Noninferiority of Orogastrically Administered Enrofloxacin Compared to Intramuscular Administration in the Olive Baboon (Papio anubis)

Abstract

Management of nonhuman primates (NHPs) in the laboratory setting is complex. Medical management often involves techniques aimed at minimizing the impact on the animals’ welfare, while considering the species-specific characteristics, research aims, and clinical needs of the patient. The current practice, at our institution, for administration of enrofloxacin has been to employ intramuscular injection, which may require brief restraint that can result in increased stress for the animal when applied throughout the course of a therapeutic regimen. Alternatively, oral dosing of standard veterinary tablet formulations has resulted in inconsistent administration, requiring supplementation with an injectable product if the animal becomes unwilling to take the medication. This inconsistency led us to investigate alternative methods of administration. Basing our efforts on previous work performed in macaques, we aimed to determine the pharmacokinetic characteristics of the injectable formulation of enrofloxacin when administered orally to one species of primate, the olive baboon (Papio anubis). Our work demonstrated that injectable enrofloxacin administered orally at 10 mg/kg resulted in higher serum levels than the intramuscular administration group for both enrofloxacin and its active metabolite, ciprofloxacin. In addition, our results support oral administration of enrofloxacin injectable product at intervals of up to once every 24 to 48 h when given at a dose of 10 mg/kg.

Introduction

NHPs present a unique challenge when managing them in a clinical setting compared with other laboratory animals such as rodents and rabbits. Olive baboons (Papio anubis) in particular are intelligent, strong, and possess complex social and psychologic interactions between individuals within a group.^1–3 They establish social hierarchies through a combination of factors, including age, health status, lineage, and regular dominance-associated behaviors, which may result in internal conflict.^1,2 With conflict may come injury, and injury may prompt the use of antimicrobial therapy to prevent or treat infection. While trained personnel working with an amenable animal can deliver an intramuscular injection relatively quickly, this process requires brief restraint if the animal has not been trained for the procedure. While many institutions employ reinforcement training toward these ends, clinicians do not have the ability to predict which animals will be injured and may, thus, be faced with an untrained animal. The use of restraint invariably results in increased stress to the animal. Further, animals are more prone to reinjuring themselves or exacerbating existing wounds during this process.

As most antimicrobials require sequential dosing,^4–6 and repeated restraint over the course of treatment presents a potential for prolonged impact on animal well-being, we sought options to improve antimicrobial efficacy and tolerance to administration procedures. Anecdotally, our team has noted that many animals receiving a therapeutic course of injectable antimicrobials display stress responses during treatment, often arising at the start of the restraint process, likley in anticipation of a distressful experience. Therefore, we aimed to refine our practices to achieve both a low-stress administration process which enables accuracy in dosing. As a therapeutic course of antimicrobials may last between 7 and 14 d, an improved, less stressful method would be of great value.^4–7

Because there are currently no published data for the efficacy of the injectable enrofloxacin compound when administered orally to baboons, evaluation of this compound was done in a manner that would minimize variables. True oral administration would optimally be performed on an awake animal, thereby eliminating the need for a needle stick for administration, and this is the overarching goal of the work described here. It is important to note, though, that this study was performed using sedated animals and orogastric intubation to establish the baseline efficacy of this compound. As the goal was to justify administration via an oral route, animals were placed under sedation to allow for direct administration into the lumen of the stomach. This action eliminated the potential for animal preference-based impact on absorption and established a standard baseline for comparison.

