Immunomodulating agents

Transcription

1 Vet Clin Small Anim 34 (2004) Immunomodulating agents Cecil P. Moore, DVM, MS Department of Veterinary Medicine and Surgery, A383 Clydesdale Hall, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA Inflammation involves a universally recognized, although incompletely understood, cascade of molecular events orchestrated by lymphokines and other innate biochemicals of immunity. Repeated or extended contact with immunogenic agents results in adaptive immunity involving antigen-induced events that stimulate downstream immune cells and result in expansion of the inflammatory cascade. For most finite insults resulting in inflammatory events, affected tissues typically heal spontaneously with resumption of normal function within a relatively defined period (ie, days or weeks). When immunogenic stimulation persists or autoregulatory immune mechanisms go awry, however, adaptive immunologic events can result in immunemediated processes detrimental to systemic or organ-specific homeostasis. Considering the eye in this context, sustained or recurring inflammatory phenomena may target a number of periocular and intraocular tissues, causing progressive eye disease. Because of the complexities of immunologic events, the potential side effects of long-term corticosteroid therapy, and the focused spectrum of most conventional nonsteroidal anti-inflammatory agents (centered on arachidonic acid related mechanisms), a variety of other chemotherapeutic immunosuppressive agents have assumed an increasingly prominent therapeutic role in veterinary ophthalmology in the management of chronic ocular inflammatory diseases. In addition, nonimmunosuppressive immunomodulating agents (ie, immunostimulants or immunorestoratives) may be used as adjunctive therapies in the management of ocular or visual system diseases. Immunosuppressive agents Immunosuppressive agents are used to manage a number of immunemediated ocular diseases, including keratoconjunctivitis sicca (KCS), address: moorec@missouri.edu /04/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi: /j.cvsm

2 726 C.P. Moore / Vet Clin Small Anim 34 (2004) chronic superficial keratitis (pannus), superficial punctuate keratitis, proliferative/eosinophilic keratitis, scleritis-episcleritis, immune-mediated uveitis, and granulomatous meningoencephalomyelitis (GME), affecting the visual system. Immunosuppressive agents applicable to the management of ocular or visual system disease are considered in this article, including modes of actions, aspects of pharmacodynamics, and ocular indications. T-cell inhibitors Background/derivation The T-cell activation inhibitors cyclosporine (cyclosporin A [CsA]) and tacrolimus were developed for their utility as systemically administered agents in preventing graft rejection in hosts after organ transplantation. Although structurally nonhomologous, the mechanism of action of these agents is similar. In target cells, each affects critical molecules that orchestrate genetically programmed events. Specifically, T-cell proliferation and activation are altered by the inhibition of interleukin (IL)-2 gene expression in CD4+ T helper lymphocytes. A variety of secondary effects lead to profound and potentially therapeutically useful influences on the activity of other cell types, most notably cells of epithelial origin [1 3]. Initially identified from culture of the fungus Tolypocladium inflatum for its potential antibiotic activity, CsA was shown to be a potent inhibitor of T- cell activation induced by mitogens or mixed lymphocyte reactions [4]. CsA was also found to be uniquely nontoxic to cells at concentrations that essentially completely blocked T-cell activation [5]. Tacrolimus, historically referred to as FK506, is a macrolide antibiotic produced by Streptomyces tsukubaensis. Rapamycin (sirolimus), a relatively new agent, is functionally similar to CsA and tacrolimus and holds promise as an additional potentially useful immunosuppressant. Molecular and cellular mechanisms These agents are highly lipophilic and readily pass through cell membranes to act on the intracellular second messenger system of activated T cells (ATs), where they exert their effects when complexed to endogenous intracellular receptors or binding proteins termed immunophilins. Intracellular complexes formed between CsA or tacrolimus and its respective receptive proteins interact with additional substrates that modulate the function of molecular targets, subsequently resulting in immunosuppressive activity (Fig. 1). CsA affects T-cell activation within several hours of lymphocyte stimulation. If administered within an hour of activation, CsA blocks T-cell proliferation, but if given more than 6 hours after the stimulus, no effect on T-cell proliferation occurs. c Fig. 1. Mechanism of action of cyclosporin A and tacrolimus inside a T cell. (Courtesy of M. Ford, PhD, DVM, Columbia, MO.)

