Tutorial Article Use of Antiviral Medications Against Equine Herpes Virus Associated Disorders D

244 EQUINE VETERINARY EDUCATION / AE / may 2010

Tutorial Article Use of antiviral medications against equine herpes virus associated disorders D. M. Wong, L. K. Maxwell*† and P. A. Wilkins‡ Department of Veterinary Clinical Sciences, Lloyd Veterinary Medical Center, Iowa State University, Ames, Iowa, 50011; †Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74078; and ‡Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Champaign-Urbana, Illinois 61802, USA.

Keywords: horse; acyclovir; valacyclovir; myeloencephalopathy; pulmonary; foal

Summary been administered to potentially expedite recovery and improve prognosis (Murray et al. 1998; Henninger Numerous types of equine herpesviruses (EHV) continue to et al. 2007; Wong et al. 2008; Lunn et al. 2009). The 2 afflict horses resulting in a variety of clinical manifestations. primary antiviral medications investigated clinically in While many of the clinical manifestations of EHV are equids are acyclic guanine nucleoside analogues, self-limiting or require only supportive care, some clinical namely acyclovir and valacyclovir. These synthetic expressions of EHV infections cause severe risk to the analogues are structurally similar to DNA and RNA horse’s overall health and can result in abortion, long-term nucleosides and are incorporated into viral DNA, thereby deficits or death. Antiviral medications are infrequently terminating the growing viral DNA chain. Recent utilised therapeutics in equine medicine and their exact investigations have described the pharmacokinetic efficacy is largely unknown. However, the use of antiviral characteristics of these drugs, but proven clinical benefit medications may potentially decrease convalescent time is, as of yet, lacking (Wilkins et al. 2005; Bentz et al. 2006; and improve outcome in horses with EHV-related diseases. Garre et al. 2007a, 2009; Maxwell et al. 2008). Despite the Thus, equine practitioners should consider the potential use lack of clear clinical benefit, the use of antiviral of antiviral medications in the future. The purpose of this medications in veterinary medicine remains appealing article is to familiarise the equine practitioner with current and is a subject of increasing interest (Garre et al. 2009). information in regard to antiviral medications and their Thus, the administration of acyclovir or valacyclovir may become more common as veterinarians attempt to potential uses in equine medicine.eve_48 244..252 provide the standard-of-care to specific EHV-associated Introduction diseases.

Significant disease entities have been associated with Mechanism of antiviral activity multiple types of equine herpesviruses (EHV) resulting in considerable morbidity and mortality in horses. Although Acyclovir has been primarily used in man for the treatment vaccines against certain types of EHV are available, they of infections caused by herpes simplex virus type 1 and 2 are not fully protective. Furthermore, while some horses and varicella zoster virus (Brigden and Whiteman 1983; infected with EHV-1 have self-limiting respiratory disease, Ormrod and Goa 2000). The mechanism of action of EHV-1 has also been associated with abortion, respiratory acyclovir (9-[2-(hydroxyethoxy)methyl]-9H-guanine) and distress and death (Del Piero and Wilkins 2001; Lunn et al. of related antiviral compounds including ganciclovir and 2009). In addition, other more severe EHV-related penciclovir, is inhibition of DNA synthesis in herpesviruses syndromes have been documented, including equine (Brigden and Whiteman 1983; Hayden 2001). After herpes myeloencephalopathy (EHM), equine administration, acyclovir is phosphorylated by thymidine multinodular pulmonary fibrosis and equine neonatal kinase to acyclovir monophosphate (Fig 1) (Brigden and respiratory disease, in which antiviral medications have Whiteman 1983; Ormrod and Goa 2000; Hayden 2001). Acyclovir monophosphate preferentially accumulates in herpesvirus-infected host cells, where it is subsequently *Author to whom correspondence should be addressed. converted by cellular guanylate kinase to acyclovir

Fig 1: Mechanism of acyclovir’s antiviral activity. Acyclovir is converted to acyclovir monophosphate by equine herpesvirus (EHV) thymidine kinase. Subsequently, host cell kinases convert acyclovir monophosphate to acyclovir triphosphate. This synthetic nucleoside competes for EHV-DNA polymerase and terminates enzyme activity within EHV. Acyclovir triphosphate is also incorporated into the DNA template preventing elongation of EHV DNA. diphosphate (Brigden and Whiteman 1983; Ormrod and the relative potency of an antiviral drug for different strains Goa 2000). A third and final phosphorylation by host cell of a virus. In vitro testing is also used to determine a enzymes results in the active acyclovir triphosphate preliminary target plasma drug concentration that can be compound that is a substrate for, and ultimately an further validated in vivo. Pharmacodynamic studies of inhibitor of, viral DNA polymerase. Specifically, this antiviral efficacy have been best studied in man, which synthetic nucleoside competes with deoxyguanosine indicate that acyclovir plasma concentrations should triphosphate, the naturally occurring nucleoside, as a remain above the IC50 for at least 12 h of each 24 h period substrate for viral DNA polymerase, and immediately (Tod et al. 2001). This dependence of efficacy on the time terminates enzyme activity (Brigden and Whiteman 1983; above the inhibitory concentration is similar to the Ormrod and Goa 2000). Acyclovir triphosphate is also pharmacokinetic and pharmacodynamic relationships incorporated in the DNA primer template, thus preventing found for some antibacterial drugs, such as penicillin, and is further elongation of the DNA chain (Brigden and also similar to the time-dependent activity reported for Whiteman 1983; Ormrod and Goa 2000). other antiviral drugs (Schmidt et al. 2008). Limited data in horses infected with EHV-1 and treated with valacyclovir Pharmacokinetic/pharmacodynamic suggest that acyclovir also exhibits time-dependent activity predictors of drug efficacy against EHV-1 (L.K. Maxwell, unpublished data).