Enrofloxacin is a fluoroquinolone antibiotic with a broad spectrum of antimicrobial activity that has a bactericidal effect and is concentration dependent; its potency is dose dependent, and peak effect coincides with peak serum levels.^7–10 Enrofloxacin begins acting on susceptible pathogens within 30 min of exposure to the antibiotic.⁵ These factors make it a good choice when working in conditions where contamination risks are high and the potential for mixed-spectrum agents exists. Enrofloxacin has long been used as a cornerstone antimicrobial in NHPs.² Acting on DNA gyrase and topoisomerase IV, it inhibits the supercoiling of DNA and subsequent DNA synthesis, resulting in bactericidal effects.^{7–8,10–12} Bioavailability data across multiple mammalian species show variable absorption rates with C_max being achieved between 30 to 60 min and T_max occurring between 2 and 10 h across various species.⁵ Oral bioavailability data range higher for animals with complex gastrointestinal systems such as ruminants, and caution is advised when using oral antimicrobials in these species.^4,5 Ciprofloxacin, the primary active metabolite of enrofloxacin, is a product of multiple metabolic changes including N-dealkylation, glucuronide activity, and oxidation reactions.^7–9 Ciprofloxacin is a commonly used antimicrobial in humans, while enrofloxacin is limited to use in veterinary species.⁷ Enrofloxacin possesses a relatively low reported incidence of adverse effects compared with other common antimicrobials such as β-lactams, sulfa therapies, and tetracyclines when administered at recommended therapeutic ranges.^4–8,10,13

While enrofloxacin impacts a wide range of susceptible agents, it does possess key characteristics to consider, which may preclude its use in clinical situations. Enrofloxacin shows little effect against anaerobic agents, in addition to a diminished effect on some Streptococcus and Enterococcus spp.^5,7 Previous studies^5,7 in canines reported adverse cartilaginous effects when administered at doses of 2 to 5 times therapeutic windows for periods lasting up to 30 d, although clinically detectable adverse effects were observed when administered 5 times therapeutic dosing only. The most significant adverse effects were reported with juvenile horses, which demonstrated enrofloxacin to be present within immature chondrocytes, supporting the conclusion that large, rapidly growing animals are most at risk of the adverse effects of enrofloxacin on cartilage.⁵ This is generally not a concern in NHPs; however, consideration should be given to the age and risk group for each case. Additional concerns exist with respect to organ function, as enrofloxacin and its metabolite ciprofloxacin require elimination via dual hepatic and renal mechanisms, and patients experiencing deficiencies in either organ system may accumulate serum levels more rapidly, thereby increasing the likelihood of adverse effects. Caution is advised when administering enrofloxacin to patients experiencing clinical dehydration for similar reasons.^4–8

Our team has used enrofloxacin for many years in NHPs due to its breadth of pathogen coverage, the dose-dependent effect, and the low frequency of side effects. Further, we have used both the injectable and oral tablet formulations in attempts to provide consistent and effective care. In our experience, while some animals reliably accept the tablets for the planned course of treatment, others have shown reluctance to take, or will discontinue, voluntary dosing, thereby requiring supplementation with injectable enrofloxacin to complete the regimen. Baboons are quite savvy; animals will frequently remove tablets “hidden” in foodstuffs, and crushed tablets result in a refusal to consume. In addition, commercially available compounded substances have limited flavor profiles that have not been preferred among our patients. Without prior knowledge of amenability, creating a treatment plan often results in a patchwork of oral or injectable formulations, which can increase stress to patients and potentially increase the risk for the rise of resistant pathogens should the regimen not be completed. While we have employed positive reinforcement training for many animals in our care, some NHP patients either have not been trained or, in the face of conflict and stress, revert away from learned behaviors. These situations may result in animals that are less inclined to participate in voluntary injections and may necessitate use of alternative options. While there are many potential antimicrobials available for use, enrofloxacin carries large benefits by requiring administration once daily and having bactericidal effects in a dose-dependent response; thus, we evaluated the efficacy of enrofloxacin when administered in novel ways. Our teams’ selection of the injectable product was due to a number of factors including availability, prior experience with taste aversion to commercially available tablets, and consultation with our Behavior Management and Enrichment Team regarding utility in a clinical setting. We also determined that the use of the injectable form for both cohorts would minimize the risk of variable drug distribution between formulations, ensuring accuracy in dosing.

Klein et al¹¹ demonstrated the efficacy of administering an injectable formulation of enrofloxacin as an oral therapy in rhesus macaques (Macacca mulatta). While both rhesus macaques and olive baboons are Old World primates, they are taxonomically separate, and caution is advised when inferring responses between the species.² This study serves to demonstrate that the observations made in rhesus macaques are consistent with those we found in the olive baboon. We hypothesized that the oral administration of injectable enrofloxacin produces serum levels equal to or greater than those measured via the intramuscular route in olive baboons. A secondary hypothesis predicts that serum levels of the active metabolite ciprofloxacin will be equal to or greater than those measured via the intramuscular route.