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4 728 C.P. Moore / Vet Clin Small Anim 34 (2004) Given blockage of activation of early T-cell genes by these agents, studies have focused on nuclear transcriptional mechanisms and protein binding. Because blocking IL-2 transcription leads to impaired proliferation of activated T helper and T-cytotoxic lymphocytes, initial investigations have targeted the IL-2 enhancer region of ATs and the inhibition of proteins that activate transcription of the IL-2 gene. A more detailed summary of intracellular mechanisms of action is provided here. The process of T-cell activation involves antigen binding to CD3 receptors on the surface of the lymphocyte, which causes an increase in intracellular calcium and stimulates activation of phosphatase activity by calcineurin (CaN), also known as phosphatase 2B. CsA binds intracellularly to a specific immunophilin, cyclophilin (CpN), whereas tacrolimus binds to immunophilins characterized as FK506-binding proteins (FKBPs). CaN is the principal intracellular target for these drugs and their respective cognate immunophilin complexes (see Fig. 1) [6]. CaN is a key rate-limiting enzyme in T-cell signal transduction acting on the nuclear transcription factors (NFs) of ATs that trigger proinflammatory cytokines, initiating an early immune response. Two NFs are operative in ATs: one cytoplasmic (NF-ATc) and one nuclear (NF-ATn). The enzymatic activity of CaN is modulated by calcium and calmodulin. CaN dephosphorylates NF-ATc in response to Ca-dependent cell activation and translocates to the nucleus, where it combines with NF- ATn, a factor essential for the transcription of the IL-2 gene. This synthesis is dependent on activation by protein kinase C (PKC). The NF-ATn complex then associates with the IL-2 promoter/enhancer, as do other transcription factors, and binds to response elements in the DNA region, regulating IL-2 gene transcription, which extends approximately 300 base pairs upstream. CaN may also activate ubiquitous transcription factors, such as cyclic adenosine monophosphate (camp) responsive element binding protein (CREB), which controls transcription of a wide variety of genes through the camp-responsive element (CRE). Disruption of CRE-mediated gene transcription represents a novel mechanism of action that may underlie pharmacologic effects and toxicities of these agents. Other CaN-regulated intracellular enzymatic events include phosphatase 1, camp-dependent protein kinase, and nitric oxide synthase activation, which affect other cellular events, such as degranulation and apoptosis in leukocytes. In summary, after CsA passes through the cell membrane, it binds to CpN (CsA-CpN); in turn, this complex binds to CaN (CsA-CpN-CaN), blocking CaN s usual role of dephosphorylation of cytoplasmic NF-AT for induction of nuclear translocation of NF-AT and, consequently, reduction of production (see Fig. 1). Besides reducing IL-2 release from lymphocytes, CsA interferes with IL-2 receptors on the surface of lymphocytes. CsA and tacrolimus have been reported to affect eosinophils, reducing production, and mast cells, blocking degranulation and transcriptional activation of

5 C.P. Moore / Vet Clin Small Anim 34 (2004) mast cell cytokine genes (IL-3 and IL-5), and tumor necrosis factor-a (TNFa) production by B cells is also suppressed [5 8]. Cyclosporine Pharmacology and ocular penetration CsA is a highly lipophilic large molecular agent with an affinity for binding to red blood cells and plasma lipoproteins. When administered systemically, CsA distributes widely, accumulating in adipose tissues and liver, but does not readily penetrate the blood-brain barrier. In animals, peak concentration times of CsA may be variable but generally occur between 2 and 4 hours after oral administration. Because of its lipophilicity, systemic absorption of CsA may be enhanced by administering it with a fatty meal. Blood levels may increase over time because of saturation of tissuebinding sites. The therapeutic level of CsA believed to be needed to achieve immunosuppression is 100 to 400 ng/ml [9,10]. CsA is usually administered topically rather than systemically for the treatment of ocular conditions. Although the half-life of topically administered CsA to the eye is unknown, when topically applied, CsA achieves high levels of drug in corneal, conjunctiva, sclera, and lacrimal tissues but not in aqueous humor, uvea, or