Similar to the use of minimum inhibitory concentrations Susceptibility of EHV to antiviral medications (MIC) used to assess bacterial sensitivity to an antibiotic, the sensitivity of viruses to antiviral drugs can be measured in Limited information is available with regard to the vitro by determining the drug concentration that inhibits susceptibility of various antiviral medications against the viral replication by 50% (IC50). Unfortunately, IC50 is not as different types and isolates of EHV. Of the information well standardised or as predictive of drug efficacy as is MIC, describing the susceptibility of EHV to various antiviral such that in vitro drug sensitivity may not correlate well with medications, it is apparent that the susceptibility is variable efficacy in the field (Weinberg et al. 2007). Despite these and dependent on the specific viral isolate tested and limitations, in vitro testing is useful to determine the relative the laboratory method used to determine antiviral sensitivity of a virus to several antiviral agents or to examine susceptibility. In one of the earliest veterinary studies

involving antiviral medications against equine pathogens, x 2 days followed by 18 mg/kg bwt per os q. 12 h for a total the authors investigated the susceptibility of EHV-1 (isolate of 7 or 14 days) prior to EHV-1 inoculation; and 3) Kentucky D) and EHV-3 (isolate 1118) to ganciclovir in vitro; administration of valacyclovir at the onset of fever, 1–2 in that study the concentration of ganciclovir required to days post inoculation (Maxwell et al. 2009). The horses that reduce viral plaque numbers by 50% (IC50) for the 2 isolates received prophylactic valacyclovir therapy demonstrated was 0.033 and 0.16 mg/ml, respectively (Smith et al. 1983). lower clinical scores 2–7 days after inoculation with EHV-1 In comparison, the reported IC50 for acyclovir against when compared to control horses. Furthermore, on Days EHV-1 (isolate Kentucky D) was 7 mg/ml in a different study 1–7 post inoculation, a statistically significant decrease in (de Clercq et al. 1986). Another nucleoside analogue, rectal temperature, viral shedding and viraemia was penciclovir, also demonstrated activity against EHV-1 detected in both groups of horses administrated (isolate AB4) with an IC50 of 1.6 mg/ml (de la Fuente et al. valacyclovir when compared to control horses (Maxwell 1992). More recently, equine embryonic lung cells were et al. 2009). Valacyclovir administration decreased the infected with 2 different isolates of EHV-1 (abortigenic severity of ataxia related to EHM but did not reduce the risk isolate N752 and neuropathogenic isolate D752) and of ataxia as compared to control horses. The investigators exposed to serial dilutions of acyclovir, ganciclovir, concluded that valacyclovir administration significantly cidofovir, adefovir and foscarnet to determine the IC50 decreased viral replication and clinical signs of EHM. (Garre et al. 2007b). The IC50 for acyclovir ranged from These positive effects were greatest when horses were 1.7–3.0 mg/ml while ganciclovir appeared to be most treated prior to EHV-1 inoculation; however, delayed potent drug tested in this in vitro system (IC50 0.1–0.4 mg/ml) administration of valacyclovir (post inoculation) (Garre et al. 2007b). A key finding in this study was that decreased viral shedding, viraemia and clinical signs of both the neuropathogenic and abortigenic isolates disease (Maxwell et al. 2009). The susceptibility of equine demonstrated similar sensitivities to the nucleoside gamma herpesviruses, such as the equine herpesvirus analogues, despite the differences between DNA associated with equine multinodular pulmonary fibrosis polymerase that occurs between these 2 strains of EHV-1. (EHV-5), to acyclovir is unknown (Williams et al. 2007; The potencies of cidofovir (IC50 1.1–6.7 mg/ml) and adefovir Wong et al. 2008). Human Epstein-Barr virus and murine (IC50 2.8–5.6 mg/ml) were moderate with forscarnet being herpesvirus 68, both gamma herpesvirus, are sensitive to the least potent compound (IC50 6.6–11.1 mg/ml) (Garre acyclovir, but specific antiviral susceptibility testing of et al. 2007b). In another investigation, viral sensitivity testing equine gamma herpesviruses is necessary (Neyts and de suggested that acyclovir at a concentration of 0.3 mg/ml Clercq 1998; Long et al. 2003; Balfour et al. 2007). was sufficient to inhibit in vitro replication of a specific EHV-1 strain from an EHM outbreak as well as 3 additional Pharmacokinetic characteristics strains of EHV-1 from different EHM outbreaks (Wilkins 2004; Henninger et al. 2007). The pharmacokinetic properties of acyclovir in horses are This variation in reported susceptibility of EHV-1 to dependent on the route of administration including various nucleoside analogues may be attributed to intravenous or oral routes. Based on 3 pharmacokinetic sensitivity differences between different EHV-1 strains or studies in adult horses, the administration of a single dose of due to interlaboratory variation, as viral IC50 values can be acyclovir orally at 10 or 20 mg/kg bwt has poor difficult to interpret (Weinberg et al. 2007). In a recent, bioavailability and low maximum measured serum drug controlled, in vivo study evaluating the therapeutic concentrations (Cmax) (Wilkins et al. 2005; Bentz et al. 2006; efficacy of valacyclovir against EHV-1, 8 naïve weanling Garre et al. 2007a). In one study, at the higher dosage of pony foals were infected intranasally and orally with a 20 mg/kg bwt, bioavailability was 2.8% with a mean Ϯ s.d. neuropathogenic isolate of EHV-1 (03P37); 4 ponies were Cmax of 0.19 Ϯ 0.10 mg/ml (Bentz et al. 2006). As systemic administered valacyclovir (40 mg/kg bwt, per os q. 8 h for acyclovir exposure did not increase with increasing doses 5 or 7 days) whereas the other 4 ponies served as of oral acyclovir, absorption of acyclovir appears to be a untreated controls (Garre et al. 2009). Despite attaining saturable process. A similar bioavailibility of 3.13% was plasma acyclovir concentrations that were expected documented by different investigators; the Cmax in that to be therapeutic, little difference between study was 0.33 Ϯ 0.14 mg/ml (Garre et al. 2007a). Plasma valacyclovir-treated and untreated ponies could be acyclovir concentrations were below the lower limits of detected in regard to clinical signs, viral shedding or detection using high-performance liquid chromatography viraemia, suggesting that valacyclovir provided no in 6 horses orally administered acyclovir at a dose of protective effect in pony foals experimentally infected with 20 mg/kg bwt in a third study (Wilkins et al. 2005). Thus, the EHV-1 (Garre et al. 2009). Conversely, another study oral bioavailability of a single administration of acyclovir in investigated the efficacy of valacyclovir in 18 aged mares horses is exceptionally poor, even compared to the limited experimentally infected with EHV-1 and demonstrated bioavailability reported in man (10–30%) (van Dyke et al. beneficial results (Maxwell et al. 2009). Treatment groups 1982; Whitley et al. 1982; Ormrod and Goa 2000). included: 1) no therapy (control); 2) prophylactic Clearly, the oral bioavailability of a single administration administration of valacyclovir (27 mg/kg bwt, per os q. 8 h of acyclovir is low and quite variable among horses.