Materials and Methods

Animals.

Animals.

All work performed was approved by the University of Oklahoma Health Sciences Center IACUC, and animals were housed and managed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and the USDA Animal Welfare Regulations. The University of Oklahoma Health Sciences Center has maintained AAALAC accreditation continuously since 1973, and animals are maintained under Assurance through the Office of Laboratory Animal Welfare.

Three male and 3 female purpose-bred, olive baboons (P. anubis) aged between 5 to 6 y were enrolled in the study. Animals had no history of health conditions that would influence the metabolism of enrofloxacin. Animals were fed twice daily rations of Lab Diet 5045 (LabDiet, Richmond, IN) and provided ad libitum tap water. All animals receive daily forage and fresh produce.

Inclusion criteria for this project included (1) age of 5 to 10 y, (2) no active identifiable disease process, (3) not presently receiving immunomodulatory or antimicrobial therapy, and (4) normal results on prestudy complete blood count (CBC) and serum biochemistry assays.

Female baboons were monitored for reproductive status throughout the study to ensure that changes in hormone profiles could be detected and evaluated for impact on results. One of 3 females was noted at peak cycling during the intramuscular administration arm, and 2 were noted to be midcycle during the oral administration arm.

Compounds.

Enrofloxacin (Baytril 100; Elanco, Greenfield, IN) was used for all dosing performed in this study. Injectable doses were administered intramuscularly via a 21-gauge needle (Covetrus, Portland, ME), attached to a 3- or 6-mL Luer-lock syringe (Covidien Medical, Minneapolis, MN). Oral dosing was performed via placement of a sterile 16-French flexible red catheter (Covidien Medical, Minneapolis, MN) attached to a syringe, followed by 3-mL sterile water flush to ensure complete deposition into the gastric lumen.

Ketamine (Ketaset III; Zoetis Animal Health, Parsippany, NJ) was used for all sedation procedures in this study. Administration of ketamine for this study was performed as a single intramuscular injection into the craniolateral thigh muscles via a 21-gauge needle attached to a Luer-lock syringe.

Acepromazine (Acepromazine Maleate Injection; Covetrus, Portland, ME) was administered to a single animal during this study to address postprocedural dysphoria via a 21-gauge needle and Luer-lock syringe intramuscularly into the cranial thigh muscle.

Study design.

Six young-adult animals aged 5 to 10 y were chosen for this crossover study. All animals underwent the intramuscular arm of the study simultaneously, followed by the oral-administration arm after a 30-d washout period (Figure 1). This allowed for comparison between individuals while requiring fewer total animals to achieve significant data.

Figure 1.

Visual representation of timeline. All animals (n = 6) were simultaneously enrolled in the intramuscular dosing cohort followed by a 30-d washout period before undergoing orogastric administration.

For all sedated procedures, animals were monitored constantly with heart rate, respiratory rate, and body temperature recorded every 5 min. Animals were continuously visually monitored until they were able to ambulate. Once ambulatory, animals received an observational examination twice more throughout the day to ensure full recovery. While the sample collection ended at 48 h for each portion of the study, an additional, end-of-study evaluation was performed at the 72-h mark to confirm no lasting effects of multiple sedations. Serum was separated, and 500-μL aliquots were created and maintained at −80 °F until processing. Save for mild postprocedural dysphoria, no complications were noted in any animals undergoing procedures in this study.

Acclimation.

Animals were single housed for 5 d before the beginning of the study to acclimate to the housing needs of the project. They maintained visual, auditory, and tactile contact with neighboring conspecifics. All animals were returned to social housing between the intramuscular and orogastric arms of this study. Total time of single housing was minimized as much as possible to maintain adherence to the recommendations given in the Guide to Care and Use of Laboratory Animals.¹⁴

Procedure timeline.

