu SC, Gukovsky I, Lugea A, et al: Role of S-adenosylmethionine in two experimental models of pancreatitis. FASEB J 77:56, 2002. 12. Laudanno OM: Cytoprotective effect of S-adenosylmethionine compared with that of misoprostol against ethanol, aspirinand stress-induced gastric damage. Am J Med 20(Suppl 5A):43, 1987. 13. El-Bassiouni EA, Abo-Ollo MM, Helmy MH, et al: Changes in the defense against free radicals in the liver and plasma of the dog during hypoxia and/or halothane anesthesia. Toxicology 128:25, 1998. 14. Hill AS, O’Neill S, Rogers QR, et al: Antioxidant prevention of Heinz body formation and oxidative injury in cats. Am J Vet Res 62:370, 2001. 15. Hill AS, Rogers QR, O’Neill SL, et al: Effects of dietary antioxidant supplementation before and after oral acetaminophen challenge in cats. Am J Vet Res 66:196, 2005. 16. Twedt DC, Webb CB, Tetrick MA: The effect of dietary vitamin E on the clinical laboratory and oxidant status of dogs with chronic hepatitis (abstract). J Vet Intern Med 17:418, 2003. 17. Boigk G, Stroedter L, Herbst H, et al: Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 26:643, 1997. 18. Wellington K, Jarvis B: Silymarin: a review of its clinical properties in the management of hepatic disorders. BioDrugs 115:465, 2001. 19. Vogel G, Tuchweber B, Trost W, et al: Protection by silibinin against Amanita phalloides intoxication in beagles. Toxicol Appl Pharmacol 73:355, 1984. 20. Paulová J, Dvorák M, Kolouch F, et al: [Verification of the hepatoprotective and therapeutic effect of silymarin in experimental liver

injury with tetrachloromethane in dogs.] Vet Med (Praha) 35:629, 1990. 21. Chon SK, Kim NS: Evaluation of silymarin in the treatment on asymptomatic Giardia infections in dogs. Parasitol Res 97:445, 2005. 22. Lukivskaya O, Zavodnik L, Knas M, et al: Antioxidant mechanism of hepatoprotection by ursodeoxycholic acid in experimental alcoholic steatohepatitis. Adv Med Sci 51:54, 2006. 23. McGrotty YL, Ramsey IK, Knottenbelt CM: Diagnosis and management of hepatic copper accumulation in a Skye Terrier. J Small Anim Pract 44:85, 2003. 24. Schoenberg MH, Muhl E, Sellin D, et al: Posthypotensive generation of superoxide free radicals—possible role in the pathogenesis of the intestinal mucosal damage. Acta Chir Scand 150:301, 1984. 25. Lantz GC, Badylak SF, Hiles MC, et al: Treatment of reperfusion injury in dogs with experimentally induced gastric dilatationvolvulus. Am J Vet Res 53:1594, 1992. 26. Lai HS, Chen WJ, Chiang LY: Free radical scavenging activity of fullerenol on the ischemia-reperfusion intestine in dogs. World J Surg 24:450, 2000. 27. Baskin CR, Hinchcliff KW, DiSilvestro RA, et al: Effects of dietary antioxidant supplementation on oxidative damage and resistance to oxidative damage during prolonged exercise in sled dogs. Am J Vet Res 61:886, 2000. 28. Piercy RJ, Hinchcliff KW, DiSilvestro RA, et al: Effect of dietary supplements containing antioxidants on attenuation of muscle damage in exercising sled dogs. Am J Vet Res 61:1438, 2000. 29. Yu S, Paetau-Robinson I: Dietary supplements of vitamin E and C and beta-carotene reduce oxidative stress in cats with renal insufficiency. Vet Res Commun 30:403, 2006.