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Pharmacokinetic, metabolism and withdrawal time of orphenadrine in camels (Camelus dromedarius) after intravenous administration
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Research in Veterinary Science 85 (2008) 563–569 www.elsevier.com/locate/rvsc
Pharmacokinetic, metabolism and withdrawal time of orphenadrine in camels (Camelus dromedarius) after intravenous administration M. Elghazali *, I.A. Wasfi, A.A. Abdel Hadi, A.M. Latum Camel Racing Forensic Laboratory, Directorate for Forensic Sciences, P.O. Box 253, Abu Dhabi, United Arab Emirates Accepted 22 January 2008
Abstract The pharmacokinetics of orphenadrine (ORPH) following a single intravenous (i.v.) dose was investigated in six camels (Camelus dormedarius). Orphenadrine was extracted from the plasma using a simple sensitive liquid–liquid extraction method and determined by gas chromatography/mass spectrometry (GC/MS). Following i.v. administration plasma concentrations of ORPH decline bi-exponentially with distribution half-life (t1/2a) of 0.50 ± 0.07 h, elimination half-life (t1/2b) of 3.57 ± 0.55 h, area under the time concentration curve (AUC) of 1.03 ± 0.10 g/h l1. The volume of distribution at steady state (Vdss) 1.92 ± 0.22 l kg1, volume of the central compartment of the two compartment pharmacokinetic model (Vc) 0.87 ± 0.09 l kg1, and total body clearance (ClT) of 0.60 ± 0.09 l/h kg1. Three orphenadrine metabolites were identified in urine samples of camels. The first metabolite N-desmethyl-orphenadrine resulted from Ndealkylation of ORPH with molecular ion m/z 255. The second N,N-didesmethyl-orphenadrine, resulted from N-didesmethylation with molecular ion m/z 241. The third metabolite, hydroxyl-orphenadrine, resulted from the hydroxylation of ORPH with molecular ion m/z 285. ORPH and its metabolites in camel were extensively eliminated in conjugated form. ORPH remains detectable in camel urine for three days after i.v. administration of a single dose of 350 mg orphenadrine aspartate. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Orphenadrine; Pharmacokinetics; Metabolism; Detection; Camels
1. Introduction Orphenadrine (N,N-dimethyl-2(o-methyl-alpha-phenylbenzyloxy) ethylamine is a close structural analogue of diphenhydramine. It is widely accepted as a skeletal muscle relaxant and is used as a therapeutic agent in the treatment of Parkinson’s disease and the neuroleptic syndrome. Orphenadrine inhibits it own elimination when administered by multiple dose regimen and prolongs the half-life of co-administered haloperidol Labout et al. (1982). ORPH is a relatively non-potent inhibitor of the cytochrome P450 (CYP) monooxygenase system that mediates drug biotransformation and undergoes CYP-dependent biotransformation within the alkylamine side chain to generate a potent inhibitory metabolite Bast and Noordhoek (1982). ORPH metabolite binds tightly to the CYP heam, thus *
Corresponding author. Tel.: +971 50 581 7969; fax: + 971 2 446 3470. E-mail address: [email protected] (M. Elghazali).
0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.01.006
generating a metabolite intermediate (M1)-complex similar to those produced by other alkylamines, such as the macrolide antibiotics Murray and Reidy (1990). In adults, orphenadrine is readily absorbed after oral administration and rapidly distributed. Up to 60% of an oral dose is excreted in the urine in three days. In the 24 h following dosage, under uncontrolled conditions, 4% of a dose is excreted as unchanged drug, 5% as N-monodesmethylorphenadrine (tofenacin), 3% as N,N-didesmethylorphenadrine, 3% as orphenadrine N-oxide, 8% as a conjugated of 2-methylbenzhdoxyacetic acid, and 6% as conjugate of 2-methylbenzhydrol. The urinary excretion appears to be dependent on urinary pH Moffat et al. (1986). Data concerning ORPH disposition, metabolism and the minimum effective plasma concentration in camels are not available. The purpose of this work was to: (1) determine the disposition of orphenadrine in camels following intravenous administration; (2) investigate the metabolism
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of orphenadrine in camels; and (3) determine the withdrawal time of orphenadrine in camels. 2. Materials and methods 2.1. Experimental animals Six healthy male camels (Camelus dormedarius), 7–14 years old, approximately 400 kg in body weight were used. The camels were kept in open pens. The camels were determined to be clinically normal before the experiment by physical examination and confirmed by serum biochemical analysis. Good quality hay and Lucerne (alfalfa) were fed to the camels once daily and water allowed ad labitum. The Ministry of Agriculture, Veterinary Department approved this study. 