Animals were sedated one day before (T to 1 d) the intramuscular arm for initial physical exam, serum collection for negative control, and samples for CBC/serum chemistry to ensure no ongoing clinical concerns. Animals received 10 mg/kg ketamine intramuscularly for collection at 0, 8, 24, and 48 h with 5 mg/kg being used for supplemental sedation at time 2 h. Immediately after reaching the point of sedation (time 0), they received 10 mg/kg enrofloxacin administered either orally or intramuscularly. One milliliter of blood was collected at each of the following time points: 0, 2, 8, 24, and 48 h. After the first 4-d study period, animals were socially housed for the 30-d washout. The −1-d collection was not repeated for the oral administration arm as this was solely to collect prestudy assessment for participation. After the second 4-d study period, animals were returned to social housing, and the data collection portion of the project was completed (Figure 1).

Intramuscular administration.

Animals received 10 mg/kg of enrofloxacin intramuscular in the craniolateral thigh. The time of administration was recorded and used as the marker for subsequent blood collections.

Orogastric administration.

Sedation was performed, and animals were placed in a seated, upright posture. A 16-French red rubber catheter attached to the preloaded syringe containing enrofloxacin was passed into the oropharynx and subsequently into the esophagus, confirmed via laryngoscope. The solution was administered over 2 min. To ensure complete administration of the calculated dose, 3 mL of sterile water for injection was flushed through the catheter following oral gavage. Animals were held in the upright posture for 1 min to ensure gravity-led passage into the stomach. Animals were then returned to lateral positioning for further monitoring and blood collection. All remaining procedures were performed identically to those in the intramuscular arm.

Mass spectrometry.

Standards and stock solutions.

All standards and internal standards were obtained from Cayman Chemical (Ann Arbor, MI); all other chemicals were from ThermoFisher Scientific (Waltham, MA). Stock solutions of enrofloxacin and enrofloxacin-d5 were prepared at a concentration of 1,000 μg/mL by dissolving the proper quantity of each compound, exactly weighted, in 10% dimethyl sulfoxide in water. Stock solutions of ciprofloxacin and ciprofloxacin-d8 were similarly prepared at 1,000 μg/mL in water. All stock solutions were stored in –20 °C freezer.

Working solutions were prepared by appropriate dilutions of the concentrated stock standard solutions with 0.1% formic acid, 30% acetonitrile in water.

Sample preparation.

An aliquot of 100 μL drug-containing rat plasma was transferred into a 1.5-mL micro-test tube with the addition of 300 μL of extraction solution (acetonitrile containing 0.1% formic acid, 0.2% TFA and internal standards of 2 μg/mL of enrofloxacin-d5, and 2 μg/mL ciprofloxacin-d8). Samples were then vortexed, placed on ice for 30 min, and then centrifuged at 14,000 × g for 15 min at 4 °C. An aliquot of 100 μL of supernatant was mixed with 125 μL of 0.1% formic acid in water. Subsequenlty, 20 μL of the sample solution was used for liquid chromatography-mass spectrometry.

Chromatographic conditions.

The separation of analytes was performed on an Acquity UPLC HSS T3 (2.1 mm × 150 mm; Waters, Inc., Framingham, MA) column with a 1.8-μm particle size, preceded by a 1.8 μm Waters Acquity UPLC HSS T3 (2.1 mm × 5 mm) guard column. The analytical column was maintained at 45 °C. Mobile phase, consisting of a gradient mixture of 0.1% vol/vol formic acid in water (solvent A) and 0.1% vol/vol formic acid in acetonitrile (solvent B), was used at a flow rate of 0.25 mL/min for separation and rapid elution of analytes within 8.0 min. During the first 3 min of the gradient, percent solvent B increased from 30% to 95% where it was kept for 0.5 min and then decreased to 30% and kept to reequilibrate the column until 8.0 min.

Mass spectrometric conditions.

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consisted of an Ultimate 3000 HPLC system coupled to a TSQ Quantis triple quadrupole mass spectrometer (ThermoFisher Scientific, Framingham, MA), which was located in the Institutional Research Core Facility. Mass spectrometric detection and quantitation of all analytes were carried out using multiple-reaction monitoring scan in electrospray positive ion mode. The following multiple-reaction monitoring transitions were selected: (m/z, Q1→Q3) of enrofloxacin (m/z, 360.2→316.1), d5-enrofloxacin (m/z, 365.2→321.2), ciprofloxacin (m/z, 332.2→288.1), and d8-ciprofloxacin (m/z, 340.2→296.1). Source temperature and gas parameters were optimized after the chromatographic conditions were finalized. All peak areas and calculations were performed using Skyline software (MacCoss Lab at the University of Washington).