vitreous humor. After oral or topical administration of CsA, intraocular absorption by the intact eye is negligible. In a rabbit model with significant breakdown of the blood-ocular barrier, oral administration of CsA resulted in detectable levels in most ocular tissues, with highest concentrations in the choroid and retina [11]. Two reports in which a relatively high topical dose (ie, 2% CsA four times daily) was applied to eyes of small animals (rabbits and cats) indicated sufficient absorption to achieve therapeutic blood levels to treat uveitis [12,13]. Ocular indications Most cases of canine KCS (dry eye) are believed to be immune mediated; therefore, CsA has been used extensively to treat this condition. Initial studies revealed 2% cyclosporine in corn oil to be effective [14] and indicated that if initial Shirmer tear test (STT) values were 2 mm/min or greater, an increase in STT of 5 mm/min was predictable. For cases with an initial STT value of 0 to 2 mm/min, STT values increased to 5 mm/min in 59% of eyes after topical treatment. Even in cases where lacrimation does not improve, a significant regression of chronic corneal neovascularization and granulation tissue has been noted in severe cases of KCS [15]. After the initiation of topical CsA treatment in cases of canine KCS, a delay in therapeutic response was noted in some cases up to approximately 2 months [14]. Clinical response was associated with histologic reduction of glandular inflammation and lacrimal acinar lobule regeneration [16]. Subsequent studies demonstrated the efficacy of topical 1% CsA in oil

6 730 C.P. Moore / Vet Clin Small Anim 34 (2004) and 0.2% CsA in a patented ointment vehicle [17,18]. Clinical response is believed to be the result of a local rather than systemic effect because of subtherapeutic blood levels in dogs administered topical CsA, although decreased CD4 and CD8 lymphocyte proliferation rates have been seen in some dogs after topical administration [19]. Topical 0.2% CsA ointment administered twice daily decreased corneal neovascularization and cellular infiltration in chronic superficial keratitis (pannus) and was determined to be as effective in managing this disease as topical 0.1% dexamethasone [20,21]. A variety of suspected superficial immune-mediated ocular conditions (ie, plasmacytic conjunctivitis of the nictitans and eosinophilic keratoconjunctivitis of cats and horses) have been treated with topical CsA, with variable response [9,22 24]. Other veterinary ophthalmic uses include adjunctive therapy for superficial keratopathies (including punctate keratitis), keratouveitis, endotheliitis, and nodular granulomatous episclerokeratitis. A slow-release implant for transscleral delivery of CsA to treat chronic uveitis in horses has recently been investigated with promising results [25]. Topical administration of a commercial ophthalmic CsA product (0.2% ointment) or compounded solutions (1% or 2%) is safe and free of side effects, such as predisposition to surface infections or adverse ocular or systemic immunosuppressant effects [26]. Tacrolimus Used in human patients as an effective alternative to CsA in the prevention of transplant rejection, tacrolimus is reportedly 10 to 100 times more potent in vitro than CsA [27]. Based on studies in experimental transplantations, when administered systemically, tacrolimus may have more adverse systemic effects than CsA, and those side effects may be severe enough to limit the usefulness of tacrolimus in preventing organ transplant rejection [8]. Tacrolimus 0.03% ointment is approved for treatment of moderate to severe atopic dermatitis in human patients. Some affected patients have a concurrent atopic blepharitis with eyelid eczema, atopic keratoconjunctivitis, conjunctival papillary hypertrophy, superficial punctate keratitis, persistent epithelial defects, neovascularization, and scarring. In a small case series of patients with bilateral severe atopic eyelid disease, topical 0.03% ointment applied to the eyelid skin twice daily resulted in marked improvement in 1 to 3 weeks [28]. Because of concerns about systemic side effects, topical application for ophthalmic use is preferred to oral or intravenous routes. Considering the lipophilicity and molecular size, tacrolimus has been incorporated into liposomes to enhance absorption when applied topically. The liposomebound drug achieved higher intraocular levels than the oil-dissolved form [29].