However, in a case series of equine multinodular 46.2 Ϯ 20.8 mg/ml; serum drug concentration rapidly pulmonary fibrosis, a recently described equine respiratory decreased immediately after discontinuation of infusion disease associated with EHV-5, acyclovir was administered (Bentz et al. 2006). In 3 subsequent studies using the same (20 mg/kg bwt, per os q. 8 h) to some horses over a i.v. dosage of acyclovir (10 mg/kg bwt), the duration of the prolonged period of time, up to 4 months (Wong et al. infusion was prolonged to 60 min, which resulted in a lower 2008). In Horse 1 of that series, blood was collected at mean Cmax concentration ranging from 10.4–12.1 mg/ml various times during treatment to measure serum acyclovir (Wilkins et al. 2005; Garre et al. 2007a; Maxwell et al. 2008). concentrations. On Day 28 of acyclovir treatment, prior to Based on the specific IC50 for acyclovir against EHV utilised, the next dosing of acyclovir (serum trough concentration), i.v. administration of acyclovir may or may not attain the serum concentration of acyclovir was 0.26 mg/ml, adequate therapeutic concentrations over the duration of based on measurement via high-performance liquid the dosing interval. In one study, utilising an IC50 of chromatography (Bentz et al. 2006). Subsequently, on Day 0.3 mg/ml,a1hi.v. infusion of acyclovir (10 mg/kg bwt) to 67 of acyclovir treatment, serum was collected prior to horses would exceed the IC50 for 8 h (Wilkins et al. 2005). The (time 0) and then 30, 60, 90 and 120 min after oral authors suggested that twice daily administration of administration of acyclovir (20 mg/kg bwt, per os). Results acyclovir (10 mg/kg bwt, i.v.) would result in adequate indicated acyclovir concentrations of 0.20, 0.40, 0.41, 0.36 acyclovir concentrations for the entire treatment interval and 0.54 mg/ml, respectively. Based on this information, it (Wilkins et al. 2005). However, more recent studies suggest appears that frequent repeated oral administration over that i.v. infusion of acyclovir (10 mg/kg bwt) over 1 h would prolonged treatment periods results in drug accumulation maintain the IC50 values (1.7–3.0 mg/ml) for only 1.5–2 h, thus and higher serum concentrations, as would be expected limiting the clinical use in cases of EHV infection in horses from the long elimination half-life of acyclovir in horses to a constant rate infusion of acyclovir to achieve (Maxwell et al. 2008). Although caution must be exercised adequate therapeutic concentrations (Garre et al. 2007a). in evaluating serum concentrations of acyclovir after Interestingly, the mean Ϯ s.d. acyclovir concentration repeated administration to a single horse, this supposition is 48 h after the discontinuation of drug infusion was supported by the acyclovir concentrations measured in a 0.34 Ϯ 12 mg/ml in another study, suggesting a prolonged, report in which median plasma acyclovir concentrations of but highly variable, elimination half-life (Maxwell et al. 0.387 mg/ml (range 0.226–0.869 mg/ml), 0.204 mg/ml 2008). The clinical utility of i.v. acyclovir has been limited in (range 0.110–0.380 mg/ml) and 0.222 mg/ml (range horses due to the high cost of the injectable formulation 0.070–0.386 mg/ml) were measured 30, 60 and 300 min, (Table 1) and the need to administer the drug as an infusion respectively, after 3–4 days of repeated administration of over1htolimit toxicity (Bentz et al. 2006). To date, no case acyclovir (10 mg/kg bwt, per os, 5 times daily) to horses with reports describing the clinical use of i.v. acyclovir EHM (Wilkins et al. 2003). Moreover, in a group of 5 horses administration for the treatment of an EHV-related disease with EHM that were treated with acyclovir (20 mg/kg bwt, have been published. per os q. 8hx5 days) the mean plasma acyclovir In efforts to increase the oral bioavailability of acyclovir concentration was 0.3 mg/ml (range 0.07–0.87) (Henninger in man, the drug has been complexed with the amino acid et al. 2007). As a result of drug accumulation with multiple L-valine by an ester linkage, resulting in the prodrug doses, steady state oral acyclovir administration may be valacyclovir, which is a substrate for carrier-mediated effective against equine herpes viruses that are highly transport into enterocytes (Ormrod and Goa 2000). This sensitive to acyclovir. However, the steady state peak and characteristic results in greatly improved and more trough concentrations of acyclovir remained below the consistent bioavailability in man and horses when acyclovir IC50 that has been reported for multiple strains of compared to oral acyclovir administration (Garre et al. EHV-1 (Garre et al. 2007b). In addition, the low and highly 2007a; Maxwell et al. 2008). After oral administration, variable concentrations of acyclovir obtained after the first valacyclovir is rapidly hydrolysed to the active moiety, dose suggest that the drug will take several days to reach acyclovir, and maintains the same mechanism of antiviral effective concentrations, limiting its utility in acute care activity as noted above (Ormrod and Goa 2000; Garre settings, such as during an EHV-1 outbreak. Thus, oral et al. 2007a). In an initial pharmacokinetic study of single acyclovir may be a more promising drug for the treatment oral administration of valacyclovir (20 mg/kg bwt), the of chronic herpes virus infection, such as EHV-5, if the viral bioavailability was 26.1 Ϯ 4.77% with a Cmax of strain is highly susceptible to acyclovir. 4.16 Ϯ 1.42 mg/ml (Garre et al. 2007a). In man, it is Intravenous administration of acyclovir has purported that maximal efficacy of acyclovir therapy is demonstrated better pharmacokinetic characteristics and reached when the length of time that acyclovir maximum serum drug concentrations when compared to concentration remains above the IC50 for more than 50% oral administration. In one study, i.v. infusion of acyclovir of the treatment period (Tod et al. 2001). Based on this over 15 min at a dose of 10 mg/kg bwt demonstrated an information, a dosing model for valacyclovir of 40 mg/kg initial rapid distribution phase, a slower secondary bwt, every 8 h, was designed to maintain drug plasma distribution phase and a more prolonged elimination concentrations above the IC50 value for the majority of the phase (Bentz et al. 2006). The Cmax in that study was dosing interval (Garre et al. 2007a). The validity of this