Analysis method.

Pharmacokinetics.

The C_max and T_max were recorded directly from the data (highest value and time at highest value, respectively). The t_1/2 was calculated using the C_max, the concentration at 48 h, and the formula N_t = N₀e^−λt, where N₀ is the initial quantity, N_t is the remaining quantity after a given time (t), and λ is the decay constant. The C_max, T_max, and t_1/2 were compared by route of administration. Normally distributed data sets were compared by multiple paired t tests; nonnormal data were compared by the Wilcoxon matched-pairs signed rank test. P values were corrected for multiple comparisons on the same animals using the Holm-Sidak method. Analyses were performed in GraphPad Prism version 10.1.2 (San Diego, CA).

Mixed-effects models.

To determine the relative effect of administration route, time, and sex on serum levels of enrofloxacin and ciprofloxacin, 2 repeated measures mixed-effect models were used. The models incorporated within-subject correlation and interaction effects were tested to discover any confounding effects. Following model testing, pairwise comparisons of groups were conducted using the Bonferroni correction to account for multiple comparisons within the same data set. In all initial models, interaction effects were not significant. Final models therefore included only administration route, time, and sex. The analysis was performed using SAS 9.4. Significance (α) was set to 0.05; all P values are reported as corrected P values.

Results

Data is sparse regarding effective serum concentrations of enrofloxacin in nonhuman primates; however, previous studies^11,12 demonstrate various potential markers for efficacy. Klein et al¹¹ cite a minimum inhibitory concentration (MIC) of less than or equal to 0.2 μg/mL for enrofloxacin across 90% of gram-negative organisms. Before this, Küng et al¹² published MIC values for enrofloxacin and ciprofloxacin against common agents in the veterinary field, which show values ranging from less than 0.015 to 1.0 μg/mL across bacterial species. Both studies^11,12 note the additive properties of enrofloxacin and its metabolite, ciprofloxacin. While some organisms are minimally susceptible to enrofloxacin, many show increased susceptibility to ciprofloxacin. For this reason, both compounds were assessed throughout this study to ensure no variations in reported metabolism were observed.

Pharmacokinetic results.

Intramuscular administration.

Intramuscular administration resulted in an initial rise within the first 8 h of the study, followed by a downward trend between 8 h and 24 h (Figure 2B and D). In contrast to oral administration, 3 (2 females and 1 male) of the 6 animals displayed peak serum levels at the 8-h time point, while 3 (1 female and 2 males) showed peak levels at the 2-h time point. Ciprofloxacin C_max levels mirrored those of enrofloxacin in each animal, achieving C_max at similar time points.

Figure 2.

Serum concentrations of enrofloxacin and ciprofloxacin in individual animals. Following both oral and intramuscular administration, serum concentrations of both enrofloxacin and its metabolite, ciprofloxacin, rose to C_max at the 2- or 8-h time point. By 48 h, enrofloxacin serum concentrations had fallen to (A) an average of 275 ng/mL when the antibiotic was administered orally and (B) an average of 185.5 ng/mL with intramuscular administration, and all animals still had serum enrofloxacin levels at least 50 times higher than the most conservative estimate of MIC (1 ng/mL; minimum enrofloxacin level: 52 ng/mL). By 48 h, ciprofloxacin levels had fallen to an averages of 25.9 and 18.2 ng/mL with (C) oral and (D) intramuscular administration, respectively. Mixed-effects modeling found average enrofloxacin levels were significantly different in all time comparisons except 0 and 48 h and between 2 and 24 h. Ciprofloxacin levels differed at all time points except between 0 and 48 h and between 2 and 8 h. Sex had no significant effect on serum concentration of either enrofloxacin (P = 0.30) or ciprofloxacin (P = 0.81). Dotted lines: male animals; solid lines: female animals.

