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Updates on Immunomodulators

Lara K. Maxwell, DVM, PhD, DACVCP Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK. [email protected]

Numerous health problems require immunosuppressive therapy. Autoimmune diseases, such as immune mediated hemolytic anemia (IMHA), immune mediated thrombocytopenia (IMT), lupoid diseases, and pemphigus foliaceus, can be life-threatening and may require profound and rapid-acting immunosuppressive therapy. Hypersensitivity conditions include diseases such as atopy, flea allergy, food allergy, and feline eosinophilic erythematosus and may respond to relatively mild immunosuppression, especially if combined with identification of the inciting antigen that can allow environmental modification, parasite control, or the use of hypoallergenic diets. As with the therapy of autoimmune disease, glucocorticoids are generally recognized as the cornerstone of immunosuppressive therapy, while often produce disappointing results. However, with the availability of alternate therapeutic agents, such as cyclosporine and oclacitinib, which were specifically developed for use in veterinary species, glucocorticoids can often be avoided for the therapy of some allergies. During this talk, we will be comparing these therapeutics head to head with respect to their time to onset of action, effectiveness, safety, and cost.

Objectives 1) The attendee will be able to weigh timeliness, effectiveness, safety, and cost of the established and newer immunomodulators. 2) The attendee will be able to identify therapeutics that are most useful for the treatment of severe autoimmune diseases, like IMHA and pemphigus. 3) The attendee will be able to identify which adjunctive immunotherapeutics are most useful for specific autoimmune diseases. 4) The attendee will identify strategies to reduce cost of immunomodulatory therapeutics will maintaining safety and efficacy.

Glucocorticoids: The Cornerstone of Therapy Glucocorticoids bind to glucocorticoid receptors, resulting in translocation of the receptor complex to the nucleus of the cell and binding to glucocorticoid response elements, with resulting activation of transcription and manufacture of lipocortin. Lipocortin inhibits phospholipase A2 and has anti- inflammatory and immunosuppressive effects, inhibiting the production of prostaglandins and leukotrienes. Fibroblast and migration, antibody production, and lymphocyte proliferation are decreased by glucocorticoid administration. As most autoimmune and hypersensitivity conditions are rapidly (hours to days) responsive to glucocorticoids, they may be considered the cornerstone of immunosuppressive therapy. Nonetheless, not all autoimmune and allergic diseases require glucocorticoid therapy, as many side effects are associated with their useThe co-administration of other immunosuppressive drugs can aid in the goal lowering the glucocorticoid dose as much as possible.

268 Specific glucocorticoid agents commonly used in dogs and cats include prednisone, , methylprednisolone, dexamethasone, and budesonide. Agents differ from one another with respect to their glucocorticoid and mineralocorticoid activity, with prednisolone having both activities whereas dexamethasone has more potent glucocorticoid activity and weak mineralocorticoid activity. Prednisone is an inactive prodrug that is converted by 11-beta-hydroxysteroid dehydrogenase (11-ɴ- HSD) to its active form, prednisolone. Dogs and people efficiently convert prednisone to prednisolone via 11-ɴ-HSD, whereas horses do not.1 On the heels of convincing evidence that horses fail to convert the majority of an administered dose of prednisone to prednisolone, there has been interest in determining whether cats are similarly poor at converting prednisone to prednisolone. Recent evidence indicates that whereas cats can interconvert prednisone and prednisolone, the administration of prednisone results in considerably lower systemic prednisolone concentrations.2 Interestingly, over-conditioned cats converted more prednisone to prednisolone than did cats with ideal body weights, which could increase both the efficacy and side effects after prednisone administration to obese cats.

Budesonide is a highly lipophilic, non-halogenated glucocorticoid with potent glucocorticoid effects. It is both rapidly absorbed in the (GIT) and rapidly metabolized to inactive metabolites. Budesonide is thought to primarily produce a localized effect in the GIT, with some systemic absorption of active drug resulting in suppression of the hypothalamic-pituitary-adrenal axis. Oral absorption is used to facilitate a local GI effect for conditions such as inflammatory bowel disease. In order for budesonide to act at the intestines, rather than being absorbed in the stomach, enteric coating prohibits dissolution until pH exceeds 5.5. Therefore, compounded products are likely to have specific activity in the small intestine. Adequate response to oral budesonide administration has been reported in a small case series of 8/11 dogs with inflammatory bowel disease.

Calcineurin Inhibitors Cyclosporine was the first calcineurin inhibitor used for immunosuppressive therapy and marked a profound improvement in the outcomes of organ transplantation in people. Cyclosporine is a large, highly lipophilic molecule that acts intracellularly to prevent several distinct downstream events of lymphocyte T-cell activation that ultimately lead to transcriptional activation of IL-2 and other inflammatory . Because cyclosporine is so lipophilic, injectable products are complexed with Cremaphor as a solubilizing agent. The intravenous administration of the resulting injectable solution is expensive and associated with anaphylactoid reactions, especially in dogs. This highly lipophilic nature of cyclosporine also necessitates specific formulation of the compound so that it can be orally absorbed, although oral of even specialized formulations is variable and moderate (~30%) at best. Each oral cyclosporine product might be absorbed differently, so human generic products are not interchangeable with the veterinary product (Atopica™). Cyclosporine is highly metabolized, primarily by CYP3A enzymes in people and dogs and is subject to many drug interactions. In order to decrease the cost of therapy, several strategies have exploited these drug interactions in order to increase the oral bioavailability or decrease the clearance of cyclosporine, so that the overall dose can be reduced in dogs. For example, administration of oral ketoconazole at a dose rate of 13 mg/kg/day decreased the rate of cyclosporine clearance to ~35% of pre-ketoconazole administration values and allowed up to a 75% reduction in cyclosporine dose.3,4 Similarly, the oral administration of clarithromycin at 10 mg/kg twice daily increased the systemic exposure of dogs to cyclosporine by

269 ~33%.5 Attempts to manipulate the disposition of cyclosporine through intentional drug interactions should be guided by therapeutic drug monitoring, as the response to the interaction will be variable.

In veterinary medicine, cyclosporine is approved for the treatment of atopic in dogs and cats, with a slow time to onset of action of approximately 10-30 days. Atopica is labeled for use in dogs greater than six months of age. Adverse effects include gastrointestinal upset, nephrotoxicity, gingival hyperplasia, and hepatotoxicity. Gingival hyperplasia and papillomas and other cutaneous masses are thought to be dose-depend side effects. As cyclosporine is an immunosuppressive agent, cats should be negative for FIV/FeLV and dogs free of neoplasms before beginning therapy. In addition to atopy, cyclosporine has been administered to dogs with keratoconjunctivis sicca (KCS), immune-mediated hemolytic anemia, and other autoimmune diseases. Other calcineurin inhibitors that have been used in dogs include tacrolimus and pimecrolimus. Pimecrolimus is available as an ophthalmic preparation and has been reported to be at least as efficacious as cyclosporine, resulting in less ocular inflammation in dogs with KCS.6 Tacrolimus has been applied locally in dogs with with apparent success and has successfully treated dogs with perianal sinuses, when combined with tapering doses of prednisone.7,8 The safety of immunosuppressive doses of prednisone combined with oral cyclosporine have been specifically studied in dogs and appear to be well-tolerated when co-administered for three weeks.