2.2. Experimental design Orphenadrine citrate (401.3 mg equivalent to 350 mg orphenadrine aspartate, Gulf Pharmaceutical Industries, Julphar) dissolved into 15 ml sterile sodium chloride 0.9% ‘‘normal saline”. 15 ml was administered to each camel as a single intravenous bolus into the jugular vein. Lack of pharmacokinetic data for orphenadrine in camels prompted us to use the manufacturer (USO veterinario, industria) clinically recommended dose of orphenadrine in horses. Blood samples (15 ml) were collected in heperinized tubes from the opposite jugular vein at zero (preinjection) and at 5, 10, 15, 30, and 45 min and 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and 24 h after drug administration. The blood samples were centrifuged at 4000g for 10 min. Separated plasma was kept at 20 °C. Samples were assayed within 10 days. Urinary detection time and metabolites were determined in two camels by collecting their urine at zero-time, (blank sample) and at 1, 2, 5, and 8 h (the first day) and at 8:00 a.m. daily for 7 days. Urine collection was done as per Wasfi et al. (1997) briefly, urine samples collected by the use of locally made plastic bags sewn to cotton cloth to give them extra strength. The bag was fastened to the camel via three straps. For male the bag was fitted directly onto the penis. For female camels, a flexible metallic bar was added to the bag. And configure to fit under the tail, separating the anus from the vagina, thus avoiding contamination of urine samples with fecal matter. When bags were half full, they were replaced by new bags. Voided urine transferred to plastic containers and kept in a freezer bending analysis. 2.3. Assay procedures The concentration of the drug in camel plasma was determined by GC/MS. Briefly, ORPH was extracted from 0.5 ml plasma by the addition of 100 ll chlorpromazine, internal standard (10 lg/ml in methanol). Then 100 ll of 1 N sodium hydroxide was added followed by 2.5 ml ethyl
acetate. Samples were agitated on horizontal mixer for 10 min and centrifuged for 10 min at 3500g. Two milliliters of the organic supernatant was removed and transferred to a clean glass tube. The organic layer was evaporated to dryness at 40 °C under a low stream of nitrogen. The residue reconstituted in 100 ll ethyl acetate. Two microliters were immediately analyzed by GC/MS. 2.4. GC/MS analysis GC/MS was carried out using an Agilent Technologies 6890N, net work GC system linked with 5973Network Mass selective detector with 7683B series injector and sample tray. Injections were made in the splitless mode onto a 30 m 0.25 mm i.d. HP-5MS column (Agilent Technologies). The initial oven temperature was 100 °C programmed to rise to 300 at 30 °C min1. Helium was used as the carrier gas. Data were acquired in the selective ion mode (SIM) monitoring the ions m/z 58, 165 and 272, 318 for orphenadrine and chlorpromazine, respectively. The interference of endogenous compounds and drugs was assessed by analyzing standard orphenadrine and chlorpromazine collect drug free plasma from the camels under investigation (six camels) before the start of experiment, plasma spiked with orphenadrine and chlorpromazine and plasma obtained from subjects given orphenadrine. Method proved to be selective for orphenadrine and the internal standard chlorpromazine. The linearity of the method was from 10 to 5000 ng/ml of ORPH in spiked plasma (r2 > 0.995). Maximum recovery was reported in this method. The intra- and inter-assay relative standard deviation and standard errors of mean were used to validate the precision and accuracy of the assay by determining standard samples of orphenadrine in camel plasma. For intra-day validation, six sets of controls at two ORPH concentrations 10 and 1000 ng/ml were assayed with one standard curve on the same run. The range of the relative standard deviation was reported. For inter-day validation six sets of two concentration of the drug in camel plasma were evaluated on three different days. The range of the relative standard deviation was also reported. The intra-coefficient of variation (CV%) for 10 and 1000 ng/ml orphenadrine spiked plasma was 7.33% and 6.59%, respectively. The inter-coefficient of variation (CV%) for 10 and 1000 ng/ml orphenadrine plasma spiked was 10.82% and 5.63%, respectively. The accuracy was determined by comparing the calculated concentration using calibration curve to known concentration. Limit of detection (LOD) and lower limit of quantitation (LLOQ) were determined according to the procedure described by Armbruster et al. (1994). The LOD, defined as the concentration at which all routine GC/MS acceptance criteria (retention time within 2% of calibrator, ion ratios within 20% of calibrator) are met at least 90% of the time, was 2.5 ng/ml. The LLOQ, defined as the lowest concentration above which quantitation can be carried out with adequate accuracy and precision, was 5 ng/ml.