Maximum serum concentrations of enrofloxacin were obtained at 5.0 ± 3.29 h (T_max) with a peak C_max of 1,802.76 ± 1,003.09 (Table 1). Average enrofloxacin levels in males and females at T_max were 1,377 and 1,720 ng/mL, respectively. Maximum serum concentrations of ciprofloxacin were observed at 5 ± 2.55 h (T_max) with the peak reaching 136.08 ± 75.9 ng/mL (C_max). The t_1/2 for both compounds when administered intramuscularly were nearly identical, with enrofloxacin halving every 13.29 ± 2.93 h and ciprofloxacin at 14.42 ± 2.55 h. C_max, T_max, and t_1/2 did not differ between oral and intramuscular administration routes (P > 0.08 in all comparisons).

Table 1.

Median data collected from each arm of the study

Pharmacokinetic parameter	Ciprofloxacin		Enrofloxacin
	Intramuscular group	Oral group	Intramuscular group	Oral group
t1/2 (h)	14.42 ± 2.55	15.07 ± 2.41	13.29 ± 2.93	11.64 ± 2.13
Tmax (h)	5.0 ± 2.55	8.0 ± 0.0	5.0 ± 3.29	7.0 ± 2.45
Cmax (ng/mL)	136.08 ± 75.90	163.43 ± 56.40	1,802.76 ± 1,003.09	2,770.21 ± 1,027.57
AUC all (h·ng/mL)	3,254.62 ± 1,815.49	4,362.02 ± 1,329.34	38,466.11 ± 19,722.61	64,747.47 ± 26,543.11

Oral administration.

Oral administration resulted in a similar trend across most animals within each time point (Figure 2). Specifically, serum levels of enrofloxacin (Figure 2A) and ciprofloxacin (Figure 2C) increased between the 0 to 8-h time frame and trended downward sometime following the 8-h mark. All animals displayed comparable decreases in serum levels by the 48-h sampling window, resulting in levels approaching 0 by the end of sampling. Five of 6 animals displayed peak serum concentrations of enrofloxacin at the 8-h sampling window, with the 1 outlier (female) showing peak levels at the 2-h time with concentrations decreasing over the course of the series. C_max trends for ciprofloxacin were like those noted with enrofloxacin (Figure 2), peaking alongside the enrofloxacin in all animals save one that showed an increase in ciprofloxacin from 2 to 8 h that correlated with a decrease in enrofloxacin concentration at that time. In our mixed-effects model, both enrofloxacin and ciprofloxacin levels were significantly different in all time comparisons except the following: enrofloxacin levels did not differ between 0 and 48 h and between 2 and 24 h; ciprofloxacin levels did not differ between 0 and 48 h and between 2 and 8 h. Sex had no significant effect on serum concentration of either enrofloxacin (P = 0.30) or ciprofloxacin (P = 0.81).

Maximal serum concentrations of enrofloxacin were achieved at 7 ± 2.45 h (T_max), with a peak serum C_max of 2,770.2 ± 1,027.6 ng/mL. If viewed by sex, males average 1,857 ng/mL and females 2,797 ng/mL. Ciprofloxacin levels showed a similar T_max of approximately 8 h and C_max of 163.43 ± 56.40. Both compounds show similar relative t_1/2 with enrofloxacin decreasing at 11.64 ± 2.13 h and ciprofloxacin at 15.07 ± 2.41 h.

Importantly, our mixed-effects models found that oral administration of enrofloxacin resulted in significantly higher levels of ciprofloxacin than intramuscular administration (Figure 3D, Bonferroni P = 0.016), even when accounting for sex and time. This effect was confined to ciprofloxacin levels; enrofloxacin serum levels did not differ by administration route (Figure 3C).

Figure 3.

Pharmacokinetic values and average serum concentrations of enrofloxacin and ciprofloxacin. The C_max, T_max, and t_1/2 values for both (A) enrofloxacin and (B) ciprofloxacin did not differ between oral and intramuscular administration. Oral administration appeared to induce greater serum concentrations of both (C) enrofloxacin and (D) ciprofloxacin, but this effect was only significant for ciprofloxacin (mixed-effects model, Bonferroni P = 0.016).

Serum concentrations from both the intramuscular group and the oral group show all animals to be in excess of the MIC (0.3 μg/mL; dotted line in Figures 2 and 3) for enrofloxacin at all sampled times with animals showing a reduction to 0.1 to 0.6 μg/mL at 48 h. Ciprofloxacin showed similar but slightly shortened duration with animals approaching levels of 0.02 to 0.1 μg/mL at 24 h and 0.005 to 0.045 μg/mL at 48 h.