JAK STAT Inhibitors There are currently two JAK STAT inhibitors approved in the United States. Oclacitinib (Apoquel™) is approved for the management of atopic dermatitis in dogs, whereas and are approved for treatment of rheumatoid arthritis and myelofibrosis in people. Several other JAK STAT inhibitors are being investigated in human clinical trials with the goal of managing rheumatoid arthritis. These agents differ with respect to the specificity of inhibition of the four members of the JAK family. Apoquel is more potent against JAK1 as compared to the other JAK enzymes, but it still inhibits all four enzymes at plasma concentrations encountered after oral administration of oclacitinib in dogs.9,10 Inhibition of JAK enzymes produces an immunomodulatory effect as activation of receptors of the immunological system act via the JAK STAT pathway to orchestrate the immune response, including the further release of inflammatory cytokines. Oclacitinib is well absorbed after oral administration to dogs, with a short elimination half-life of approximately 4 hours.9 Feeding did not affect oral administration, so oclacitinib can be administered with or without a meal. There were also no pharmacokinetic differences attributed to gender or to beagle versus mixed breed dogs. Oclacitinib appears to have a rapid onset of action, with pruritus decreasing by at least 50% within a day of beginning administration, and pruritus decreasing in 70% of treated atopic dogs by one week of beginning therapy.11 The efficacy of oclacitinib in dogs with atopic dermatitis is similar to that of prednisolone after one month of therapy.12 Minimal gastrointestinal side effects were reported in the clinical trials of Apoqel that supported its submission to the FDA, although possible self-limiting GI signs, decreased leukocytes, and decreased globulins were reported. Long term toxicity studies at one to five times the labeled dose in laboratory beagles resulted in interdigital furunculosis, lymphadenopathy, bone marrow and GALT suppression, and mild interstitial . Similar testing in 4-6 month old puppies resulted in bacterial pneumonia, sepsis, and demodicosis, all signs that point to immunosuppression. Due to these results, Apoquel should not be administered to dogs under one year of age or to dogs with demodicosis.

270 Monoclonal Antibody to Canine IL-31 The use of biological methods to produce therapeutics has been used for some time but has become increasingly sophisticated in medicine. Recent years have seen considerable expansion in the investigation and use of biological therapies as veterinary therapeutics. Advance in species-specific engineering of antibodies, with a resulting decrease in antigenicity, have brought monoclonal antibodies (mAbs) to many fields of human medicine, and mAbs are now entering the veterinary field as well. Therapeutic antibodies are usually raised in vitro in cell systems, especially hybridomas. After the success of oclacitinib in controlling signs of hypersensitivity in dogs, a mAb targeting a specific portion of the same pathway has been introduced to veterinary medicine. Lokivetmab (Cytopoint™) specifically targets IL-31, a cytokine that has been implicated in pruritus in dogs and other species. In late, 2016 the USDA granted a license for lokivetmab in the therapy of atopic dermatitis in dogs. In a placebo-controlled trial of 211 client-owned dogs with allergic dermatitis, lokivetmab was administered at doses ranging from 0.125 to 2 mg/kg subcutaneously. There was a dose-dependent effect on pruritus, but the highest dose rate had the most profound effect, beginning at 1 day after initiating therapy and continuing for 49 days.13 Ultimately, the product was approved at a dose rate of 2 mg/kg SC and frequency of 4-8 weeks. Only histologically apparent injection site reactions were reported as side effects in preclinical or clinical studies at dose rates of up to 10 mg/kg monthly for 7 months. Lokivetmab appears to rapidly and profoundly decrease pruritus in dogs with atopic dermatitis, supporting the key role of IL-31 in the pathogenesis of this condition in dogs. Limitations of its use include little experience with the use of lokivetmab in the therapy of other hypersensitivities in dogs, and the reported high immunogenicity of this caninized mAb in cats.14

Cytotoxic Agents Several cytotoxic agent are used for the management of autoimmune disease. As such, these agents primarily act by impairing DNA synthesis, thereby affecting the most rapidly dividing cells, such as white blood progenitor cells. Azathioprine is an antimetabolite that is a purine analog, ultimately inhibiting DNA synthesis. It’s generally considered a second or third-line immunosuppressant in dogs, where it has a well-established track record as an immunosuppressant agent. Azathioprine is not as well-tolerated in cats. As is common to cytotoxic drugs, bone marrow suppression can occur, as well as gastrointestinal disturbances. Hepatotoxicity of azathioprine is also a concern. Azathioprine is metabolized to 6-mercaptopurine (6-MP), which is itself metabolized to active metabolites, the 6- thioguanine nucleotides (6-TGNs) that are incorporated into DNA and are cytotoxic. 6-MP is also inactivated by several enzymes, thiopurine methyltransferase (TPMT) and xanthine oxidase. TPMT is of particular interest, since in people, this enzyme is genetically polymorphic and correlates will with the risk of myelotoxicity. In people, TPMT activity in red blood cells is routinely determined in order to assess the potential risk of azathioprine therapy. Interestingly, dogs have higher TPMT activities than do cats, suggesting that lower TPMT in cats is responsible for lower tolerance for azathioprine in this species as compared with dogs.15 TPMT activity appears to also differ between dog breeds; for example, Giant Schnauzers may have low TPMT activity whereas that of Alaskan Malamutes is high.16 However, the ultimate consequence of TPMT activity in dogs is unclear, as when TPMT was measured in 299 that were receiving azathioprine therapy, dogs with azathioprine myelotoxicity did not have low TMPT activity.17 On the other hand, dogs with intermediate TMPT activity had lower neutrophil counts than did dogs with high activity, which does support the important role of this enzyme.

271 Mycophenolate mofetil (MMF) is another cytotoxic antimetabolite that inhibits the enzyme inosine monophosphate dehydrogenase, which is need for guanosine synthesis. Lymphocytes are particularly sensitive to MMF because they must synthesize purines. This mechanism of action makes MMF more specific to inhibition of T-cells as compared to white blood cells, so MMF is not expected to have the broader effects of azathioprine. Nonetheless, gastrointestinal toxicity appears to be a real concern in dogs.18 The use of MMF as an immunosuppressant has increasingly supplanted azathioprine in people, but is still relatively new to veterinary medicine. However, MMF shows promise for several challenging immune mediated diseases in veterinary medicine, and has been investigated in a study of 52 dogs with idiopathic immune-mediated hemolytic anemia (IMHA).19 Here, MMF was administered with glucocorticoids and compared to standard glucocorticoid and one other immunosuppressant. While both groups of dogs showed similar survival, with 23/30 MMF dogs surviving to discharge and 20/30 surviving at 60 days, adverse events were higher in the standard combination group. MMF has also been investigated in a small number of cats with IMHA, with favorable outcomes.20 A retrospective comparison of MMF versus cyclosporine in combination with a glucocorticoid in dogs with immune- mediated thrombocytopenia found similar survival with both drug regimens, but a lower cost with the MMF as compared with cyclosporine.21 Numerous human health oriented studies have tested MMF in dogs and found it efficacious, so further veterinary applications for immune mediated disease, such as disease,22 are likely, but gastrointestinal side effects must be carefully monitored. Other cytotoxic agents include leflunomide and chlorambucil and will also be discussed.