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2.5. Pharmacokinetic analysis
2.7. Extraction and identification of the major metabolites of orphenadrine
Pharmacokinetic analysis was performed, using standard methods and equations Gibaldi and Perrier (1982) and was aided by use of a computer program (PC Modfit, Cambridgeshire, UK). Plasma concentrations were first plotted against time on a semilogarithmic graph to select initial models. Plasma concentrations over time after i.v. administration were analyzed via compartmental pharmacokinetic method. Examination of the semilogarithmic graphs suggested that a one- or two-compartment model, with first order output and bolus input, would be appropriate. All data were fit to these two models. Fitting parameters selected were weighted least squares method using 1/conc weighting scheme, measurement of Akaikes’s information criterion (AIC) Yamoka et al. (1978) was used to determined the model that was best for the data from each specific camel. For the two-compartment open model a biexponential equation was used: Ct ¼ Aeat þ Bebt
ð1Þ
where C is the drug plasma concentration at time equal t, e is the base of the natural logarithm, A is the intercept, and a is the rate constant for the initial steep phase (distribution) portion of the curve; B is the intercept, and a is the rate constant of the terminal (elimination) portion of the curve. For one-compartment open model, a mono-exponential equation was used: Ct ¼ C 0 ekt
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ð2Þ
where C is the plasma concentration at time t, C0 is the initial plasma concentration at time 0, and k is the elimination rate constant (equivalent to kel). After performing this analysis it was discovered that data for all camels could best fit with 2-compartment model. Also, for data fit to a two-compartment model, there were some instances in which the data defined the distribution phase showed problem with few data points on the distribution portion of the curve. Because of these problems, noncompartmental analysis was used in all camels to minimize the need for compartmental assumptions. The primary pharmacokinetics parameters estimated were intercepts A and B, and the rate constants a and b. Secondary variables were calculated by standard equations Gibaldi and Perrier (1982). Area under the plasma concentration versus time curve (AUC) was calculated using the trapezoidal method, with area from the last time point to infinity estimated from the terminal elimination rate constant. 2.6. Determination of drug withdrawal time The detection time for orphenadrine in racing camels was determined by collecting daily urine samples from the treated camels at about 8:00 a.m. for seven days. These urine samples were then subjected to a routine method for screening drugs in post-race urine samples Wasfi et al. (1998a).
The extraction and identification of the major metabolites of orphenadrine was performed on urine samples collected at 0-time (pre-injection sample; blank), 1, 5, and 8 h after drug administration by the method reported elsewhere Wasfi et al. (2000a). 2.8. Statistical analysis of data The value of the pharmacokinetic parameters is expressed as mean ± SEM. 3. Results The GC chromatographic method used in this study showed good separation between the drug and internal standard. Retention times were 7.1 and 9.2 min for orphenadrine and the internal standard chloropromazine respectively (Fig. 1). The total analysis time for each run was 10 min. We were able to analyze more than 60 samples per day. The best recovery of orphenadrine was obtained when the pH of the sample was adjusted to above 10 and this was achieved by addition of 100 ll of 1 N sodium hydroxide solution. The plasma profile of ORPH following i.v. administration is presented in Fig. 2. Pharmacokinetics parameters (mean ± SEM) are presented in Table 1. Pharmacokinetic results for camel six are not consistence with the other five. Camel six showed large distribution phase, with large distribution half-life (t1/2a) 1.5 h, terminal elimination half-life (t1/2b) > 3 d, Vdss was 4.29 l kg1 and total body clearance ClT 9.5604 l h1 kg1. Although, all camels were treated the same and kept under the same conditions. This may be attributed to misestimating in age and weight. A bi-exponential model best fits the plasma concentration after intravenous administration of orphenadrine in camels. The curve (Fig. 2) of the mean ± SEM plasma concentrations after i.v. orphenadrine administration in camels indicated an initial rapid phase, corresponding to the distribution of the drug with distribution half-life (t1/2a) 0.50 ± 0.07 h. The distribution phase of orphenadrine was followed by a slower elimination phase with elimination half-life (t1/2b) of 3.57 ± 0.55 h. Identification of orphenadrine metabolites was based on the presence of chromatographic peaks in the urine samples collected after drug administration in comparison with urine samples obtained before drug administration. According to our procedure, the acidic–neutral fractions, with or without derivatization, showed no signs of metabolites. The enzymatically hydrolyzed sample, basic fraction of camel’s urine showed three major metabolites in addition to orphenadrine. In this study it appears that the drug and its metabolites were excreted mainly as conjugates, glucournide and/or sulphate: the GC/MS peaks of the drug and its metabolites in the non-hydrolyzed urine samples
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M. Elghazali et al. / Research in Veterinary Science 85 (2008) 563–569 Orphenadrine
TIC: ca-spl3a.D\data.ms
400000
350000
Abundance
300000
250000
Chlorpromazine
200000
150000
100000
50000
6.60
6.80
7.00
7.20
7.40
7.60
7.80
8.00
8.20
8.40
8.60
8.80
9.00
9.20
9.40
9.60
9.80
Time-->
Orphenadrine Concentration μg ml-1
Fig. 1. Reconstructed selected ion chromatogram obtained from extracted camel plasma 15 min after intravenous administration of orphenadrine. Ions m/z 58, 165 were monitored for orphenadrine and ions m/z 272, 318 were monitored for chlorpromazine (internal standard).