Discussion

A successful antibiotic regimen must be carefully developed with consideration for species, age, target region, suspected agents of risk, and overall physical condition of the animal.^4,5 Bacterial culture and sensitivity results should be obtained to verify the efficacy of any plan.^2,4–7 Failure to adequately plan or using arbitrary antimicrobials that are ineffective for a given agent may result in increased complication rates, presence and proliferation of resistant bacterial strains, and negative welfare to the animal undergoing care.^2,4,5,8,13 Judicious use of antibiotics requires a thorough history and understanding of the particular factors for a given case, and recommendations are to ensure efficacy of a given therapeutic before administration. In cases of rapid need, selection of a broad-spectrum antibiotic may serve to provide initial intervention while awaiting results of a bacterial culture and sensitivity testing.

For a concentration-dependent antibiotic, a standout characteristic for evaluation of suitable administration is the C_max.^5,6,9,10,15 Values exceeding the MIC for a given agent correlate with antimicrobial efficacy and attest to the viability of interventional use, presuming supporting sensitivity data is present. The duration that C_max exceeds MIC translates to the duration that a potential pathogen is exposed to the antimicrobial and increases the overall success of therapy. Our work shows that both intramuscular administration and oral administration resulted in serum levels exceeding the generally accepted MIC for many pathogens.^11,12 Beyond that, when compared with intramuscular administration, oral dosing resulted in equal or greater serum concentrations of both enrofloxacin and ciprofloxacin at all time points evaluated, save for one animal that displayed higher serum enrofloxacin in the intramuscular group at 2 h. While the increase in serum levels of enrofloxacin was not determined to be significant, it meets the criteria for noninferiority. This may indicate higher oral bioavailability of this product compared with intramuscular injection, though our data do not possess the breadth to confirm that here. Overall, these data support our hypothesis that oral administration of the injectable enrofloxacin compound is noninferior to intramuscular administration of this same compound.

One factor that was considered to be potentially confounding was the reproductive status of the females. As is typical of many female mammals, hormonal changes that occur during ovulation or reproductive cycling may influence homeostasis.^1,2 This may result in variations to antimicrobial metabolism and skew data when compared with males or noncycling females. Throughout procedures on this study, all female cycling was tracked by means of a visual scoring system. During the intramuscular arm of the study, 1 female was noted to be at the peak of her cycle (coinciding with the peri-ovulatory stage) between days 1 and 2 with the remaining 2 females existing in a noncycling state. During the oral administration arm, these 2 noncycling females were cycling while the individual from the intramuscular study was not. This allowed us the benefit of seeing comparative numbers in similar individuals between hormonal states. Data reflected no obvious impact on the serum levels noted across females on either arm of the study. All animals displayed some variance in serum concentrations and T_max between samples from the oral and intramuscular administration groups, but this did not correlate with reproductive status.

We aimed to show through our work that administration of the injectable formulation of enrofloxacin via oral dosing was noninferior to administration via intramuscular injection. A major benefit of our work is that it informs acceptability of both oral or intramuscular dosing of enrofloxacin, in contrast to the limited previous studies which showed intramuscular administration to be the only effective known option. While our team has incorporated positive reinforcement training for the animals, this task would be more effective if with the ability to remove the noxious stimulus (needle stick) from the desired activity. In cases where a long antibiotic regimen is necessary, use of novel food items containing this antibiotic may be considered as a refinement to injectable administration. While this study required the use of intramuscular injection to facilitate comparison of these 2 administration routes, the resulting data will suppport use or oral enrofloxacin administration versus a procedure requiring needle-sticks (intramuscular administration). This would also allow development of treatment options tailored to an individual animal based on preference testing.