REFERENCES

1. Peroni DL, Stanley S, Kollias-Baker C, Robinson NE. Prednisone per os is likely to have limited efficacy in horses. Equine Vet J. 2002;34(3):283-287. 2. Center SA, Randolph JF, Warner KL, Simpson KW, Rishniw M. Influence of body condition on plasma prednisolone and prednisone concentrations in clinically healthy cats after single oral dose administration. Res Vet Sci. 2013;95(1):225-230. 3. D'Mello A, Venkataramanan R, Satake M, et al. of the cyclosporine- ketoconazole interaction in dogs. Res Commun Chem Pathol Pharmacol. 1989;64(3):441-454. 4. Dahlinger J, Gregory C, Bea J. Effect of ketoconazole on cyclosporine dose in healthy dogs. Vet Surg. 1998;27(1):64-68. 5. Katayama M, Kawakami Y, Katayama R, Shimamura S, Okamura Y, Uzuka Y. Preliminary study of effects of multiple oral dosing of clarithromycin on the pharmacokinetics of cyclosporine in dogs. J Vet Med Sci. 2014;76(3):431-433. 6. Ofri R, Lambrou GN, Allgoewer I, et al. Clinical evaluation of pimecrolimus eye drops for treatment of canine keratoconjunctivitis sicca: A comparison with cyclosporine A. Vet J. 2009;179(1):70-77. 7. Olivry T, Foster AP, Mueller RS, McEwan NA, Chesney C, Williams HC. Interventions for atopic dermatitis in dogs: a systematic review of randomized controlled trials. Vet Dermatol. 2010;21(1):4-22. 8. Stanley BJ, Hauptman JG. Long-term prospective evaluation of topically applied 0.1% tacrolimus ointment for treatment of perianal sinuses in dogs. J Am Vet Med Assoc. 2009;235(4):397-404. 9. Collard WT, Hummel BD, Fielder AF, et al. The pharmacokinetics of oclacitinib maleate, a inhibitor, in the dog. J Vet Pharmacol Ther. 2014;37(3):279-285.

272 10. Gonzales AJ, Bowman JW, Fici GJ, Zhang M, Mann DW, Mitton-Fry M. Oclacitinib (APOQUEL((R))) is a novel with activity against cytokines involved in allergy. J Vet Pharmacol Ther. 2014;37(4):317-324. 11. Cosgrove SB, Wren JA, Cleaver DM, et al. A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis. Vet Dermatol. 2013;24(6):587-597, e141-582. 12. Gadeyne C, Little P, King VL, Edwards N, Davis K, Stegemann MR. Efficacy of oclacitinib (Apoquel ) compared with prednisolone for the control of pruritus and clinical signs associated with allergic dermatitis in client-owned dogs in Australia. Vet Dermatol. 2014. 13. Michels GM, Ramsey DS, Walsh KF, et al. A blinded, randomized, placebo-controlled, dose determination trial of lokivetmab (ZTS-00103289), a caninized, anti-canine IL-31 monoclonal antibody in client owned dogs with atopic dermatitis. Vet Dermatol. 2016;27(6):478-e129. 14. Zoetis. Canine Atopic Dermatitis Immunotherapeutic: A Caninized Anti-cIL-31 Monoclonal Antibody U.S. FAQ’s for External Use. 15. White SD, Rosychuk RA, Outerbridge CA, et al. Thiopurine methyltransferase in red blood cells of dogs, cats, and horses. J Vet Intern Med. 2000;14(5):499-502. 16. Kidd LB, Salavaggione OE, Szumlanski CL, Miller JL, Weinshilboum RM, Trepanier L. Thiopurine methyltransferase activity in red blood cells of dogs. J Vet Intern Med. 2004;18(2):214-218. 17. Rodriguez DB, Mackin A, Easley R, et al. Relationship between red blood cell thiopurine methyltransferase activity and myelotoxicity in dogs receiving azathioprine. J Vet Intern Med. 2004;18(3):339-345. 18. West LD, Hart JR. Treatment of idiopathic immune-mediated hemolytic anemia with mycophenolate mofetil in five dogs. J Vet Emerg Crit Care (San Antonio). 2014;24(2):226-231. 19. Wang A, Smith JR, Creevy KE. Treatment of canine idiopathic immune-mediated haemolytic anaemia with mycophenolate mofetil and glucocorticoids: 30 cases (2007 to 2011). J Small Anim Pract. 2013;54(8):399-404. 20. Bacek LM, Macintire DK. Treatment of primary immune-mediated hemolytic anemia with mycophenolate mofetil in two cats. J Vet Emerg Crit Care. 2011;21(1):45-49. 21. Cummings FO, Rizzo SA. Treatment of presumptive primary immune-mediated thrombocytopenia with mycophenolate mofetil versus cyclosporine in dogs. J Small Anim Pract. 2017;58(2):96-102. 22. Ackermann AL, May ER, Frank LA. Use of mycophenolate mofetil to treat immune-mediated skin disease in 14 dogs - a retrospective evaluation. Vet Dermatol. 2017;28(2):195-e144.

273 Multimodal Control of Pain and Disease

Lara K. Maxwell, DVM, PhD, DACVCP Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK. [email protected]

There are several different points in the pain pathway, each responsive to different modulators. When analgesic agents act at different points in the pain pathway, overall analgesic effects can be additive or even synergistic. Even if a similar level of analgesia could be achieved by higher doses of a single therapeutic agent, multimodal analgesia is still likely to be superior in several ways. 1) The use of several drugs at lower doses is likely to produce fewer side effects than a single, higher dose of one agents. Analgesic agents that act at different points in the pain pathway will generally also exhibit differing toxicity profiles. 2) The use of several analgesic agents can allow preemptive control of pain. As an example, some patients, such as the hypotensive patient, should probably not receive a pre- or inter-operative NSAID, as hypotension predisposes patients to a decrease in renal perfusion and NSAID associated nephrotoxicity. However, the preemptive use of an opioid agent can prevent pain and central wind-up, so that the overall analgesic plan is more successful when the NSAID is later administered after correction of hypotension. 3) Analgesic monotherapy, even with a highly efficacious drug like morphine, may not sufficiently control severe pain. If monotherapy is attempted in such a situation, side effects such as nausea and are more likely to occur due to the use of escalating doses. The use of multimodal analgesia can be useful for both surgery patients and those with chronic diseases, like osteoarthritis. However, some patients will benefit more from the multimodal approach than others. The application of multimodal principles will be applied to both the perioperative setting and to the long-term management of chronic pain.

Objectives 1) The attendee will be able to describe the pathophysiology of pain. 2) The attendee will assess how different classes of drugs, from NSAIDs to opioids, fit into the multimodal control of pain. 3) The attendee will identify alternate therapies for patients that respond poorly to a particular drug or drug class.

When injury occurs, such as a surgical incision, a chain of nocioceptive events begin even in the anesthetized patient. Depolarization of the afferent myelinated (A-delta) and unmyelinated (C) nerve fibers is transmitted to the dorsal horn in the spinal cord and on to the thalamus and cortex of the brain. In the awake patient, perception of pain is the immediate outcome of injury. However, even in the anesthetized patient, local release of inflammatory and vasodilatory cytokines augment the nocioceptive response and set the stage for the later perception of pain. Inflammatory mediators activate the afferent nerve fibers directly but also decrease their threshold for depolarization, resulting in peripheral sensitization and increased perception of pain. Central sensitization in the spinal cord may also occur. Sympathetic stimulation further augments the peripheral and central response. Repetitive C fiber stimulation activates N-methyl-D-aspartate (NMDA) receptors and glutamate sensitivity, contributing to central windup, hyperalgesia, and allodynia. In counterpoint to these pathways of pain escalation, descending supraspinal modulation pathways can help to decrease the perception of pain, with serotonin and endorphins inhibiting the spinal nocioceptive response.

