PHARMACOKINETICS OF KETOPROFEN IN HEALTHY HORSES IN PAKISTAN

INTRODUCTION

NSAID’s  are  frequently  used  and  commonly prescribed in humans and animals for reduction in pain, fever and inflammation in rheumatic problems (Geof et al., 2008). Chemically, ketoprofen (KTP) is 2-(3-benzoyl phenyl) propionic acid. Molecular formula is C16H14O3. KTP exists in two enantiomeric forms S and R, which have different half lives. KTP is formulated as racemic mixture.  The  S(+)  enantiomer  is  associated  with prostaglandin  inhibition  and  toxicity  and  the  R(-) enantiomer is linked with analgesic action and protection of  gastrointestinal tract. The S(+) isomer, due to chiral inversion dominated in cats, dogs and horses. The R(-) isomer, dominated in sheep. Ketoprofen is approved for use in horses and advised in the inflammatory and pain conditions  associated  with  musculo  skeletal  problems (Huskisson  et  al.  1996).  Ketoprofen  has  a  wide  safety margin and low toxicity compared to phenylbutazone and flunixin  meglumine  in  horses  (Mozaffari  et  al.,  2010). Ketoprofen  has  comparatively  a  better  gastro  intestinal safety  and  has  less  adverse  events  than  aspirin, phenylbutazone  and  flunixin  meglumin  in  horses (MacAllister  et  al.  1993).  Favourable  pharmacokinetic profile of ketoprofen in humans made it a suitable and effective NSAID for veterinary use. The present project was  designed  to  investigate  the  pharmacokinetic parameters  of  KTP  in  local  Pakistani  horses  after intravenous administration at the dose of 3mg/kg body weight so  that  we could recommend dosage regime in

this particular specie.

MATERIALS AND METHODS

Animals:  Eight  healthy  male  horses  with  an  average weight of 315kg were used in this study. All the horses were  tagged  and  acclimatized  to  the  experimentation area. Food was provided to animals along with water ad libitum. Experimental animals were monitored regularly regarding their health throughout the experiment.

Drugs and Chemicals: Ketoprofen standard (Sigma) was purchased for use as external standard from the company. HPLC grade water, di ethyl ether, acetonitrile (E. Merck Germany)  was  used.  Injection  (20ml)  of  Ketoprofen manufactured by Selmore Pharmaceuticals (Pvt) Limited, Lahore, Pakistan  were used during experiment. All the chemicals used were of analytical grade.

Treatment protocol, sampling and drug analysis: An I/V  dose  of  Ketoprofen  @  3  mg/kg  body  weight  was administered via jugular vein into horses. Blood samples (5 ml) were collected in test tubes prior to medication and then at 0.08, 0.17, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0,  10,  12,  24,  48,  60,  72,  84  and  96  hours  post medication.  Separation  of  plasma  was  made  by centrifugating  blood  at  4000  rpm  for  10  minutes  and stored at −80°C till analyzed.

Extraction of drug: 1.0 ml of 1.0 M hydrochloric acid was added to 1.0 ml of plasma. Vortexed it for 1 minute and then 1.0 ml of HPLC grade di-ethyl ether was added. Vortexed the material again at high speed vortex (4000 rpm) for 10 minutes. The clear supernatant was taken and evaporated  to  dryness.  The  dried  portion  was

reconstituted  with  1.0  ml  of  mobile  phase  (buffer: acetonitrile 75: 25). Filtered it through 0.22 μm syringe filter and analysis was made.

HPLC analysis: HPLC analysis of ketoprofen in plasma was  made  with  already  developed,  standardized  and validated  HPLC  method  by  using Shimadzu  LC2000, equipped with a LC-20AT VP pump, a SIL-20AC HT auto-sampler, SPD-M20A, CTO 20 AC and CBM 20A control  unit.  20  μl  of  the  sample  was  run  into  HPLC system via auto sampler with flow rate of 1 ml/minute. Mixture  of  phosphate  buffer  (Di-potassium  hydrogen phosphate)  and  acetonitrile  (75:25  v/v)  was  used  as mobile phase. A reversed phase C18 column  (Thermo, BDS  Hypersil.  5 μm;  4.6 mm × 250 mm)  was  used  as stationary phase. Separation was achieved at 7.0 minutes (retention  time)  at  wavelength  of  254nm.  Oven temperature was set at 30°C. Ketoprofen in sample was compared with ketoprofen external standard. The discrete peak  found  in  chromatograms  of  plasma  samples  of horses  was  similar  to  the  peak  in  chromatogram  of ketoprofen (external standard) at 7.0 minutes of retention time.  The  limit  of  quantification  was  0.125μg.  The plasma  concentration  (μg/ml)  versus  time  profile  of ketoprofen in horses was prepared semilogarithmically.