TABLE 1: Proposed acyclovir and valacyclovir dosages for horses based on veterinary studies along with specific dosages utilised in clinical reports

Route of Cost for 450 kg Medication Dosage administration Frequency Reference horse per day* Study or case remarks

Acyclovir 20 mg/kg bwt per os t.i.d. Bentz et al. 2006 $20.25 Poor oral bioavailability and low Cmax Garre et al. 2007a Cmax 0.19 Ϯ 0.10 mg/ml (Bentz et al. 2006) Cmax 0.33 Ϯ 0.14 mg/ml (Garre et al. 2007a) Acyclovir 10 mg/kg bwt i.v. b.i.d. Wilkins et al. 2005 $172.80 Predicted to exceed IC50 (0.3 mg/ml) for 8 h Valacyclovir 40 mg/kg bwt per os t.i.d. Garre et al. 2008, 2009 $558.90 Therapeutic plasma acyclovir concentration attained; little difference in clinical variables between treatment and control horses with EHV-1 Valacyclovir 27 mg/kg bwt per os t.i.d. Maxwell et al. 2009 $377.25 Horses treated with valacyclovir had improved clinical (loading) per os b.i.d. $167.67 variables compared to control horses with EHV-1 18 mg/kg bwt (maintenance) Acyclovir‡ 8–16 mg/kg bwt per os t.i.d. Murray et al. 1998 NA EHV-1 associated pulmonary disease in 3 foals; no definitive benefit of acyclovir administration documented Acyclovir 20 mg/kg bwt per os t.i.d. Friday et al. 2000 NA Unable to determine any benefit of acyclovir EQUINE VETERINARY EDUCATION /AEmay 2010 administered in outbreak of EHM Acyclovir 10 mg/kg bwt per os 5 doses/day Wilkins et al. 2003 NA Serum acyclovir concentrations in 5 treated horses measured (0.204–0.287 mg/ml); unable to determine any benefit of acyclovir administration in outbreak of EHM Acyclovir 20 mg/kg bwt per os t.i.d. Henniger et al. 2007 NA Subjective clinical improvement in clinical signs of EHM in horses treated with acyclovir; confounding factors present Acyclovir 20 mg/kg bwt per os t.i.d. Wong et al. 2008 NA EHV-5 associated equine multinodular pulmonary fibrosis in horses; no definitive benefit of acyclovir administration documented

* Average wholesale prices in USA, US dollars; Prices will vary depending on distributor and country of origin. Cmax, maximum measured serum drug concentrations; IC50, concentration of medication required to reduce viral plaque numbers by 50%; ‡ Not a complete list of clinical reports utilising acyclovir in horses; NA, not applicable; EHM, equine herpes myeloencephalopathy. EQUINE VETERINARY EDUCATION / AE / may 2010 249