7 C.P. Moore / Vet Clin Small Anim 34 (2004) Tacrolimus has been used experimentally for the topical treatment of canine KCS and seems promising. Twice-daily administration of 0.02% tacrolimus in aqueous suspension effectively increased tear production in dogs with KCS [30]. In this short-term study, tacrolimus was effective as a substitute for CsA, and dogs that fail to respond to CsA may respond positively to topical 0.02% tacrolimus in aqueous suspension [30]. An olive oil compounded 0.003% tacrolimus has been demonstrated to be safe in normal dogs; when it was used to treat KCS-affected dogs, no difference in tear values or clinical signs was noted compared with 2% CsA-treated KCS eyes [31]. Cytotoxic agents In the management of serious immune-mediated ocular diseases, use of potent cytotoxic agents may be considered in cases refractory to empiric anti-inflammatory therapy or to reduce side effects associated with persistent high-dose corticosteroid therapy. Azathioprine Azathioprine (AZA), a purine analogue originally developed as a prodrug of the cytotoxic agent 6-mercaptopurine, is used systemically as an immunosuppressive agent for various human immune-mediated ocular disorders [32]. In veterinary ophthalmology, AZA has been used most commonly in combination with corticosteroid agents to treat intractable scleritis or uveitis cases nonresponsive to conventional anti-inflammatory therapy [33,34]. Uveitis manifesting with canine uveal-dermatologic syndrome (VKH-like syndrome) may be a particularly challenging form of panuveitis that is often difficult to manage with corticosteroid therapy alone but can usually be effectively managed with a combination of AZA and topical and systemic corticosteroids. An additional indication for the use of AZA in ophthalmic disease is for treating nodular granulomatous episclerokeratitis, the epibulbar proliferative disorder occurring most commonly in Collie dogs [35]. The recommended initial dosage of AZA for dogs is 2 mg/kg/d for 3 to 5 days with reduction to 1 mg/kg/d for 10 days and then, if needed, 0.5 mg/kg/ d as a maintenance dose [36]. Dose and frequency of AZA for ocular disease are tailored to each patient based on clinical response and tolerance to therapy. AZA has also been used in the cat at a dose of 1.1 mg/kg every other day. Posttreatment hematologic evaluations should be performed on patients receiving systemic AZA. Because of potential myelosuppressive and hepatotoxic effects, a complete blood cell count (including platelet count) and liver function tests are recommended 10 to 14 days after the initial course of treatment and subsequently every 2 to 3 months.

8 732 C.P. Moore / Vet Clin Small Anim 34 (2004) Cytosine arabinoside Cytosine arabinoside (cytarabine, Ara-C) is an antimetabolite that acts on mitotically active cells by becoming incorporated into DNA molecules, causing premature chain termination via inhibition of DNA polymerase. This immunosuppressive agent has the ability to cross the blood-brain barrier and thus can be used to treat central nervous system (CNS) diseases, including lymphomas and GME [37,38]. Ara-C is relevant to a discussion of therapeutics relating to the visual system because of the frequent association of optic neuritis with GME in dogs [39]. Ara-C may be injected subcutaneously at a dosage of 50 mg/m 2 twice daily for 2 consecutive days with repeated dosing every 3 weeks. As with AZA, myelosuppression may occur, and complete blood cell and platelet counts should be performed in 10 to 14 days after initiation of therapy and repeated at 2- to 3-month intervals. For best results, Ara-C is usually combined with prednisone (1 mg/kg administered orally twice daily), with reduction of the prednisone dose after the second round of cytosine injections. Ara-C has been used occasionally as the sole agent for treating dogs with GME [38]. High-dose administration of Ara-C may be associated with cytotoxic concentrations within the CNS. Chlorambucil A nitrogen mustard derivative, chlorambucil is an alkylating agent used originally in veterinary medicine to treat chronic lymphocytic leukemia [40], with subsequent use to treat autoimmune skin diseases. Compared with other alkylating agents, it is slow-acting and relatively nontoxic. Use of chlorambucil to treat ophthalmic disease has primarily been for control of canine pemphigus foliaceous associated periocular dermatitis and blepharitis. It is also an alternative agent for the treatment of refractory cases of immune-mediated uveitis in dogs, when, for example, it may be used in combination with systemic corticosteroids if AZA is not well tolerated. Chlorambucil is readily absorbed by