274 Multimodal Analgesia: Who Needs That? As we can see, there are several different points in the pain pathway, each responsive to different modulators. When analgesic agents act at different points in the pain pathway, overall analgesic effects can be additive or even synergistic. Even if a similar level of analgesia could be achieved by higher doses of a single therapeutic agent, multimodal analgesia is still likely to be superior in several ways. 1) The use of several drugs at lower doses is likely to produce fewer side effects than a single, higher dose of one agents. Analgesic agents that act at different points in the pain pathway will generally also exhibit differing toxicity profiles. Therefore, even if the analgesic effect of multimodal therapy is additive, the side effects will be less than additive. For example, NSAIDs tend to be most closely associated with gastrointestinal toxicity as their major side effect. On the other hand, opioids tend to be associated with respiratory depression and constipation, depending on their use and the status of the patient. As the toxicity spectrum of NSAIDs and opioids do not substantially overlap, typical therapeutic doses are likely to produce only minor side effects with adequate analgesia. 2) The use of several analgesic agents can allow preemptive control of pain. As an example, some patients, such as the hypotensive patient, should probably not receive a pre- or inter-operative NSAID, as hypotension predisposes patients to a decrease in renal perfusion and NSAID associated nephrotoxicity. However, opioids may be used if the intravenous route is avoided, allowing opioid use before, during, or immediately after the surgery. The preemptive use of the opioid agent can prevent pain and central wind-up, so that the overall analgesic plan is more successful when the NSAID is later administered after correction of hypotension. 3) Analgesic monotherapy, even with a highly efficacious drug like morphine, may not sufficiently control severe pain. If monotherapy is attempted in such a situation, side effects such as nausea and vomiting are more likely to occur due to the use of escalating doses.

The use of multimodal analgesia can be useful for both surgery patients and those with chronic diseases, like osteoarthritis. However, some patients will benefit more from the multimodal approach than others. For example, an uncomplicated ovariohysterectomy in a cat may benefit from the use of an opioid preanesthetic agents combined with peri-operative administration of a NSAID. However, even if the preanesthetic protocol did not use an opioid, the NSAID as monotherapy would probably provide sufficient post-operative analgesia for relatively mild pain such as this. However, the patient with severe pain, such as from limb amputation, will benefit much more from appropriate multimodal therapy that hits several points in the pain pathway. For example, the amputation patient may benefit from an opioid preanesthetic, peri-operative NSAID administration, local anesthetic infiltrated around the bisected nerve plexus, and a long-acting post-operative opioid, such as a fentanyl patch. The use of the pre- and intra-operative analgesics would serve to dampen transmission and transduction of nerve impulses, with a concomitant decrease hyperalgesia and windup of spinal nerves. Post-operative analgesics would then be more effective, and agitation due to severe pain in the immediate post- operative period minimized. Several excellent reviews of the use of multi-modal analgesia for specific types of surgical patients are available.1,2

Treatment Combinations that Can Work over the Long Haul The recent emphasis on multimodal analgesic techniques for surgical patients has also heightened interest in applying such techniques for the control of chronic pain. Osteoarthritis is a common example of chronic pain that can be debilitating and affect the quality of life of both cats and dogs, with other common examples including cancer and neuropathic pain. NSAIDs have been the mainstay of therapeutics used in chronic pain in dogs for some years, even before the newer NSAIDs became

275 available. With the widespread occurrence of osteoarthritis in dogs, combined with larger proportions of geriatric pets, and the availability of highly efficacious, safer drugs, NSAIDs have become very popular for long-term administration in this species. NSAID monotherapy is often sufficient to improve owner perception of comfort in dogs with osteoarthritis and the toxicity profile is favorable in most pets, even geriatric pets. In contrast, cats have not benefited nearly as much from the increased availability of NSAIDs, with only two, meloxicam and robenacoxib, currently approved for U.S. use in cats. While the risk versus benefit ratio of NSAID use in dogs is generally positive, NSAIDs alone are insufficient to control pain in some arthritic dogs and in many pets with more severe or more centralized pain, such as neuropathic pain. In these animals, multimodal analgesia has made strides in providing comfort, and may practitioners maintain a list of drugs that can be added to standard NSAIDs if necessary.

For osteoarthritis, the use of chondroprotective nutraceutical agents, such as glucosamine, chondroitin, and omega-3-fatty acids has become popular. Although the hard evidence to support the efficacy of many of these nutraceutical agents is somewhat underwhelming, they continue to be first- line drugs for many practitioners in the therapy of osteoarthritis for several reasons: 1) popular perception within the U.S. public is that nutraceutical agents are effective in supporting normal function in humans with osteoarthritis. 2) There is a widespread perception in pet owners and practitioners that common nutraceutical agents provide some degree of pain relief, particularly in dogs with osteoarthritis. 3) Although the efficacy of many nutraceutical agents is open to debate, nutraceuticals appear to be safe, with very few side effects reported in veterinary species.3 Although these nutraceutical products are generally given without NSAIDs or other established analgesics in published efficacy studies, the patient that is even moderately painful due to newly diagnosed osteoarthritis can greatly benefit from including an NSAID when beginning nutraceutical chondroprotective administration, as the nutraceutical cannot be expected to provide relief in the short term. The rationale for giving the chondroprotective agent alone is that its efficacy can be determined in that specific patient, to facilitate a decision of whether to continue nutraceutical administration. However, given the greater spectrum of side effects associated with NSAIDs as compared to nutraceutical agents, the eventual goal of combination therapy is often to decrease the NSAID dose to the lowest effective dose after control of clinical signs has been achieved. A weaning of NSAID dose is particularly important in treating cats with osteoarthritis long-term, since the safety profile of NSAID use in cats is more questionable than it is in dogs.

The popularity of opioid drugs for surgical pain can be explained by their excellent analgesia operating at several different points of the pain pathway, especially when administered with a second analgesic agent. However, only recently has the use of multimodal analgesics has become common in the therapy of osteoarthritis. Although NSAIDs and nutraceutical supplements are usually the first agents employed in the therapy of osteoarthritis, additional pain relievers often become necessary during short-term flare-ups, as the osteoarthritis progresses, or for the treatment of other types of chronic pain, such as cancer and neuropathic pain. Tramadol (and its metabolites) is an opioid-like drug that is not a controlled substance but acts as a partial mu receptor agonist at opioid receptors. Unlike most opioids, tramadol is distinguished by distinct effects at other points in the pain pathway, functioning centrally to increase both serotonin and norepinephrine availability. The activity of tramadol at a variety of points in the pain pathway may act in a multi-modal fashion, affecting transduction, perception, and modulation of pain. Unfortunately, the pharmacokinetic behavior of tramadol appears

276 to be somewhat short-lived and variable, resulting in a range of suggested doses and a short dosing interval of 6-8 hours in dogs.4,5 Despite these shortcomings and the need for caution in giving multiple serotoninergic drugs simultaneously, in order to avoid serotonin syndrome, tramadol may be particularly useful in combination with other analgesics for both chronic and surgical pain.6

Other drugs that are increasingly popular for the treatment of chronic pain, particularly of neuropathic pain, include gabapentin and amantadine. Both drugs have interesting histories that include discovery of analgesic properties quite apart from their original purposes of antiseizure and antiviral indications. As a centrally acting drug, gabapentin penetrates into the central nervous system well and acts by poorly understood mechanisms at calcium channels in the treatment of epilepsy. As an analgesic, gabapentin is thought to act by decreasing the availability of excitatory neurotransmitters, decreasing the “wind-up” process described above. In addition, gabapentin may affect modulation of pain. Thus, gabapentin can be expected to decrease hyperalgesia and increased response to nervous transmission of pain, which can be a major factor in continual neuropathic pain. Despite a disappointing lack of analgesia in several dog and cat experimental studies, gabapentin continues to enjoy clinical use as part of a multimodal analgesic plan, especially where neuropathic pain is involved.7 Amantadine is also thought to decrease central windup and allodynia through a different mechanism of action at the NMDA receptor. As with gabapentin, the analgesic efficacy of amantadine is similarly unclear, with some studies finding a beneficial analgesic effect whereas others do not. However, at least one study suggests that amantadine may be a useful adjuvant to NSAIDs in the therapy of osteoarthritis in dogs.8 Similarly to the multimodal effects of tramadol, tricyclic antidepressants, such as amitriptyline, may also act at multiple points of the pain pathway, but are thought to promote modulation of pain via the central actions of serotonin and norepinephrine. Amitriptyline has similarly been promising in the treatment of neuropathic pain in people. Taken together, the studies conducted to date in humans and veterinary species indicate that NSAIDs are still the most effective, first choice agent for inflammatory pain, but that the addition of agents that act at separate sites in the pain pathway may further decrease chronic pain and may be necessary if neuropathic pain exists. Failure of chronic pain to respond adequately to conventional therapy with NSAIDs justifies a trial of adjuvant therapy, which can provide safe analgesia to the painful patient.

