Challenges in oral administration of clioquinol dispersed in gel to pair-housed rhesus monkeys (Macaca mulatta)
Bakker J1§, Klomp R1§, te Poele EEM2, Vis J1, Langermans JAM1 ,van Geijlswijk IM2
1Biomedical Primate Research Centre (BPRC), Lange Kleiweg 161, 2288GJ Rijswijk, THE NETHERLANDS;
2Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, THE NETHERLANDS
§ authors share first authorship
Summary
Entamoeba spp. is a common protozoa in rhesus monkeys (Macaca mulatta) and humans (Johnson-Delaney, 2009; Levecke et al., 2007). This parasite can affect the health status of macaques as the infection can cause severe diarrhoea (Johnson-Delaney, 2009). Therefore, in some cases, treatment can be mandatory. Metronidazole is the most common treatment for Entamoeba spp. in veterinary medicine (Jiang et al., 2008; Wright 2012). To handle the animals as little as possible, oral administration of the medication in e.g. drinking water would be optimal. However, the administration of metronidazole in drinking water was not successful in rhesus monkeys due to low fluid intake attributed to undesirable taste (Labberton et al., 2013). We investigated whether another anti amoebic medicine, clioquinol, administered via drinking water could be used to treat rhesus monkeys. A cross-over study was designed in which water, medicated fluid and non medicated fluid was provided to macaques. The normal daily water intake of 16 macaques over a 27-day period was determined. Secondly, it was investigated whether the addition of clioquinol dispersed in gel influenced the daily fluid intake. Furthermore, it was determined if the monkeys drank enough medicated fluid to receive a dose of 20 mg/kg/day clioquinol. The mean water uptake was 78.8 ml/kg. Clioquinol addition resulted in a statistically significant decrease of 15.7 ml/kg in fluid intake. The mean intake of the non-medicated fluid compared with water intake did not significantly differ. The mean intake of clioquinol for five days was 22.4 mg/kg. This study shows that it is possible to administer clioquinol in gel to rhesus monkeys in the desired dose.
Introduction
Entamoeba histolytica and Entamoeba dispar are well described in primates (Toft and Eberhard, 1998). 1 These amoebae are facultative pathogenic and asymptomatic carriage is common. 1, 6 Faecal samples from rhesus monkeys housed at the Biomedical Primate Research Centre (BPRC, Rijswijk, The Netherlands) are yearly screened for Entamoeba spp. The number of positive faecal samples for Entamoeba spp. in 2010, 2011, 2012 and 2013 were 5% (n=628), 11% (n=1020), 18% (n=1134) and 2 % (n=926) respectively (unpublished data). In some cases, infected rhesus monkeys experienced acute severe diarrhoea requiring immediate treatment.
There is no authorized veterinary medicine product for the treatment of intestinal protozoa in non-human primates. The selection of medicine is based on experience and on literature reports, although evidence-based information on effectiveness and dosages is often lacking. Treatment options for Entamoeba spp. infections include clioquinol, tinidazole and the most common used metronidazole (Johnson and Delaney, 2009; Wright, 2012). However, medicine compliance via drinking water of the latter is difficult to ensure because of the unpleasant metallic taste of metronidazole. A decrease has been described in fluid intake after metronidazole addition in rhesus monkeys even after flavouring with syrup (Labberton et al., 2013). Although it was possible to administer metronidazole to some rhesus monkeys, it was concluded that this was not a clinically effective treatment (Labberton et al., 2013). Clioquinol could be a better option for the treatment of Entamoeba spp. in group-housed rhesus monkeys (Bareggi and Cornelli, 2012; Schure et al., 2013) because of the more neutral taste. Clioquinol is available as a pharmaceutical compounding oral suspension of 100mg/ml. However, it was decided to prepare a clioquinol medicated fluid of 0.3 mg/ml. As clioquinol is practically insoluble in water it was decided to prepare another formulation by pharmaceutical compounding: the viscosity of the drinking water was increased by the use of carboxymethylcellulose 0.5%.
This study investigated whether clioquinol dispersed in a gel could be successfully administered to pair-housed rhesus monkeys. Firstly, the normal water intake of 16 rhesus monkeys over a 27-day period was determined. Secondly, non-medicated fluid (carboxymethylcellulose 0.5%) and clioquinol (0.30 mg/ml) dispersed in a gel (carboxymethylcellulose 0.5%) was offered and daily intake was measured. It was determined whether the addition of clioquinol dispersed in gel influenced the daily fluid intake and if the amount of fluid intake was high enough to ensure the dose of 20 mg/kg/day clioquinol.