passive diffusion and should not be given with food. Regimens vary with daily, alternate-week, or 1 week in every 4 weeks therapy [37]. Chlorambucil is available as 2-mg tablets and may be given orally at a dosage of 0.1 to 0.2 mg/kg daily for 4 to 7 days and then 0.1 mg/kg daily until remission occurs. Alternatively, it may be administered once every other week at a dose of 0.4 mg/kg with, after remission, a maintenance protocol of 0.1 mg/kg/d for 7 consecutive days for 1 week, followed by a 3-week rest period. Bone marrow suppression may occur but is reversible if detected early. Hematologic parameters should thus be monitored beginning 1 month after initiation of therapy. Procarbazine (Matulane) Procarbazine is a monamine oxidase inhibitor that alkylates DNA and affects RNA and protein synthesis. Because it crosses the blood-brain

9 C.P. Moore / Vet Clin Small Anim 34 (2004) barrier, it is promising as an alternative treatment of GME [41]. Procarbazine may be compounded into a liquid (ie, oil-based solution at a dose of 10 mg/ml, active for up to 30 days) and administered orally at a dose of 25 to 50 mg/m 2 /d. Compounding should be done by a pharmacist under controlled conditions, because the powder form is toxic. Gloves should be worn when handling procarbazine. Procarbazine may cause neurotoxicity and myelosuppression, with thrombocytopenia and leukopenia representing significant limiting factors. A complete blood cell count should be checked once a week for the first month and monthly thereafter. After the first month of treatment, the frequency of the dose is reduced to every other day. Antibiotic antiproliferative agents Mitoxantrone Because immune-mediated optic neuritis occurs with multiple sclerosis (MS) in human beings and idiopathic presumed immune-mediated forms of optic neuritis occur in companion animals, particularly in dogs, agents used to treat human MS are of interest to veterinarians. Mitoxantrone, an antitumor antibiotic related to doxorubicin, has recently been shown to be beneficial when used to treat human patients with MS [42]. In veterinary medicine, mitoxantrone has been used to treat resistant lymphomas [37] and may be considered in the therapy of progressive idiopathic canine optic neuritis resistant to conventional anti-inflammatory therapy. Mitoxantrone seems to be less cardiotoxic than doxorubicin. As an interesting ophthalmologic side note, blue-green coloration of the sclera may be observed in animals treated with mitoxantrone. Tetracycline and niacinamide The combination of tetracycline and niacinamide has been used successfully in dogs to treat sterile pyogranulomatous disease, including periocular granulomas resistant to systemic corticosteroid therapy [43]. Tetracycline (500 mg administered orally every 8 hours) and niacinamide (500 mg administered orally every 8 hours) are administered until remission is noted, when frequency of treatment is tapered to every 12 hours and then to every 24 hours over several weeks. The therapeutic aim is eventually to discontinue this combination treatment. Although the specific mechanism of action is not understood, the combination of these agents is believed to have immunomodulatory and anti-inflammatory effects. Miscellaneous biologic response modifiers Biologic response modifiers include natural or synthetic preparations administered to increase host responses to pathogens or proliferative lesions. Biologic response modifiers alter physiologic responses by influencing

10 734 C.P. Moore / Vet Clin Small Anim 34 (2004) regulatory pathways. These agents include cytokines that may be derived naturally or can be molecularly cloned. A number of recombinant cytokines are commercially available in pharmacologic quantities and may be prescribed (extralabel use) for animal patients. Interferons Interferons (IFNs) are cytokines particularly relevant to veterinary ophthalmology because of their antiviral immunologic activity. Viral infection stimulates secretion of IFNs into extracellular spaces by affected cells. IFNs bind to specific receptors on neighboring cells, inducing antiviral proteins and protecting those cells from spread of infection. Topical IFN therapy has been shown to be effective in preventing herpes simplex virus infection; although with established infections, IFN treatment alone has shown little efficacy in most studies. Cross-species antiviral efficacy seems operative with human recombinant IFNs. The combination of IFNa and antiviral therapy has been demonstrated to be synergistic against feline herpes virus-1 (FHV-1) in vitro [44]. In experimental FHV-1, the effects of systemic IFNa administered subcutaneously twice daily