References 1. Lascelles B. Preoperative analgesia - opioids and NSAIDs. Waltham Focus 1999;9(4):2-9. 2. Robertson SA. Managing pain in feline patients. Vet Clin North Am Small Anim Pract 2008;38(6):1267-90, vi. 3. Vandeweerd JM, Coisnon C, Clegg P, et al. Systematic review of efficacy of nutraceuticals to alleviate clinical signs of osteoarthritis. J Vet Intern Med 2012;26(3):448-56. 4. Clark JS, Bentley E, Smith LJ. Evaluation of topical nalbuphine or oral tramadol as analgesics for corneal pain in dogs: a pilot study. Vet Ophthalmol 2011;14(6):358-64. 5. Kukanich B, Papich MG. Pharmacokinetics and antinociceptive effects of oral tramadol hydrochloride administration in Greyhounds. Am J Vet Res 2011;72(2):256-62. 6. Rychel JK. Diagnosis and treatment of osteoarthritis. Top Companion Anim Med 2010;25(1):20-5. 7. Vettorato E, Corletto F. Gabapentin as part of multi-modal analgesia in two cats suffering multiple injuries. Vet Anaesth Analg 2011;38(5):518-20. 8. Lascelles BD, Gaynor JS, Smith ES, et al. Amantadine in a multimodal analgesic regimen for alleviation of refractory osteoarthritis pain in dogs. J Vet Intern Med 2008;22(1):53-9.

277 Herpes to Flu: Antivirals Lara K. Maxwell, DVM, PhD, DACVCP Affiliation: Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, [email protected] Description and Objectives Tremendous progress has been made in the development of drugs for the therapy of human viral diseases, and veterinary species are slowly gaining from these discoveries. However, there are still many uncertainties in the appropriate use of antiviral therapies in veterinary medicine. Although antiviral therapies share some traits with antibacterial agents, the antiviral drugs are also uniquely different. This talk will discuss the selection of therapeutics used in the treatment of common viral diseases, including feline herpesvirus, feline retroviruses, influenza, and parvovirus. Objective 1. The attendee will be able to identify the similarities and differences between antiviral and other anti-infective drugs. Objective 2. The attendee will be able to list the antiviral drugs used for different manifestations of common viral diseases of dogs and cats. Objective 3. The attendee will be able to determine whether antiviral interventions can be justified for the therapy of FHV, FIV, FeLV, canine influenza, and canine parvovirus. Feline Herpes Virus-1 (FHV-1) FHV-1 is ubiquitous in cats. However, clinical disease is more prevalent in high density conditions, such as pet stores, shelters, catteries, etc. Clinical signs include rhinitis, conjunctivitis, stomatitis, and dermatitis. The FVRCP vaccination is used to reduce disease associated with FHV-1. Vaccines include both modified live and killed vaccines. As with other herpes viruses, some vaccinated cats may have disease associated with FHV-1. Intranasal vaccination with FHV-1 may be useful in some cats with chronic rhinitis. Herpes conjunctivitis can be self-limiting and resolve without treatment. Chronic conjunctivitis (>3 weeks?) or recurrence merits antiviral therapy. Corneal ulceration merits both antiviral and antibacterial therapy. Antiviral therapy is unlikely to be curative. Typical goals of therapy include control of clinical signs, prevention of further damage, and reduction of pain. Therapeutic options for herpetic conjunctivitis and keratitis include idoxuridine, trifluridine, vidarabine, cidofovir, famciclovir, L-lysine, and . Specific Agents: Acyclovir is a nucleoside (guanosine) analog. Phosphorylation by viral thymidine kinase allows inhibition of DNA polymerase and termination of the DNA chain. Acyclovir (the prototypic drug of this class) is the most selective of these antiviral drugs due to the need for viral TK phosphorylation. Of the commonly used nucleoside analogs, acyclovir is also the least effective against many types of herpesviruses. Acyclovir is likely to be effective only against herpes viruses and has been used in birds, cats, horses, and dogs. The timing of therapy is important as it is not active against latent virus. Acyclovir can be administered IV, orally, or topically. Oral absorption is poor in humans (<30%) and probably poorer in veterinary species. Acyclovir is primarily eliminated by the , with some hepatic involvement. The IV formulation has the greatest risk of toxicity, with renal insufficiency and CNS signs possible.

278 Toxicity is rarely associated with oral dosing, since so little of the drug is absorbed. Since the oral bioavailability of acyclovir is so poor, the ester prodrug of valacyclovir has been developed to markedly enhance the bioavailability of the active drug, acyclovir. Oral absorption is much improved over that of acyclovir. Since valacyclovir itself has minimal activity, side effects are similar to high dose acyclovir therapy. At high doses (60 mg/kg q 6 hr), oral valacyclovir was toxic to cats, with renal tubular necrosis and bone marrow suppression reported.1 As FHV-1 also appears to be insensitive to acyclovir, even this high dose of valacyclovir had negligible effects on FHV-1 viral loads in cats. Therefore, neither acyclovir nor valacyclovir are generally indicated for the therapy of FHV-1 infections. Famciclovir is the oral prodrug of penciclovir with enhanced absorption as compared to the active agent, penciclovir. In vitro testing indicates that FHV-1 is more sensitive to penciclovir as compared with acyclovir. Famciclovir is currently the only systemically administered antiviral agent that is routinely recommended for use in cats with signs of FHV-1 infection. A dose of 90 mg/kg administered every 8 hr for 21 days appears safe in cats,2 but a lower dose is more commonly used.3 It is available as 125, 250, 500 mg tablets, with ¼ -1 of a 125 mg administered every 8-12 hours most often selected. Oral dosing is generally well tolerated in cats, with and polydipsia possible. In people, side effects include GI distress and neutropenia. Idoxuridine is a popular ophthalmic treatment for herpetic keratitis and conjunctivitis in cats. It is only available as a compounded formulation, as the ophthalmic preparation was withdrawn. Both solutions (0.1%) and ointments (0.5%) are available, but the solution is cleared rapidly from the eye. Effects don’t persist after drug is cleared, due to virostatic action. However, idoxuridine is usually well-tolerated by cats and is popular.4 Idoxuridine may need to be administered every 2-3 waking hours at the beginning of therapy for best efficacy. One suggested alternative dosing schedule involves, initial therapy with one drop q 5 min for 30 min, then 3-6 times daily (Morgan, Small Animal Drug Handbook). Effective therapy may require prolonged dosing, for at least one week past resolution of signs. Trifluridine is available as a 1% solution (Viroptic™), that is cleared rapidly from the eye. As with other antiviral solutions, effects don’t persist after the drug is cleared, due to virostatic action. Although FHV-1 appears to be sensitive to trifluridine, its utility in cats is limited by irritation, as it may sting upon administration. Trifluridine may need to be administered every 2- 3 waking hours at beginning of therapy and require prolonged dosing, past the resolution of signs. Cidofovir is available as a 0.5%, compounded solution. Cidofovir is generally more expensive than compounded idoxuridine, but can be administered less frequently.5 Due to its long intracellular half-life, cidofovir may be administered every 12 hr. As with other antiviral drugs, successful treatment may require prolonged dosing, past resolution of clinical signs. Vidarabine is yet another antiviral drug that is no longer commercially produced, so is compounded as a 3% ointment. Solutions may also be available, but are cleared rapidly from the eye. Vidarabine is generally well- tolerated. Good compliance is necessary, since the drug is often administered every 2-3 waking hours at the beginning of therapy.4 L-Lysine is an amino acid, nutritional supplement that is structurally related to arginine and can displace arginine in many chemical reactions. Biologically, L-lysine acts as a competitive