Materials and methods
Animals, housing and care
This study included sixteen rhesus monkeys (Macaca mulatta) that originated from and were housed at BPRC: eight healthy adult females (aged 5-13 years, weighing 5.35-12.39 kg) and eight healthy males (6-9 years, weighing 7.4-11.67kg). Males and females were housed in separate rooms. All macaques were pair-housed in the experimental facility. The rhesus monkeys had no history of oral treatment with clioquinol. Their diet consisted of one slice of bread around 8.30 a.m., 150 g of commercial monkey pellets (Sniff-Spezialitäten Gmbh, Soest, Germany) around 11 a.m., enrichment at 1 p.m. (Vernes and Louwerse, 2010) and 150 g fruits or vegetables around 3 p.m.
During this study the drinking nipples in the cages were shut off and 600 ml drinking bottles (crystal de luxe 600 ml, Caldex ltd, England) were placed in custom-made holders attached to the animals cages, as monkeys are known to destroy standard holders (Labberton et al., 2013).
Drugs
Clioquinol (5-chloro-7-iodo-quinolin-8-ol, iodochlorhydroxyquin) is a luminal amoebicide. It has been shown to possess antiprotozoal activity. The exact mechanism is unknown (Mao and Schimmer, 2008; Nagata et al., 2012; Schure et al., 2013). The oral dose of clioquinol in humans varies between 15 mg/kg body weight (mg/kg bw) per day for children and 750 mg per day for adults (Geneesmiddel Informatie Centrum van de KNMP, 2013). Clioquinol is used in rhesus monkeys with a dose of 20 mg/kg bw per day over a period of 5 days (Personal communication H. Bolhuis). As daily fluid intake among rhesus monkeys varies between 49 ml and 115 ml/kg per day with an average of 75 ml/kg per day (Labberton et al., 2013), it was decided to prepare a clioquinol medicated fluid of 0.3 mg/ml so that an average fluid intake of 66.7 (0.3*66.7=20) ml/kg per day would already be adequate to administrate the desired dose of 20 mg/kg clioquinol.
Clioquinol is available as a pharmaceutical compounding oral suspension of 100mg/ml (Geneesmiddel Informatie Centrum van de KNMP, 2013). Clioquinol is practically insoluble in water (Martindale and Reynolds, 1996). Therefore, dissolution in drinking water is impossible and dispersion is challenging because of immediate precipitation of the clioquinol. It was decided to prepare another formulation by pharmaceutical compounding. The viscosity of the drinking water was increased by the use of carboxymethylcellulose 0.5% (carmellosum natricum, Spruyt Hillen, Netherlands) (Geneesmiddel Informatie Centrum van de KNMP, 2013). Due to the higher viscosity the clioquinol remained homogeneously distributed in suspension for at least 16 hours, as demonstrated in a precipitation experiment (unpublished data). The non-medicated fluid consisted of 0.5% carmellosum natricum and 99.5% drinking water. The final medicated fluid consisted of 0.5% carmellosum natricum and 0.03% clioquinol (clioquinolum, Spryut Hillen, Netherlands) and 99.47% drinking water. To ensure that the fluid consisted the exact amount of 0.3 mg/ml clioquinol the 5% gel was diluted per 50 ml into a 1 litre bottle.
Experimental design
Normal water intake of all animals was determined for 27 days. Each cage housing two animals received at 9.00 a.m. and 17.00 a.m. two bottles of 600 ml fresh drinking water. At 12.00 a.m. the bottles were checked on content: if the volume was less or equal to 200 ml in one bottle than the bottle was refilled with 600 ml fresh drinking water.
A cross-over study was performed in which the animals were provided with medicated fluid and non-medicated fluid. The monkeys were divided into two groups. Four couples received medicated fluid and four couples received non-medicated fluid for the first consecutive five days and thereafter vice versa. Between these two different trials the rhesus monkeys received two days normal drinking water in drinking bottles. The monkeys received twice a day at 9.00 a.m. and 17.00 p.m. fresh filled bottles with the specific fluid. At 12.00 a.m. the bottles were checked on the content, if the volume was less or equal to 200 ml the bottle was filled with the specific fluid for that particular couple. All administered fluids were measured with a 1000 ml measuring cup (OXO Good Grips, New York).
In addition, faecal samples were collected on three consecutive days and examined one week before the medication and one week after receiving the medicated fluid. The faecal samples were directly fixed in Sodium Acetate-Acetic Acid Formalin (SAF). From the fixed SAF faeces DNA was isolated. This DNA was screened with the PCR for Entamoeba spp. (Fotedar et al., 2007; Jiang et al., 2008; Petri, 2003).
During the study, the rhesus monkeys were under close veterinary supervision. The procedures performed in this study were in agreement with the regulations for animal handling as described in the EU Directive 63/2010 and with the Weatherall report (2006).