on 2 consecutive days were studied, whereby animals were inoculated after the first two doses were administered. Although disease was not prevented, cumulative clinical scores were lower for cats treated with IFNa [45]. Results of giving oral IFN 24 and 48 hours after virus challenge suggest that orally administered IFNa is effective in limiting the severity of FHV-1 infection even if given after exposure to the virus. Further studies on experimental FHV-1 are warranted to determine optimal IFNa dosage. Besides IFNa, other commercially available recombinant IFNs are IFNb (1a and 1b), IFNc, and IFNx. In human medicine, IFNb has been approved for the treatment of MS and IFNc has been approved for the treatment of chronic granulomatous disease. Possible clinical applications of other human recombinant IFNs (eg, IFNb, IFNc, IFNx) in veterinary ophthalmology remain to be investigated. For additional discussion of the antiviral effects of IFNs in feline patients, the reader is referred to the article on antiviral agents in this issue. A recent report indicates that oral administration of IFNa at a dosage of 80 IU/d for 6 months resulted in remission of refractory idiopathic ocular granulomatous disease in four Collie dogs [46]. The author suggests IFNa as an alternative therapy to other medical (corticosteroids or AZA) or surgical (excision or cryosurgery) approaches to treating refractory ocular Collie granulomas. Human immunomodulating agents Immunomodulatory disease-modifying agents used in human medicine for remitting-relapsing immune-mediated diseases may merit consideration and investigation in small companion animals. In particular, two agents

11 C.P. Moore / Vet Clin Small Anim 34 (2004) used as anti-ms agents in human patients may offer an alternative or adjunctive approach to the treatment of canine neurologic conditions, including remitting-relapsing optic neuritis associated with GME. Glatiramer acetate (formerly known as copolymer-1) is a synthetic protein resembling a natural myelin protein that has been administered with IFNb as a means of effectively managing MS in human patients [47]. Although its precise mechanism is not understood, patients with MS have been treated with glatiramer acetate using the rationale that it alters autoimmune mechanisms responsible for attacking innate myelin. Glatiramer acetate administered subcutaneously once daily has reduced relapses of MS in human patients by 30%; however, it has not been shown to significantly delay or reduce disability. Natalizumab is a recombinant monoclonal antibody against a-4 integrins also administered for treatment of MS [48]. Affected patients receiving monthly injections of natalizumab had significantly fewer new inflammatory CNS lesions and relapses than placebo-treated patients [48]. These agents are reportedly more effective than corticosteroids in preventing progressive disability from MS in human patients. References [1] Furue M, Gaspari AA, Kaz SI. The effect of cyclosporine A on epidermal cells II. Cyclosporine A inhibits proliferation of normal and transformed keratinocytes. J Invest Dermatol 1988;90: [2] Phillips TE, McHugh J, Moore CP. Cyclosporine has a direct effect on the differentiation of a mucin-secreting cell line. J Cell Physiol 2000;184: [3] Moore CP, McHugh J, Thorne JG, Phillips TE. Effect of cyclosporine on conjunctival mucin in a canine keratoconjunctivitis sicca model. Invest Ophthalmol Vis Sci 2001;42: [4] Schreiber SL, Crabtree CR. The mechanism of action of cyclosporin A and FK506. Immunol Today 1992;13: [5] Borel JF, Feurer C, Magnee C, Stahelin H. Effects of the new anti-lymphocytic peptide cyclosporin A in animals. Immunology 1977;32: [6] Thomson AW. The effects of cyclosporin A on non-t cell components of the immune system. J Autoimmun 1992;5: [7] Kohyama T, Takizawa H, Kawasaki S, Akiyama N, Sato M, Ito K, et al. A potent immunosuppressant FK506 inhibits IL-8 expression in human eosinophils. Mol Cell Biol 1999;1:72 7. [8] Vaden SL. Cyclosporine and tacrolimus. Semin Vet Med Surg 1997;12(3): [9] Gilger BC, Allen JB. Cyclosporine A in veterinary ophthalmology. Vet Ophthalmol 1998; 1: [10] Grevel J, Kahan BD. Pharmacokinetics of cyclosporin A. In: Thompson AW, editor. Cyclosporin: mode of action and clinical application. Dordrecht: Klumer Academic Publishers; p [11] Ben Ezra D, Maftzir B. Ocular penetration of cyclosporin A, the rabbit eye. Invest Ophthalmol Vis Sci 1990;31: [12] Foets B, Missotten L, Vanderveeren P, et al. Prolonged survival of allogenic corneal grafts in rabbits treated with topically applied cyclosporin A. Systemic absorption and local immunosuppressive effect. Br J Ophthalmol 1985;69:600 3.