model was subsequently confirmed in a follow-up study in Adverse effects ponies administered 40 mg/kg bwt of valacyclovir every 8 h for 5 or 7 days in which the IC50-value of 1.7 mg/ml could Adverse side effects to the administration of acyclovir or be maintained for 76% of the treatment period and valacyclovir in people are generally minimal and mild remained above the IC50-value of 3.0 mg/ml for 46% of the and include nausea, headache, vomiting, diarrhoea, treatment period (Garre et al. 2009). Alternatively, in a dizziness, anorexia and abdominal pain (Brigden and different study, valacyclovir administered at a single dose Whiteman 1983; Ormrod and Goa 2000). More serious of 9.0 or 26.6 mg/kg bwt demonstrated a mean Ϯ s.d. adverse effects include renal toxicity and rarely, bioavailability of 60 Ϯ 12% and 48 Ϯ 12%, respectively, and neurotoxicity (Schreiber et al. 2008; Asahi et al. 2009). a mean Ϯ s.d. Cmax of 1.45 Ϯ 0.38 and 5.26 Ϯ 2.82, Renal insufficiency is believed to result from an intrarenal respectively (Maxwell et al. 2008). obstructive nephropathy secondary to precipitation of As the respiratory and central nervous systems are acyclovir crystals in renal tubules if the maximum solubility target areas for different types of EHV-1 infections, is exceeded or during bolus administration (Garre et al. investigation into the concentration of acyclovir in these 2007a; Schreiber et al. 2008). Pre-existing renal tissues has been measured after multiple dose insufficiency, volume depletion, administration of other administration of valacyclovir (40 mg/kg bwt, per os,q. potentially nephrotoxic medications and high acyclovir 8 h) (Garre et al. 2008, 2009). In one study, low dosage are predisposing factors associated with the concentrations of acyclovir could be detected in nasal development of renal toxicity in patients treated with mucus samples (mean 0.72 mg/ml; range 0.36–1.17 mg/ml) acyclovir (Schreiber et al. 2008). No adverse side effects as well as cerebrospinal fluid samples (mean 0.16 mg/ml; have been reported in the majority of controlled studies range 0.11–0.23 mg/ml) (Garre et al. 2008). This suggests or clinical cases of acyclovir/valacyclovir administration that there may not be an effective concentration of in horses (Wilkins et al. 2005; Garre et al. 2007a). In one acyclovir in these tissues that would affect viral replication study, a horse was reported to demonstrate clinical signs and shedding and is supported by one in vivo study (Garre of sweating, colic and generalised muscle tremors during et al. 2008). However, unpublished data has measured i.v. infusion of acyclovir (Bentz et al. 2006). In that horse, acyclovir in peripheral blood mononuclear cells (PBMC) at acyclovir was administered both more rapidly (15 min) a concentration of approximately 57% of the and in a greater concentration than is recommended for corresponding plasma concentration (i.e. approximately human i.v. treatment or used in other equine studies. In 5 mg/ml acyclovir in PBMC), which is higher than the addition, transient elevation in serum creatinine IC50-values for numerous isolates of EHV-1 (Garre et al. concentration in one horse with EHM that was treated 2008). This information, coupled with the fact that with acyclovir was documented despite i.v. fluid therapy neuropathic isolates of EHV-1 are endotheliotropic rather and a lack of pre-existing renal dysfunction; azotaemia than neurotropic, suggest that valacyclovir may have an resolved upon cessation of acyclovir (Friday et al. 2000). inhibitory effect on the development of EHM (Donaldson No other evidence of renal injury or toxicity has been and Sweeney 1998; Wong and Scarratt 2006; Garre et al. reported in horses when monitored (Wong and Scarratt 2008). Confounding matters more, however, in a follow-up 2006; Henninger et al. 2007; Garre et al. 2007a). In study by the same group of investigators, higher general, administration of acyclovir and valacyclovir concentrations of acyclovir were measured in nasal mucus appears to be safe in horses at the published samples (0.5–6.5 mg/ml) ranging from 50–100% of the recommended dosages. corresponding plasma concentrations after multiple oral valacyclovir administration (Garre et al. 2009). Other pharmacokinetic information garnered from Case reports various studies includes the finding that there is no difference in plasma peak concentrations in fasted vs. non Currently, there is no prospective randomised clinical trial fasted horses nor in ponies vs. horses (Garre et al. 2007a). that has investigated the efficacy of acyclovir or This suggests that the presence of food with drug valacyclovir in horses with EHV-related disorders. Therefore, administration nor breed has any influence on the the use of acyclovir is empiric with modest scientific absorption of valacyclovir (Garre et al. 2007a). In addition, evidence to substantiate its use at this time. Despite this, the plasma protein binding of acyclovir is relatively low and acyclovir and valacyclovir remain attractive therapeutic has been calculated to be 10–20% (Garre et al. 2007a). In options in horses with EHV-related disorders because of regard to elimination, acyclovir produces 2 other their theoretic antiviral properties against herpesviruses pharmacologically inactive compounds in man that are that may potentially decrease convalescent time as well primarily eliminated via the renal route, along with as decrease viral shedding and facilitate control of disease valacyclovir and acyclovir (Brigden and Whiteman 1983; transmission. This perceived benefit is supported by Ormrod and Goa 2000). Small amounts of valacyclovir and anecdotal evidence documented in clinical case reports acyclovir are also excreted in the faeces in man (Brigden and retrospective case series of acyclovir administration to and Whiteman 1983; Ormrod and Goa 2000). horses with EHV-related diseases (Table 1). However, one

must remain cautious in the interpretation of these reports foals treated with acyclovir, one died and 2 survived as many confounding factors may have contributed to (Murray et al. 1998). In a case series of equine the outcome of the cases described. In light of this, the multinodular pulmonary fibrosis, 4 horses were successful treatment of neonatal pulmonary disease administered acyclovir orally for variable lengths of time related to EHV-1 as well as a reduction in the incidence of (Wong et al. 2008). In this report, 2 of 4 horses treated with EHM in horses prophylactically treated with acyclovir acyclovir survived but the use of acyclovir was entirely during a recent outbreak has been reported (Murray empirical, and its efficacy could not be established in this et al. 1998; Henninger et al. 2007). Acyclovir has been small group of horses (Wong et al. 2008). most frequently implemented in the treatment of EHM. In In women, the acquisition of herpes simplex virus during a large case series involving 135 horses at a university pregnancy has been associated with spontaneous equestrian centre, 119 horses (88%) displayed clinical signs abortion, intrauterine growth retardation, premature of EHV-1 infection of which 46 developed EHM (Henninger labour and congenital and neonatal herpes infections et al. 2007). The mortality rate of those with EHM was 30% (Brown et al. 1997). Although acyclovir and valacyclovir (14/46). Acyclovir (20 mg/kg bwt, per os q. 8 h x 5 days) are not approved for treatment of pregnant women, these was administered to 99 horses on the premises. Of these medications have been administered to treat primary 99 horses, 19 that received acyclovir had neurological clinical episodes or recurrences of herpes simplex virus disease for 1–2 days before treatment was instituted infection without any ascribed short-term side effects to whereas the remaining 80 horses were prophylactically the fetus (Anzivino et al. 2009). Randomised studies have administered acyclovir. Subjectively, positive clinical shown that suppressive treatment with acyclovir or efficacy of acyclovir therapy was observed based on the valacyclovir during late gestation significantly reduced the fact that 11 of 22 horses with EHM that either did not frequency of clinical manifestations and virus shedding as receive acyclovir or received it at least 24 h after the well as the risk of vertical transmission in women (Anzivino onset of neurological deficits became recumbent and et al. 2009). Similarly, EHV-1 has been associated with did not survive (Henninger et al. 2007). In contrast, 1 of 24 abortion and congenital and neonatal infections in foals horses with EHM became recumbent when treatment was (Murray et al. 1998; Del Piero and Wilkins 2001; Lunn et al. administered prophylactically or within 24 h of the onset 2009). An inactivated EHV-1 vaccine is available and has of neurological deficits (Henninger et al. 2007). The demonstrated decreased incidence of abortion in authors believed that the prophylactic use, early vaccinated mares in one study (Bryans and Allen 1982). therapeutic use, or both prophylactic and therapeutic Interestingly, 2 subsequent studies failed to identify a administration of acyclovir was associated with survival difference in abortion rates with the use of EHV-1 vaccine but confounding factors, such as limited acyclovir supplies (Burrows et al. 1984; Burki et al. 1990). Therefore, in certain that prevented most treated horses from receiving the situations in which a mare and her progeny are particularly drug until later in the course of the outbreak, could not be valuable, the administration of antiviral medication is a excluded (Henninger et al. 2007). However, these findings consideration as abortigenic isolates of EHV-1 are do show that oral acyclovir therapy at 20 mg/kg bwt q. susceptible to acyclovir and, therefore, to valacyclovir 8 h does not appear to be associated with any signs of (Garre et al. 2007b). Furthermore, the use of antiviral toxicity in horses. In another report involving 46 horses, 19 medications should be considered in the face of EHV horses developed EHM of which 6 horses were treated abortion outbreaks to potentially limit the spread of with acyclovir beginning 3–4 days after the onset of disease. Investigation of the use of acyclovir or valacyclovir neurological deficits (Friday et al. 2000). However, the in the prophylaxis or treatment of pregnant mares exposed authors were unable to divulge any benefit, or lack to EHV-1 has not been undertaken but may be a prudent thereof, with the use of acyclovir in this report (Friday et al. venture. 2000). All 5 horses with EHM treated with oral acyclovir survived in a further report (Wilkins et al. 2003). Other case reports of acyclovir administration to horses with EHM Conclusions have been published but provide no clear information as to usefulness in the treatment of EHM (Wilkins et al. 2003; Currently, the use of acyclovir is limited to its poor Wong and Scarratt 2006). Less commonly, acyclovir has bioavailability when administered orally and short been administered to foals with an EHV-1 associated elimination half-life when administered i.v. However, pulmonary disease and in horses with equine multinodular repeated oral administration of acyclovir may have a pulmonary fibrosis (Murray et al. 1998; Wong et al. 2008). cumulative effect and result in higher plasma In the report describing neonatal foals with EHV-1 concentrations when compared to single dose associated pulmonary disease, characterised by administration. Acyclovir may have potentially decreased weakness, lethargy, fever and laboured breathing, 3 foals the severity of disease and improved outcome based on a were treated with acyclovir (Murray et al. 1998). Again, no large outbreak of EHM in which acyclovir was administered definitive evidence of the effectiveness of acyclovir in the (Henninger et al. 2007). Nonetheless, the collective treatment of these foals could be established but of the 3 evidence suggests that the efficacy of oral acyclovir