Pharmacokinetic  Analysis:  The  computer  software APO pharmacological analysis MW /PHARM,  Version 3.02,  (Holland,  1987)  was  used  for  calculation  of pharmacokinetic  parameters  through  standard  two compartmental  analysis.  Pharmacokinetic  parameters were calculated for horses by use of statistical moment theory. The following equations were used for different calculations: Dose= Cl x AUC

Cl = Dose/AUC; AUMC = MRT x AUC; 

Where, AU C = Area under the curve, Cl = Clearance,

MRT = Mean residence time and AUMC =Area under the first moment curve.

Statistical  analysis:  The  software  SPSS  (Statistical Package  for  the  Social  Scientists)  13.0  was  used  for statistics. The values in the raw data were expressed as individual  as  well  as  range,  mean,  SEM,  median  and standard deviation.

RESULTS AND DISCUSSION

The  group  means  for  plasma  concentrations (μg/ml)-time (h) data and pharmacokinetic parameters of ketoprofen  were determined  at dose of 3mg.kg-1 body weight in horses after intravenous administration. The I/V dose of 3mg/kg body weight of KTP was selected from literature for administration in buffalo calves to achieve that KTP plasma concentration which was likely to have an  anti-inflammatory,  analgesic  and  antipyretic  effect (Fosse et al., 2010, Neirinckxa et al., 2010, Koshi, 2008, Arifah et al., 2003, Boothe, 2001, Landoni et al., 1999, Paul et al., 1997, Banting  et al.,2008).  The results are given in Table 1and 2. Plasma concentrations (μg/ml)- time profile of ketoprofen was also prepared and is given in Figure 1.

KTP  in  horses  after  I/V  administration  @  3mg/kg body weight. (N=8): The Mean + SEM pharmacokinetic parameters of KTP determined in our local healthy horses in this study are, Area under conc. time curve (AUC)= 9.62  ±  1.580g.h/ml,  body  clearance  (Cl)  =  0.304  ± 0.054 l/h/kg, Volume of distribution (VD) =1.25±0.476 l/kg,  Volume  of  distribution  at  steady  state  (VDss)  =

0.693 ± 0.190 l/kg, plasma half life (t1/2)  =2.665 ±0.658 h, and Elimination constant (Kel)  =2.129 ±0.366 l/hrs.  

Table 1: Group means for plasma conc. time data (µg/ml) of KTP in horses after I/V administration @3mg/kg

body weight. (N=8).

Rehman et al.,  J. Anim. Plant Sci. 22(3):2012

Table 2: Group means for PK parameters of KTP in horses after I/V administration @ 3 mg/kg body weight.

(N=8).

Figure  1:  Semilogarithmic  plot  of  group  means  for

plasma conc. time data (µg/ml) of

The  pharmacokinetic  parameters  like  body clearance (Cl) are comparable with donkeys and mares (Oukessou  et  al.,  1996,  Sams  et  al.,  2008).  The comparatively  large  value  of  Volume  of  distribution (VD) and Volume of distribution at steady state (VDss) may  result  in  fast  elimination  of  drug  in  horses.  The higher drug metabolizing enzyme activities in the liver and other organs of horses  have been linked with fast elimination of KTP like antipyrine, ampicillin (Elsheikh, 1997),  sulphadimidine  (Nouws  et  al.,  1988), isometamidium (Wesongah et al., 2004) and meloxicam (Mehmood  et  al.,2011)  in  goats  as  compared  to  other species. Heavily protein bound drugs have long plasma half  lives  (Sindhu  and  Ram  Pal.,2007).The pharmacokinetic  parameters  observed  in  horses  are different  from  those  reported  in  literature  for  horses, dogs, buffalo calves, humans, and laboratory animals. 

In this study, dose was also calculated by putting values of AUC and Clearance in formula (Dose= AUC x Cl). In horses it was found to be 2.92mg/kg body weight. The dose was rounded off to 3mg/kg body weight. In the present  study,  I/V  dose  of  3mg/kg  body  weight  is suggested in horses. The dose should be repeated after 12

hours in horses. In this study, no toxicity was observed in any animal. 

However,  it  is  suggested  that  clinical  trials  of ketoprofen  should be conducted in  horses to assess its MEC (minimum effective concentration) to calculate its activity  as  an  analgesic  and  anti  inflammatory  agent. Government  of  Pakistan  should  bound  pharmaceutical manufacturers  to  mention  specific  dosage  regimens  of ketoprofen for different species on labels of formulations instead of mentioning a generalized dosage regime.

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