12 736 C.P. Moore / Vet Clin Small Anim 34 (2004) [13] Gregory CR, Hietala SK, Pedersen NE, et al. Cyclosporine pharmacokinetics in cats following topical ocular administration. Transplantation 1989;47: [14] Kaswan RL, Salisbury MA, Ward D. Spontaneous canine keratoconjunctivitis sicca. A useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops. Arch Ophthalmol 1989;107: [15] Salisbury MA, Kaswan RL, Ward DA, Martin CL, Ramsey JM, Fischer CA. Topical application of cyclosporine in the management of keratoconjunctivitis sicca in dogs. J Am Anim Hosp Assoc 1990;26: [16] Bounous DI, Carmichael KP, Kaswan RL, Hirsh S, Stiles J. Effects of ophthalmic cyclosporine on lacrimal gland pathology and function in dogs with keratoconjunctivitis sicca. Vet Comp Ophthalmol 1995;5(1):5 12. [17] Olivero DK, Davidson MG, English RV, et al. Clinical evaluation of 1% cyclosporine for topical treatment of keratoconjunctivitis sicca in dogs. J Am Vet Med Assoc 1991;199: [18] Sansom J, Barnett KC, Newmann W, et al. Treatment of keratoconjunctivitis sicca in dogs with cyclosporine ophthalmic ointment: a European clinical field trial. Vet Rec 1995;137: [19] Gilger BC, Andrews J, Wilkie DA, Wyman M, Lairmore DM. Cellular immunity in dogs with keratoconjunctivitis sicca before and after treatment with topical 2% cyclosporine. Vet Immunol Immunopathol 1997;49: [20] Jackson P, Kaswan R, Merideth RE, Barrett PM. Chronic superficial keratitis in dogs: a placebo controlled trial of topical cyclosporine treatment. Prog Vet Comp Ophthalmol 1991;1: [21] Williams DL, Hoey AJ, Smitherman P. Comparison of topical cyclosporin and dexamethasone for the treatment of chronic superficial keratitis in dogs. Vet Rec 1995; 137: [22] Read RA. Treatment of canine nictitans plasmacytic conjunctivitis with 0.2% cyclosporin ointment. J Small Anim Pract 1995;36:50 6. [23] Read RA, Barnett KC, Sansom J. Cyclosporine-responsive keratoconjunctivitis in the cat and horse. Vet Rec 1995;137: [24] Allgoewer I, Schaffer EH, Stockhaus C, Vogtlin A. Feline eosinophilic conjunctivitis. Vet Ophthalmol 2001;4: [25] Roberge K, Papich M, Krysburg L, Ehelhauser HF, Robinson M, Gilger BC. In vitro studies of trans-scleral delivery of cyclosporine to the equine eye: ocular parameters, drug diffusion, and analysis of a new delivery system [abstract 19]. Presented at the 32nd Annual Meeting of the American College of Veterinary Ophthalmologists, Sarasota, October 10 13, Vet Ophthalmol 2001;4(4):292. [26] Salisbury MR, Kaswan RL, Brown J. Microorganisms isolated from the corneal surface before and during topical cyclosporine treatment in dogs with keratoconjunctivitis sicca. Am J Vet Res 1995;56: [27] Morris R. Modes of action of FK506, cyclosporin A, and rapamycin. Transplant Proc 1994;26: [28] Rikkers SM, Holland GN, Drayton GE, Michel FK, Torres MF, Takahasi S. Topical tacrolimus treatment of atopic eyelid disease. Am J Ophthalmol 2003;135: [29] Pleyer U, Lutz S, Jusko WJ, Nguyen K, Narawane M, Ruckert D, et al. Ocular absorption of topically applied FK506 from liposomal and oil formulations in the rabbit eye. Invest Ophthalmol Vis Sci 1993;34: [30] Berdoulay A, English RV, Nadelstein B, Weigt A. The effect of topical 0.02% tacrolimus aqueous suspension on tear production in dogs with keratoconjunctivitis sicca. Presented at the 34th Annual Conference of the American College of Veterinary Ophthalmologists, Coeur D Alene, ID, October 22 25, 2003, p. 33. [31] Adkins EA, Hendrix DVH, Stuffle JL, Ward DA, Skorobohadv BJ. An investigation of the safety and efficacy of topical ophthalmic application of tacrolimus in dogs [abstract 42].