against EHV is very low. Conversely, valacyclovir, a prodrug Donaldson, M.T. and Sweeney, C.R. (1998) Herpesvirus of acyclovir, appears to have better oral bioavailability and myeloencephalopathy in horses: 11 cases (1982-96). J. Am. vet. med. Ass. 213, 671-675. reach higher and potentially more therapeutic plasma Friday, P.A., Scarratt, W.K., Elvinger, F., Timoney, P.J. and Bonda, A. (2000) concentrations against EHV but administration of Ataxia and paresis with equine herpesvirus type 1 infection in a herd valacyclovir in clinical cases has not been reported. Despite of riding school horses. J. vet. intern. Med. 14, 197-201. contradictory findings between studies, one experimental Garre, B., Gryspeerdt, A., Croubels, S., De Backer, P. and Nauwynck, H. study utilising valacyclovir for the treatment of EHM appears (2009) Evaluation of orally administered valacyclovir in to be encouraging (Maxwell et al. 2009). At present, a experimentally EHV1-infected ponies. Vet. Microbiol. 135, 214-221. valacyclovir dosage of 27 mg/kg bwt, per os q. 8 h as a 2 Garre, B., Baert, K., Nauwynck, H., Deprez, P., Debacker, P. and Croublels, S. (2008) Multiple oral dosing of valacyclovir in horses and day loading dose, followed by a maintenance dose of ponies. J. vet. Pharmacol. Ther. 32, 207-212. 18 mg/kg bwt, per os, q. 12 h for a total of 7–14 days is Garre, B., Shebany, K., Gryspeerdt, A., Baert, K., van der Meulen, K., recommended based on current studies (Maxwell et al. Nauwynck, H., Deprez, P., De Backer, P. and Croubels, S. (2007a) 2008). However, in light of the shortage of veterinary studies Pharmacokinetics of acyclovir after intravenous infusion of acyclovir involving antiviral medications in horses, the reader should and after oral administration of acyclovir and its prodrug valacyclovir in healthy adult horses. Antimicrob. Agents Chemother. be cautious in using the information presented here and be 51, 4308-4314. attentive to future published studies in regard to antiviral Garre, B., van der Meulen, K., Nugent, J., Neyts, J., Croubels, S., De medications in equine practice. Much of the future of Backer, P. and Nauwynck, H. (2007b) In vitro susceptibility of six antiviral therapy in equine medicine remains to be isolates of equine herpesvirus 1 to acyclovir, ganciclovir, cidofovir, adefovir, PMEDAP and foscarnet. Vet. Microbiol. 122, 43-51. explored. Hayden, F. (2001) Antimicrobial agents (continued) Antiviral agents (nonretroviral). In: Goodman & Gillman’s the Pharmacological Basis of Therapeutics, 10th edn., Eds: J. Hardman and L. Limbird, References McGraw-Hill, New York. pp 1313-1348. Henninger, R.W., Reed, S.M., Saville, W.J., Allen, G.P., Hass, G.F., Kohn, Anzivino, E., Fioriti, D., Mischitelli, M., Bellizzi, A., Barucca, V., Chiarini, F. C.W. and Sofaly, C. (2007) Outbreak of neurologic disease caused and Pietropaolo, V. (2009) Herpes simplex virus infection in by equine herpesvirus-1 at a university equestrian center. J. vet. pregnancy and in neonate: status of art of epidemiology, diagnosis, intern. Med. 21, 157-165. therapy and prevention. Virol. J. 6, 40. Long, M.C., Bidanset, D.J., Williams, S.L., Kushner, N.L. and Kern, E.R. Asahi, T., Tsutsui, M., Wakasugi, M., Tange, D., Takahashi, C., Tokui, K., (2003) Determination of antiviral efficacy against lymphotropic Okazawa, S. and Okeuera, H. (2009) Valacyclovir neurotoxicity: herpesviruses utilizing flow cytometry. Antiviral Res. 58, 149-157. clinical experience and review of the literature. Eur. J. Neurol. 16, 457-460. Lunn, D.P., Davis-Poynter, N., Flaminio, M.J., Horohov, D.W., Osterrieder, K., Pusterla, N. and Townsend, H. (2009) Equine herpesvirus-1 Balfour, H.H., Jr, Hokanson, K.M., Schacherer, R.M., Fietzer, C.M., consensus statement. J. vet. intern. Med. 23, 450-461. Schmeling, D.O., Holman, C.J., Vezina, H.E. and Brundage, R.C. (2007) A virologic pilot study of valacyclovir in infectious Maxwell, L.K., Bentz, B.G., Bourne, D.W. and Erkert, R.S. (2008) mononucleosis. J. clin. Virol. 39, 16-21. Pharmacokinetics of valacyclovir in the adult horse. J. vet. Pharmacol. Ther. 31, 312-320. Bentz, B.G., Maxwell, L.K., Erkert, R.S., Royer, C.M., Davis, M.S., MacAllister, C.G. and Clarke, C.R. (2006) Pharmacokinetics of Maxwell, L.K., Bentz, B.G., Gilliam, L.L., Ritchey, J.W., Eberle, R.W., acyclovir after single intravenous and oral administration to adult Holbrook, T.C., Mcfarlane, D., Rezabek, G.B., MacAllister, C.G., horses. J. vet. intern. Med. 20, 589-594. Goad, C.L. and Allen, G.P. (2009) Efficacy of valacyclovir against disease following EHV-1 challenge. Proc. Am. Coll. vet. intern. Med., Brigden, D. and Whiteman, P. (1983) The mechanism of action, 176. pharmacokinetics and toxicity of acyclovir–areview. J. Infect 6, Suppl. 1, 3-9. Murray, M.J., Piero, F., Jeffrey, S.C., Davis, M.S., Furr, M.O., Dubovi, E.J. and Mayo, J.A. (1998) Neonatal equine herpesvirus type 1 infection Brown, Z.A., Selke, S., Zeh, J., Kopelman, J., Maslow, A., Ashley, R.L., on a thoroughbred breeding farm. J. vet. intern. Med. 12, 36-41. Watts, D.H., Berry, S., Herd, M. and Corey, L. (1997) The acquisition of herpes simplex virus during pregnancy. N. Engl. J. Med. 337, 509-515. Neyts, J. and De Clercq, E. (1998) In vitro and in vivo inhibition of murine Bryans, J.T. and Allen, G.P. (1982) Application of a chemically gamma herpesvirus 68 replication by selected antiviral agents. inactivated, adjuvanted vaccine to control abortigenic infection of Antimicob. Agents Chemother. 42, 170-172. mares by equine herpesvirus I. Dev. Biol. Stand. 52, 493-498. Ormrod, D. and Goa, K. (2000) Valaciclovir: a review of its use in the Burki, F., Rossmanith, W., Nowotny, N., Pallan, C., Mostl, K. and Lussy, H. management of herpes zoster. Drugs 59, 1317-1340. (1990) Viraemia and abortions are not prevented by two Schmidt, S., Barbour, A., Sahre, M., Rand, K.H. and Derendorf, H. (2008) commercial equine herpesvirus-1 vaccines after experimental PK/PD: new insights for antibacterial and antiviral applications. Curr. challenge of horses. Vet. Q. 12, 80-86. Opin. Pharmacol. 8, 549-556. Burrows, R., Goodridge, D. and Denyer, M.S. (1984) Trials of an Schreiber, R., Wolpin, J. and Koren, G. (2008) Determinants of inactivated equid herpesvirus 1 vaccine: challenge with a subtype acyclovir-induced nephrotoxicity in children. Paediatr. Drugs 10, 1 virus. Vet. Rec. 114, 369-374. 135-139. De Clercq, E., Holy, A., Rosenberg, I., Sakuma, T., Balzarini, J. and Smith, K.O., Galloway, K.S., Hodges, S.L., Ogilvie, K.K., Radatus, B.K., Maudgal, P. (1986) A novel selective broad-spectrum anti-DNA virus Kalter, S.S. and Heberling, R.L. (1983) Sensitivity of equine agent. Nature 323, 464-467. herpesviruses 1 and 3 in vitro to a new nucleoside analogue, De la Fuente, R., Awan, A.R. and Field, H.J. (1992) The acyclic 9-[[2-hydroxy-1-(hydroxymethyl) ethoxy] methyl] guanine. Am. J. nucleoside analogue penciclovir is a potent inhibitor of equine vet. Res. 44, 1032-1035. herpesvirus type 1 (EHV-1) in tissue culture and in a murine model. Tod, M., Lokiec, F., Bidault, R., De Bony, F., Petitjean, O. and Aujard, Y. Antiviral Res. 18, 77-89. (2001) Pharmacokinetics of oral acyclovir in neonates and in infants: Del Piero, F. and Wilkins, P.A. (2001) Pulmonary vasculotropic EHV-1 a population analysis. Antimicrob. Agents Chemother. 45, 150- infection in equids. Vet. Pathol. 38, 474. 157.