279 inhibitor for processes requiring arginine. This inhibitory action results in a relative deficiency in arginine that has been shown to produce an antiherpetic effect in some settings. The usual rationale for this antiherpetic effect is that herpesviruses require arginine for replication, so the administration of relatively large doses of L-lysine may inhibit herpesvirus replication. Lysine administration has been used for years for prophylactic therapy in human HSV carriers. Oral lysine supplementation (e.g., 500 mg/cat every 12 hr) has also been used in cats with recurrent FHV infections. Unfortunately, one prospective study of lysine supplementation in cats with FHV failed to find a benefit to lysine administration.6 On the other hand, other studies have found a positive effect.7 Given these contradictory results and the mild response expected from supplementation alone, lysine shouldn’t be considered for monotherapy of affected animals. However, lysine may help to reduce recrudescence with stress and may be useful as an adjunct. While little detriment is expected with short-term therapy, long-term lysine administration with high doses could conceivably result in a nutritional deficiency in arginine, so caution is warranted if long-term therapy is utilized. are biological response modifiers that are cytokine stimulators of cell mediated immunity. They stimulate the activity of cytotoxic T-cells, which are thought to be the major immunological defense against herpesviruses. Unlike specific antiviral drugs, interferon is a general immunostimulant and does not target specific viruses. Interferons have been suggested as adjunct therapy for the treatment of FHV. The use of interferons for FHV is empirical. Interferons of both human and feline origin have been used in cats. Alpha interferon, a human recombinant product, is commercially available in the U.S. and has generally been given SC. Omega interferon of feline origin has also been produced, but is not manufactured in the U.S. In a recent study, this product provided minimal benefits to cats with acute upper respiratory viral disease.(Ballin et al.) As a protein, it is generally thought that parenteral administration of interferon is necessary to prevent proteolysis by the gut. Several studies have used very high doses (e.g., 10,000-1 million units/kg) parenterally. However, other studies suggest that low, oral doses of interferon have immunomodulatory effects in cats. Therapy is usually reserved for short-term management, whichever interferon is used. Feline Immunodeficiency Virus/ Feline Leukemia Virus (FIV, FeLV) FIV and FeLV are lentiviruses, and are RNA virus that uses reverse transcriptase. Clinical signs include fever, lymphadenopathy, stomatitis, immunosuppression, leukemia, and lymphoma. Although antiviral therapy has made considerable progress and been quite successful in maintaining people with Human Immunodeficiency Virus, improvements in the therapy of feline retroviral infections has been frustratingly sparse. Dose-limiting side effects, lack of intense efficacy and toxicity monitoring, and scarce investigation of combination therapy may partly explain the lack of pharmacological progress in cats. Zidovudine (AZT) is a thymidine analog that inhibits viral reverse transcriptase. AZT is primarily used to improve stomatitis and the CD4+:CD8+ ratio for FIV positive cats. AZT is also used in to improve stomatitis and has been shown to improve the p27 antigen in FeLV cats. AZT is well absorbed after oral administration to cats and is rapidly eliminated by hepatic and renal mechanisms. One reported dose of AZT in cats is 5 mg/kg given orally every 12 hr.8 Toxicities include Heinz body anemia (non-regenerative) in cats, so serial complete blood counts are used to monitor side effects, especially at the higher end of the dose range.

280 Interferon: Feline omega-interferon (IFN) has been administered to cats with FeLV or FeLV/FIV. Studies have reported lower mortality with IFN therapy (30% vs 59 at 4 months) and improved clinical scores at a dose rate of 1 million U/kg/day given subcutaneously.9 Alpha interferon has also been administered, but in cats ill with FeLV, treatment with oral IFN-ĮIDLOHG to increase survival or clinical signs at a dose of 30 U orally, once per day.10 In contrast, a previous study did find an increase in survival with oral IFN-ĮXVLQJDQ)H/9FKDOOHQJHPRGHO at a dose of 0.5 or 5 U IFN-ĮRUDOO\11 Influenza Virus Influenza is an enveloped RNA virus that has recently been associated with hemorrhagic pneumonia and acute death in dogs, particularly in Greyhounds. Fever, cough, and nasal discharge may also occur with influenza infection. Oseltamivir (Tamiflu™) is a competitive inhibitor of neuraminidase that prevents virus budding, and may have other actions. Tamiflu™ is labeled for the therapy of seasonal influenza in people (only labeled indication) and has also been used to treat zoonotic influenza, such as avian influenza in humans. Oseltamivir phosphate is a prodrug, that is converted to its active form, oseltamivir carboxylate, which is primarily eliminated by the kidney. The use of Tamiflu™ in dogs with influenza is generally not recommended, because there is scant evidence for efficacy. In humans, therapy must begin early (within 48 hours after infection) to be successful. There is little chance that such early therapy could be successfully instituted in dogs. Given these limitations and its considerable importance in human pandemics, most infectious disease experts believe that Tamiflu™ should be reserved for human influenza. References

1. Nasisse MP, Dorman DC, Jamison KC, et al. Effects of valacyclovir in cats infected with feline herpesvivus 1. Am J Vet Res 1997;58:1141-1144. 2. Thomasy SM, Lim CC, Reilly CM, et al. Evaluation of orally administered famciclovir in cats experimentally infected with feline herpesvirus type-1. Am J Vet Res 2011;72:85-95. 3. Malik R, Lessels NS, Webb S, et al. Treatment of feline herpesvirus-1 associated disease in cats with famciclovir and related drugs. J Feline Med Surg 2009;11:40-48. 4. Stiles J. Treatment of cats with ocular disease attributable to herpesvirus infection: 17 cases (1983-1993). J Am Vet Med Assoc 1995;207:599-603. 5. Davidson GSBRFD. Compounding for Feline Herpetic Keratitis. Int J Pharm Compd 2006;10:411-414. 6. Rees TM, Lubinski JL. Oral supplementation with L-lysine did not prevent upper respiratory infection in a shelter population of cats. J Feline Med Surg 2008;10:510-513. 7. Maggs DJ, Nasisse MP, Kass PH. Efficacy of oral supplementation with L-lysine in cats latently infected with feline herpesvirus. Am J Vet Res 2003;64:37-42. 8. Stuetzer B, Brunner K, Lutz H, et al. A trial with 3'-azido-2',3'-dideoxythymidine and human interferon-alpha in cats naturally infected with feline leukaemia virus. J Feline Med Surg 2013;15:667- 671. 9. Domenech A, Miro G, Collado VM, et al. Use of recombinant interferon omega in feline retrovirosis: from theory to practice. Vet Immunol Immunopathol 2011;143:301-306. 10. McCaw DL, Boon GD, Jergens AE, et al. Immunomodulation therapy for feline leukemia virus infection. J Am Anim Hosp Assoc 2001;37:356-363. 11. Cummins JM, Tompkins MB, Olsen RG, et al. Oral use of human alpha interferon in cats. J Biol Response Mod 1988;7:513-523.