13 C.P. Moore / Vet Clin Small Anim 34 (2004) Presented at the 34th Annual Meeting of the American College of Veterinary Ophthalmologists, Coeur D Alene, ID, October 22 25, Vet Ophthalmol 2003; 6(4):358. [32] Hemady R, Tauber J, Foster CJ. Immunosuppressive drugs in immune and inflammatory ocular disease. Surv Ophthalmol 1991;35: [33] Wilkie DA. Control of ocular inflammation. Vet Clin North Am Small Anim Pract 1990; 20: [34] Schadler HJ. Azathioprine in treatment for ocular nodular episcleritis. Vet Med 1985;80: [35] Paulsen ME, Lavach JD, Snyder SP, Severin G, Eichenbaum JD. Nodular granulomatous episclerokeratitis in dogs;19 cases ( ). J Am Vet Med Assoc 1987;190(12): [36] Moore CP. Ophthalmic pharmacology. In: Adams HR, editor. Veterinary pharmacology and therapeutics. 8th edition. Ames: Iowa State University Press; p [37] Barton CL. Chemotherapy. In: Boothe DM, editor. Small animal clinical pharmacology and therapeutics. Philadelphia: WB Saunders; p [38] Nuhsbaum MT, Powell CC, Gionfriddo JR, Cuddon PA. Treatment of granulomatous meningoencephalomyelitis in a dog. Vet Ophthalmol 2002;5(1): [39] Knollinger AM, Moore PA, Dietrich U, Dey T, Vidyashankar A. Canine optic neuritis: 25 cases ( ) [abstract 37]. Presented at the 33rd Annual Meeting of the American College of Veterinary Ophthalmologists, Denver, October 9 13, Vet Ophthalmol 2002;5(4): [40] MacEwen EG, Hurvitz A, Hayes A. Hyperviscosity syndrome associated with lymphocytic leukemia in three dogs. J Am Vet Med Assoc 1997;170: [41] Cuddon PA, Coates JR, Murray M. New treatments for granulomatous meningoencephalomyelitis. Presented at the 20th American College of Veterinary Internal Medicine Forum, Dallas, May 29 June 1, p [42] Polman CH, Uitdehaag BM. New and imaging treatment options for multiple sclerosis. Lancet Neurol 2003;2(9): [43] Rothstein E, Scott D, Riis RC. Tetracycline and niacinamide for the treatment of sterile pyogranuloma/granuloma syndrome in a dog. J Am Anim Hosp Assoc 1997;33: [44] Weiss RC. Synergistic antiviral activities of acyclovir and recombinant human leukocyte (alpha) interferon on feline herpesvirus replication. Am J Vet Res 1989;50: [45] Nasisse MP, Halenda RM, Luo H. Efficacy of low dose oral natural human interferon alpha in acute feline herpesvirus-1 (FHV-1) infection. A preliminary dose determination trial. Proc Am Coll Vet Ophthalmol 1996;27:79. [46] Riis RC. Interferon treatment of idiopathic ocular granulomatous disease of collies [abstract 001]. Presented at the 31st Annual Meeting of the American College of Veterinary Ophthalmologists, Montreal, October 11 15, Vet Ophthalmol 2000;3(4):247. [47] Rudick RA. Disease-modifying drugs for relapsing-remitting multiple sclerosis and future directions for multiple sclerosis therapeutics. Arch Neurol 1999;56: [48] von Andrian UH, Engelhardt B. Integrins as the therapeutic targets in autoimmune disease. N Engl J Med 2003;348:68 72.