Van Dyke, R.B., Connor, J.D., Wyborny, C., Hintz, M. and Keeney, R.E. Wilkins, P.A., Papich, M. and Sweeney, R. (2005) Pharmacokinetics (1982) Pharmacokinetics of orally administered acyclovir in patients of acyclovir in adult horses. J. vet. emerg. crit. Care. 15, 174- with herpes progenitalis. Am. J. Med. 73, 172-175. 178. Weinberg, A., Leary, J.J., Sarisky, R.T. and Levin, M.J. (2007) Factors that Williams, K.J., Maes, R., Del Piero, F., Lim, A., Wise, A., Bolin, D.C., Caswell, affect in vitro measurement of the susceptibility of herpes simplex J., Jackson, C., Robinson, N.E., Derksen, F., Scott, M.A., Uhal, B.D., Li, virus to nucleoside analogues. J. clin. Virol. 38, 139-145. X., Youssef, S.A. and Bolin, S.R. (2007) Equine multinodular pulmonary Whitley, R.J., Blum, M.R., Barton, N. and de Miranda, P. (1982) fibrosis: a newly recognized herpesvirus-associated fibrotic lung Pharmacokinetics of acyclovir in humans following intravenous disease. Vet. Pathol. 44, 849-862. administration. A model for the development of parenteral antivirals. Am. J. Med. 73, 165-171. Wong, D.M. and Scarratt, W.K. (2006) Equine herpes myeloencephalopathy in a 12-year-old American Quarter horse. Wilkins, P.A. (2004) Acyclovir in the treatment of EHV-1 Vet. Clin. N. Am.: Equine Pract. 22, 177-191. myeloencephalopathy. Proc. Am. Coll. vet. intern. Med., 170- 172. Wong, D.M., Belgrave, R.L., Williams, K.J., Del Piero, F., Alcott, C.J., Bolin, Wilkins, P.A., Henninger, R.W. and Reed, S. (2003) Acyclovir as a S.R., Marr, C.M., Nolen-Walston, R., Myers, R.K. and Wilkins, P.A. (2008) treatment for EHV-1 myeloencephalopathy. Proc. Am. Ass. equine Multinodular pulmonary fibrosis in five horses. J. Am. vet. med. Ass. Practnrs. 49, 394-396. 232, 898-905.