Statistical analysis
Data were collected in Excel spread sheets (Microsoft Corporation, Redmond, USA) and average intake per pair of monkeys per week was determined. These averages were transferred for graphing and statistical evaluation in Prism 6 for Mac OS X, version 6.0c (Graph Pad Software). To determine if water intake reached a baseline, a regression line was drawn for all couples and inclination was calculated. The differences in fluid intake were initially compared using paired t-tests. To test whether there was a difference between the mean intake of medicated fluid over five days and the desired volume for 20 mg/kg a one-sample t-test was used. P values of <0.05 were considered statistically significant.
Results
Daily intake of water was measured over a period of 27 days. The mean water intake was 78.8 ml/kg bw. However, the water intake showed a significant increase of 9.6 ml per week over those 27 days. It was decided to use only the last 5 days of these 27 days to calculate the mean water intake (90.5±29.1 ml/kg bw) and compare these values with the intake of medicated and non-medicated fluid.
The mean intake of medicated fluid was 74.7±37.5 ml/kg bw and the mean intake of non-medicated fluid was 82.6±41.28 ml/kg bw. Monkeys consumed on average 15.7 ml/kg less medicated fluid in comparison to water intake (p=0.0329). Monkeys drank on average 7.9 ml/kg bw less non-medicated fluid vs. water (p=0.1875). Monkeys drank on average 7.8 ml/kg bw less medicated fluid vs. non-medicated fluid (p=0.2307) (figure 1, 2).
The average intake of medicated fluid was 74.7 ml/kg. The average intake of clioquinol was 22.4 mg/kg bw. This mean value did not significantly differ from 20 mg/kg (p=0.5624) (figure 3).
All faecal samples taken before and after clioquinol treatment were negative for Entamoeba spp.
Fig. 1: Comparison of water and non-medicated fluid intake ml per kg bodyweight. The couples showed a decrease of 7.9 ml/kg bw between water and non-medicated fluid.
Fig. 2: Comparison of average non-medicated fluid and medicated fluid intake ml per kg bodyweight. The couples showed a decrease of 7.8 ml/kg bw between non-medicated and medicated fluid.
Fig. 3: Mean daily intake clioquinol (mg/kg bw) per couple for five days. Circle sign: female macaques, square sign: male macaques.
Discussion
This study investigated whether replacement of drinking water by medicated fluid resulted in sufficient intake of the medication in rhesus monkeys. It showed that it is possible to administer 20 mg/kg for five consecutive days to rhesus monkeys. The monkeys had a mean intake of 22.4 mg/kg bw clioquinol in five days however the inter- and intra variation between monkeys make it difficult to ensure that all individual monkeys drank enough of medicated fluid for the therapy of Entamoeba spp.
Switching from drinking nipples to drinking bottles is possible. However, the rhesus monkeys may require a longer adaptation period to reach a steady water uptake per kg body weight per day. Our observations suggest that more than four weeks are required for habituation to drinking bottles. However, after four weeks the animals still did not reach a steady state phase. Instituting a longer acclimation period for the macaques to become accustomed to the drinking bottles before beginning the study could have addressed the lack of familiarity. In the case of acute illness demanding immediate treatment, such as severe diarrhea resulting from intestinal protozoan infection, such a long acclimation period is not feasible.
The daily fluid intake of the eight pairs during the last five days of the 27 days water intake measurement was higher than expected on the basis of literature reports ±90 ml/kg versus 75 ml/kg bw. This discrepancy could result from differences in diet, housing conditions and other variables or from the macaques’ unfamiliarity with the drinking bottles.
Further research is needed to evaluate the effective dose of clioquinol in rhesus monkeys. In our study a doses of 20 mg/kg/day is used for 5 days. However, to what extent achieving 20 mg/kg is relevant is not clear, because there are no pk/pd studies performed for clioquinol in rhesus monkeys.
The results showed that it is possible to administer clioquinol dispersed in a gel in drinking bottles to rhesus monkeys. This strategy is not yet a reliable method because of wide variation of fluid and clioquinol intake between pairs. However, drinking water medication leads by definition to concessions to the individual dosing because of the inter- and intra variation. The risk on side-effects due overdosing is low as neurological toxicity has only been observed in animals when treated with clioquinol at doses exceeding 200 mg/kg per day for a month and doses of 400 mg/kg per day for a week (Mao and Schimmer, 2008; Worden et al., 1978).
Acknowledgement
The authors gratefully thank the help of the caretakers.
References
BAREGGI SR, CORNELLI U (2012): Clioquinol: Review of its Mechanisms of Action and Clinical Uses in Neurodegenerative Disorders. CNS Neuroscience & Therapeutics 18, 41-46.
FOTEDAR R, STARK D, BEEBE N, MARRIOTT D, ELLIS J, HARKNESS J (2007): Laboratory diagnostic techniques for Entamoeba species. Clin. Microbiol. Rev. 20, 511-32, table of contents.