281 Therapy of Thromboembolic Diseases Lara K. Maxwell, DVM, PhD, DACVCP Affiliation: Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University [email protected] City, State, Country: Stillwater, OK, USA Thromboembolic agents are indicated for a variety of conditions, ranging from disseminated intravascular coagulation to pulmonary thromboembolism in both dogs and cats. The selection of appropriate thromboembolic therapy will depend on the underlying condition and the goal of treatment. For example, if a thromboembolic obstruction has been diagnosed, then the goal of therapy is generally dissolution of the thrombus. However, a second goal of therapy for a primary thrombus is the prevention of additional thromboembolism due to fragmentation of the original lesion with subsequent blockage of the smaller, down-stream vessels. Similarly, prophylaxis of thromboembolic disease is frequently the primary goal of anticoagulant therapy in patients that are predisposed to thrombus formation, such as cats with hypertrophic cardiomyopathy. Thromboembolic agents can be broadly classified into agents that are thrombolytic and those that are anticoagulant. It is important to recognize that while anticoagulant therapy can indirectly support the lysis of pre-existing thrombi, anticoagulants cannot directly lyse a clot.

Learning Objectives 1) Attendees will be able to distinguish between thrombolytic vs. anticoagulant therapies

2) Attendees will be familiar with the latest treatments for the prevention and management of thromboembolic diseases in cats and dogs

Thrombolytic agents include urokinase, streptokinase, and recombinant tissue plasminogen activators (rt-PA). The present thrombolytic agents share a common mechanism of action, serving to stimulate the endogenous system of thrombus removal. Normally, the initiation of thrombus degradation is by release of t-PA from endothelial cells that respond to signals such as vessel occlusion. The t-PA first binds to fibrin in the clot, than converts free plasminogen to the enzyme plasmin, which in turn will digest fibrin. The action of the thrombolytic agents tends to be nonspecific, such that both pathological fibrin clots and those at the sites of vascular injury are lysed. In addition, plasmin can degrade other plasma proteins, including coagulation cofactors, worsening the possibility of hemorrhage. As a consequence, hemorrhage is the most worrisome side effect associated with the use of thrombolytic agents and can occur at sites of catheter placement and trauma. For this reason, recent surgery, gastrointestinal bleeding, and hemorrhagic disorders are all contraindications for thrombolytic therapy. However, the thrombolytic agents do differ with respect to their specificity for fibrin. Streptokinase is the oldest member of this class of drugs and is produced from beta-hemolytic Streptococcus. Streptokinase facilitates the cleavage of plasminogen to plasmin. Because Streptococcus spp. exposure generally produces antibodies to streptokinase, streptokinase is immunogenic. High doses may

282 be necessary to bind streptokinase antibody and allow high enough circulating concentrations of streptokinase for drug efficacy. In addition to the risk of hemorrhage due to the lack of specificity for fibrin degradation, streptokinase may also be associated with hypersensitivity reactions. Only the low molecular weight form of urokinase is presently available in the U.S., as an injectable drug approved for the treatment of pulmonary emboli in humans. Urokinase is derived from human donor neonatal kidney cells, making it an expensive preparation with major supply issues. Like streptokinase, urokinase is also not selective for fibrin, making it unattractive from the standpoint of both side effects and cost. The use of t-PA has largely supplanted that of streptokinase and urokinase in human medicine. By acting similarly to endogenous t-PA, rt-PA products (such as alteplase and reteplase) provide a greater measure of safety as compared to the streptokinase and urokinase. Because endogenous t-PA only converts plasminogen to plasmin in the presence of fibrin, systemic lysis of proteins does not occur. However, physiological thrombi at the site of vascular injury will still be affected by low concentrations of t- PA. In addition, supra-physiological concentrations of rt-PA occur during drug therapy, such that hemorrhage is still the major side effect associated with the administration of rt-PA. Therapy with rt-PA is also very expensive. Alteplase has been used in companion animals with thromboembolic disease,1,2 although evidence for its safety and efficacy in presently incomplete.3 Similarly, retrospective studies of streptokinase administration in companion animals with arterial thromboembolic disease have not provided strong evidence of increase survival.3,4 Overall, clinical evidence for the efficacy of thrombolytic therapy for the treatment of thromboembolic disease in veterinary species is primarily retrospective and inconclusive, but does not support a robust treatment effect. Anticoagulant, antiplatelet agents include aspirin, ticlopidine, and clopidogrel. These agents interfere the formation of thrombi and as such may be used both to keep thrombi from forming or worsening. Clot formation requires numerous factors and co-factors, presenting numerous pharmacological targets for an anticoagulant effect. Upon exposure of collagen by endothelial cell injury, activation of von Willebrand factor allows binding of collagen to platelet adhesive glycoprotein receptors (e.g., GPIa/IIa, GPIb) which produces several downstream events, such as the formation of thromboxane (TXA2) and adenosine diphosphate (ADP). In addition to other release factors, the localized actions of TXA2 and ADP amplify hemostasis by recruiting and activating additional platelets to the growing thrombus. The eicosanoids TXA2 and prostacyclin (PGI2) are eicosanoid products of COX-1 that are balanced against one another to affect appropriate homeostasis. Platelets generate TXA2 for a pro-coagulant effect, whereas endothelial cells produce PGI2 for an anti-coagulant effect. Although other nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) besides aspirin also inhibit the formation of eicosanoid products, aspirin is distinct in its irreversible inhibition of cyclooxygenase-1 (COX-1). As platelets do not synthesize new proteins, COX-1 is effectively inhibited for the lifespan of the platelet, thus losing its ability to produce TXA2. This inhibition of platelet COX-1 occurs at low aspirin concentrations. In contrast, the endothelial cell COX-1 is not similarly inhibited or is able to be rapidly turned over, such that the vasodilatory and anticoagulant eicosanoid PGI2 is still active. It is the relative difference in TXA2 and PGI2 concentrations associated with low-doses of aspirin that appears to be responsible for aspirin’s anticoagulant effect. This effect may be decreased by high doses of aspirin or by the concomitant administration of additional NSAIDs. Although aspirin has been used with apparent safety in cats with a history of thrombotic episodes, its efficacy at preventing future episodes has been questionable in several retrospective studies.5,6 A second class of antiplatelet drugs act by inhibiting purinergic receptors

283 (P2Y1/P2Y12) that respond to localized ADP release by activated platelets. Inhibition of these purinergic receptors by ticlopidine and clopidogrel prevents the activation and recruitment of additional platelets. Like aspirin, the biological half-lives of ticlopidine and clopidogrel are longer than their pharmacokinetic half-lives, due to irreversible inhibition of the purinergic receptor. Clopidogrel appears to inhibit platelet aggregation more strongly than does aspirin, but the combination of clopidogrel and aspirin produces a synergistic effect.7 This synergy has been utilized to prevent thromboembolic disease in high-risk populations of humans with heart disease, but is also more likely to cause hemorrhage and is detrimental in some disease states.8 Whether such a synergistic effect would be beneficial in veterinary species at high risk of thromboembolic disease, such as cats with hypertrophic cardiomyopathy and thrombi, has not been determined. Although preliminary studies of clopidogrel and ticlopidine efficacy have been encouraging in veterinary species, ticlopidine also appears to be associated with unacceptable gastrointestinal toxicity in cats.9-11 In cats with thromboembolism, either the administration of aspirin or clopidogrel was associated with better odds of survival past the first day of diagnosis.12 A more definitive clinical trial (Fat Cat) comparing the efficacy of clopidogrel to that of aspirin in preventing a second recurrence of thromboembolism in cats indicated that clopidgrel was superior to aspirin, with survival times of >365 days in the clopidogrel group and 192 days in the aspirin group.13 Other anticoagulant agents include unfractionated heparin, low molecular weight heparin, and warfarin. Like the antiplatelet anticoagulants, these agents are used for the therapy of both existing thromembolism and for the prophylaxis of thrombi formation. However, the rationale for using these drugs to treat an existing thrombus is the prevention of further thrombus expansion and the formation of new thrombi downstream from a fragmented thrombus. In this case, limitation of thrombus expansion may allow endogenous thrombolytic mechanisms to dissolve the existing clot. Both unfractionated heparin and low molecular weight heparin are used for this purpose. Unfractionated heparin is derived from porcine tissue and is a mixture of sulfated, anionic, and polysufated glycosaminoglycans. This heterogeneous mixture consists of glycosaminoglycans with differing molecular weights, action, and pharmacokinetic properties. Because there may be batch to batch variation in heparin composition, the response to heparin administration may also be unpredictable. Due to this heterogeneity, only a fraction of the heparin may bind to antithrombin III (ATIII). It is by binding to and activating ATIII that heparin acts as an anticoagulant, as ATIII inactivates thrombin (Factor IIa) and Factor Xa. Intact heparin is poorly absorbed from the gastrointestinal tract, so it is given parenterally by intravenous of subcutaneaous injection. The major adverse effect associated with heparin administration is hemorrhage, although protamine can be given as an antidote to heparin overdose. Both the activated partial thromboplastin time (APTT) and activated clotting time (ACT) test the intrinsic and common pathway, and so can be used to monitor the anticoagulant activity of heparin therapy, although the APTT test is superior.14 However, even with the use of APTT monitoring, the response to unfractionated heparin therapy can be unpredictable, especially at high doses of heparin (e.g., 900 U/kg/day by constant rate infusion).15 In addition to the predictable increase in bleeding and hemorrhage expected from heparin administration, recent recalls due to anaphylaxis from unfractionated heparin preparations are particularly troubling. Hypersensitivity appeared to be associated with oversulphation of the related glycosaminoglycan, chondroitin sulfate, which contaminated the preparations.16 The FDA responded to numerous anaphylactoid adverse events in humans by increasing the