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PRECAUTIONS At least one episode of diarrhea, loose, soft, or cowpie Prescribing antibacterial drugs in the absence of a proven stools were observed in 25 of 278 (9%) of the EXCEDE-treated or strongly suspected bacterial infection is unlikely to horses and 7 of 95 (7%) of the placebo-treated horses. The provide benefit to the treated animal and may increase the duration of episodes in EXCEDE-treated horses ranged from risk of development of drug-resistant animal pathogens. a single observation of loose stool to observations lasting 6 For intramuscular injection in the horse. days. All cases were self-limiting and resolved with minimal The administration of antimicrobials to horses under (a single dose of loperamide) or no treatment. CAUTION conditions of stress may be associated with acute diarrhea Federal (USA) law restricts this drug to use by or on the that can be fatal. If acute diarrhea is observed, additional Table 1. Number of Horses with Adverse Reactions order of a licensed veterinarian. doses of EXCEDE should not be administered and appropriate During the Field Study with EXCEDE INDICATION therapy should be initiated. EXCEDE Placebo Adverse Reaction (n=278) (n=95) EXCEDE Sterile Suspension is indicated for the treatment Due to the extended exposure in horses, based on the of lower respiratory tract infections in horses caused by drug’s pharmacokinetic properties, adverse reactions may Diarrhea/Soft Stool 25 (9%) 7 (7%) susceptible strains of Streptococcus equi ssp. zooepidemicus. require prolonged care. EXCEDE is slowly eliminated from the Injection Site Swelling 10 (4%) 1 (1%) body, with approximately 17 days needed to eliminate 97% CONTRAINDICATIONS of the dose from the body. Animals experiencing adverse The material safety data sheet (MSDS) contains more de- EXCEDE Sterile Suspension is contraindicated in horses reactions may need to be monitored for this duration of time. tailed occupational safety information. To obtain a material with known allergy to ceftiofur or to ß-lactam (penicillins and The use of ceftiofur has not been evaluated in horses less safety data sheet, please call 1-800-733-5500. To report any cephalosporins) group antimicrobials. Due to the extended than 4 months of age and in breeding, pregnant, or lactating adverse event please call 1-800-366-5288. exposure in horses, based on the drug’s pharmacokinetic horses. The long term effects on injection sites have not been STORAGE CONDITIONS properties, adverse reactions may require prolonged care. evaluated. Store at controlled room temperature 20° to 25°C (68° WARNINGS ADVERSE REACTIONS to 77°F). Shake well before using. Contents should be used Not for use in humans. For use in animals only. Keep The injection of EXCEDE Sterile Suspension in the horse within 12 weeks after the first dose is removed. this and all drugs out of reach of children. Consult a physi- may cause firmness, swelling, sensitivity, and/or edema at HOW SUPPLIED cian in case of accidental human exposure. the injection site. EXCEDE Sterile Suspension is available in the following Do not use in horses intended for human consumption. A total of 373 horses of various breeds, ranging in age from package size: 100 mL vial Penicillins and cephalosporins can cause allergic reactions in 4 months to 20 years, were included in the field study safety U.S. Patent No. 5,721,359 and other patents pending. sensitized individuals. Topical exposure to such antimicrobials, analysis. Adverse reactions reported in horses treated with NADA #141-209, Approved by FDA including ceftiofur, may elicit mild to severe allergic reactions EXCEDE and the placebo control are summarized in Table 1. in some individuals. Repeated or prolonged exposure may Injection site swelling (edema) was reported in 10 of lead to sensitization. Avoid direct contact of the product with 278 (3.6%) EXCEDE-treated horses and 1 of 95 (1%) of the the skin, eyes, mouth and clothing. Sensitization of the skin placebo-treated horses. Of the 10 EXCEDE-treated horses may be avoided by wearing protective gloves. Persons with with injection site swelling, 8 horses had swellings of 4 cm or www.EXCEDE.com or call 1-866-387-2287 a known sensitivity to penicillin or cephalosporins should less in diameter, one horse had a 10 cm diameter swelling avoid exposure to this product. In the case of accidental and one horse had injection site reactions to both injections Revised October 2009 10423900 eye exposure, flush with water for 15 minutes. In case of measuring 25 x 12 cm each. The injection site reactions in EXEQ0110010 accidental skin exposure, wash with soap and water. Remove EXCEDE-treated horses resolved over 1 to 20 days. contaminated clothing. If allergic reaction occurs (e.g. skin rash, hives, difficult breathing) seek medical attention.