While we selected a standardized group of animals, aiming to reflect “healthy, normal,” young adults, the small sample size for our study may limit certain conclusions from being drawn. Specifically, additional animals and/or time points may have allowed statistical significance to be obtained for serum enrofloxacin levels in the oral administration group compared to those in the intramuscular administration group, as these animals routinely showed higher serum levels but not to the point of significance. In addition, both groups displayed a T_max of 5 ± 3 h, which may have been more accurate had we collected samples hourly across the first 12 h, followed by larger gaps to determine the end of the peak and subsequent clearance of the compounds. Due to the welfare impact of sedation lasting 12 h, the authors chose to approach in a more conservative manner, aiming to prove noninferiority rather the determining the full extent of superiority. Use of indwelling vascular access ports or tether systems would have facilitated direct sampling across a larger number of time points without additional sedation; however, this was beyond the scope of the present project.

Further work on this subject may include determinations of pharmacokinetic effects in young or aging baboons and the impact of pregnancy on enrofloxacin serum levels. One consideration might be ts look into specific preparations of novel food items containing enrofloxacin and the effect on absorption, biodistribution, and serum levels. There is little literature evaluating the interactions of specific food items on the bioactivity of enrofloxacin; however, it has been noted⁷ that an increase in interference in absorption occurs when interacting with cations (Ca²⁺, Mg²⁺, etc). General recommendations are for enrofloxacin to be administered on an empty stomach; however, clinical guidance denotes the potential for nausea, and small food quantities are supported in the face of this effect.⁷ Food items containing high levels of cations should be avoided to minimize disruption of systemic absorption.⁷ Disruption to absorption occurs as charged cations serve as chelating (binding) agents for the administered drug and, thus, prevent metabolism by the body and reducing total levels received. When crafting a therapeutic containing enrofloxacin, whole food items should be given preference as the level of free, charged cations is generally low. Foods with added salts, preservatives, or chemicals may increase the levels of free cations, thereby increasing binding and decreasing absorption. Common produce items such as bananas, apples, and melons are considered safe to give in conjunction with enrofloxacin. While potassium is a cation, it is not considered as a risk for chelation. Other potential options would be whole grains such as oatmeal, which can be flavored with fresh or frozen fruit to mitigate the taste of the enrofloxacin.

Additional information regarding prolonged administration and incidence of long-term physiologic or metabolic effects may be beneficial for identifying markers of toxicity in case of this need, but as this was performed with standard clinical application in mind, it was not addressed in the current work. Serum biochemistry markers showed no variation in evaluated metrics for short-term use. This does not verify the lack of adverse effects associated with enrofloxacin administration in baboons; however, prior work has identified the potential for gastrointestinal irritation in horses when administered orally.⁵ Placement directly into the gastric lumen via oral gavage likely would have avoided any potential for this to occur, but use in a general setting may consider this possible effect.

While we did not obtain data showing statistically significant differences for the oral administration group, the trend indicates that oral administration may result in higher serum levels for an individual. Additional work would be needed to verify this conclusion. Regardless, the knowledge of the similarities in C_max, T_max, and susceptibility profiles for enrofloxacin and ciprofloxacin allows potential users to create valid conclusions from this work. Enrofloxacin is effective when administered at dosing of 10 mg/kg orally to members of the P. anubis species. Ciprofloxacin, potentially the more impactful of the 2 compounds, shows a significant increase in serum concentrations following oral administration compared with intramuscular administration, which may be of benefit when complex infections are present, or if agents only possess moderate susceptibility to enrofloxacin. The clearance period for each compound was similar, which allows for inference of effective dosing timeline, suggesting that one dose, administered every 24 to 48 h dosing is adequate for maintaining serum levels above reported MIC values. While this may support decreased frequency of dosing, additional testing of this methodology using food or fluid-based, conscious administration should be performed to assess potential variation in absorption. Once daily dosing is still recommended by these authors.

In summary, this work demonstrates options for effective enrofloxacin administration to P. anubis. Enrofloxacin is a powerful tool to be used in clinical care of NHPs, and our results support effective methods of enrofloxacin administration which promote animal welfare. By demonstrating noninferiority between intramuscular and oral injection of an injectable enrofloxacin preparation, we anticipate improved nonhuman primate care that will incorporate creative thinking and species-specific considerations when designing patient care plans.

Funding

This work was internally funded by University of Oklahoma Health Sciences Center Division of Comparative Medicine with aims of improving patient care and clinical efficacy. This team has no conflicts of interest to declare associated with this work.