284 inspection of the overseas suppliers of heparin, but this incident has exposed some of the safety issues involved in the remote manufacture and importation of these tissue-derived drugs. An alternative to unfractionated heparin is the use of low molecular weight heparin (LMWH). LMWH is purified from unfractionated heparin to produce a heparin with more uniform molecular size. This uniformity is associated with more predictable pharmacokinetic behavior and activity than unfractionated heparin, although LMWH has also been contaminated with oversulphated chondroitin sulfate. LMWH primarily inactivates factor Xa, such that the APTT test is not accurate for monitoring response to therapy. Factor Xa inhibitory activity (anti-Xa activity) can instead be measured to assess the response to LMWH therapy.17 Although LMWH products are uniform within preparations, these agents differ considerably between different manufacturers and trade names, so direct substitution requires monitoring and dose adjustment. In addition the shorter biological half-life of LMWH as compared to unfractionated heparin necessitates more frequent dosing of LMWH to cats, and the response to two agents, dalteparin and enoxaparin, can be highly variable between cats.17,18 Dogs at risk for thromboembolic disease have been treated with dalteparin with apparent success (29/38 survival to discharge), rare (3/38) bleeding complications, and inconsistent success in reaching the targeted anti-Xa range.19 Warfarin is used as an anticoagulant that is similar to products used as rodenticides because it is capable of producing a profound anticoagulant effect at high enough doses. Warfarin acts by inhibiting the hepatic synthesis of vitamin K-dependent clotting factors (II, VII, IX, X). Vitamin K is necessary for postribosomal carboxylation of precursors to active clotting factors. Warfarin is orally administered and highly protein bound (>90%). It is eliminated by hepatic cytochrome P450 enzymes, and is subject to potentially dangerous drug interactions.20 Hemorrhage is the major adverse effect associated with warfarin administration and its activity can be monitored using prothrombin time (PT) or international normalized ratio (INR).21 The target PT is 1.5-2 times normal and the typical onset of action occurs at 8-12 hours after administration. If an overdose of warfarin is given, vitamin K1 can be used as an antidote. Warfarin administration has been reported to successfully manage aortic thromboembolism in dogs.22

REFERENCES

1. Clare AC, Kraje BJ. Use of recombinant tissue-plasminogen activator for aortic thrombolysis in a hypoproteinemic dog. J Am Vet Med Assoc 1998;212:539-543. 2. Pion PD. Feline aortic thromboemboli and the potential utility of thrombolytic therapy with tissue plasminogen activator. Vet Clin North Am Small Anim Pract 1988;18:79-86. 3. Lunsford KV, Mackin AJ. Thromboembolic therapies in dogs and cats: an evidence-based approach. Vet Clin North Am Small Anim Pract 2007;37:579-609. 4. Moore K, Morris N, Dhupa N, et al. Retrospective study of streptokinase administration in 46 cats with arterial thromboembolism. J Vet Emerg Crit Care 2000;10:245-257. 5. Fox PR. Evidence for or against efficacy of beta-blockers and aspirin for management of feline cardiomyopathies. Vet Clin North Am Small Anim Pract 1991;21:1011-1022. 6. Schoeman JP. Feline distal aortic thromboembolism: a review of 44 cases (1990-1998). J Feline Med Surg 1999;1:221-231.

285 7. Moshfegh K, Redondo M, Julmy F, et al. Antiplatelet effects of clopidogrel compared with aspirin after myocardial infarction: enhanced inhibitory effects of combination therapy. J Am Coll Cardiol 2000;36:699-705. 8. Fisher M, Folland E. Acute ischemic coronary artery disease and ischemic stroke: similarities and differences. Am J Ther 2008;15:137-149. 9. Hogan DF, Andrews DA, Green HW, et al. Antiplatelet effects and pharmacodynamics of clopidogrel in cats. J Am Vet Med Assoc 2004;225:1406-1411. 10. Hogan DF, Ward MP. Effect of clopidogrel on tissue-plasminogen activator-induced in vitro thrombolysis of feline whole blood thrombi. Am J Vet Res 2004;65:715-719. 11. Hogan DF, Andrews DA, Talbott KK, et al. Evaluation of antiplatelet effects of ticlopidine in cats. Am J Vet Res 2004;65:327-332. 12. Borgeat K, Wright J, Garrod O, et al. Arterial thromboembolism in 250 cats in general practice: 2004-2012. J Vet Intern Med 2014;28:102-108. 13. Hogan D FP, Jacob K , B Keene, N Laste, S Rosenthal. Analysis of the Feline Arterial Thromboembolism: Clopidogrel vs. Aspirin Trial (FAT CAT) ACVIM 2013. 14. Green RA. Activated coagulation time in monitoring heparinized dogs. Am J Vet Res 1980;41:1793-1797. 15. Scott KC, Hansen BD, DeFrancesco TC. Coagulation effects of low molecular weight heparin compared with heparin in dogs considered to be at risk for clinically significant venous thrombosis. J Vet Emerg Crit Care 2009;19:74-80. 16. Kishimoto TK, Viswanathan K, Ganguly T, et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med 2008;358:2457-2467. 17. Alwood AJ, Downend AB, Brooks MB, et al. Anticoagulant effects of low-molecular-weight heparins in healthy cats. J Vet Intern Med 2007;21:378-387. 18. Vargo CL, Taylor SM, Carr A, et al. The effect of a low molecular weight heparin on coagulation parameters in healthy cats. Can J Vet Res 2009;73:132-136. 19. Lynch AM, deLaforcade AM, Sharp CR. Clinical experience of anti-Xa monitoring in critically ill dogs receiving dalteparin. J Vet Emerg Crit Care (San Antonio) 2014;24:421- 428. 20. Trepanier LA. Cytochrome P450 and its role in veterinary drug interactions. Veterinary Clinics of North America-Small Animal Practice 2006;36:975-+. 21. Monnet E, Morgan MR. Effect of three loading doses of warfarin on the international normalized ratio for dogs. Am J Vet Res 2000;61:48-50. 22. Winter RL, Sedacca CD, Adams A, et al. Aortic thrombosis in dogs: presentation, therapy, and outcome in 26 cases. J Vet Cardiol 2012;14:333-342.

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