were very small compared with enzymatically hydrolyzed urine samples. The main orphenadrine metabolites detected in this study had molecular ion fragment at m/z 255 (M1, Fig. 3B) and m/z 241 (M2, Fig. 3B) suggestive N-desmethylation, and N,N-didesmethyl-orphenadrine, respectively. N-desmethylorphenadrine known as tofenacin is an active orphenadrine metabolite and it acts as antidepressant. The third metabolite with molecular ion fragment m/z 285 (M3, Fig. 3B) suggests hydroxy-orphenadrine. The present of the hydroxyl metabolite was confirmed by LC/ MS/MS chromatograph, method not validated. Using our routine method for screening post-race urine samples, we were able to detect orphenadrine for up to three days.
1.00
0.10
0.01
0
5
10
15
4. Discussion
Time (h)
Fig. 2. Orphenadrine concentration (mean ± SEM) in plasma after administration of 350 mg orphenadrine aspartate/camel intravenously (n = 6).
Orphenadrine is a widely used anticholinergic drug in the treatment of Parkinson’s disease. However, to date,
Table 1 Orphenadrine PK parameters (mean ± SEM) following administration of a single i.v. dose of 350 mg orphenadrine aspartate to five male camels Camel No. PK Parameter
Camel 1
Camel 2
Camel 3
Camel 4
Camel 5
Mean ± SEM
t1/2a (h) t1/2b (h) ClT (l h1 kg1) Vdss (l kg1) AUC (g h1 l1) VC (l kg1)
0.38 2.24 0.50 1.29 1.17 0.73
0.58 4.33 0.52 1.75 1.12 0.73
0.62 4.79 0.52 2.54 1.11 1.10
0.30 2.26 0.95 2.31 0.61 1.07
0.60 4.21 0.51 1.70 1.13 0.74
0.50 ± 0.07 3.57 ± 0.55 0.60 ± 0.09 1.92 ± 0.22 1.03 ± 0.10 0.87 ± 0.09
Where t1/2a and t1/2b, half-lives of the distribution and elimination phase, respectively; ClT, total body clearance; Vdss, volume of distribution at steady state; AUC, area under the curve from time 0 to infinity; Vc, volume of central compartment.
Abundance
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567
Ion 255.00 (254.70 to 255.70): ca-blk-b.D\data.ms
14000 12000 10000 8000 6000 4000 2000 0
A
10.00
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
11.60
11.80
Abundance
Time--> 14000 12000 10000 8000 6000 4000 2000 0
Ion 241.00 (240.70 to 241.70): ca-blk-b.D\data.ms
10.00
10.20
10.40
10.60
10.80
11.00
11.20
11.40
Abundance
Time--> 14000 12000 10000 8000 6000 4000 2000 0 10.00
Ion 285.00 (284.70 to 285.70): ca-blk-b.D\data.ms
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
Time-->
Abundance
Ion 255.00 (254.70 to 255.70): ca-spl3-b.D\data.ms 600000 500000 400000 300000 200000 100000 0 10.00
CH 3 O
M1
Mwt=255
B
H N CH 3
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time-->
Abundance
Ion 241.00 (240.70 to 241.70): ca-spl3-b.D\data.ms 600000 500000 400000 300000 200000 100000 0 10.00
CH 3
H
O
M2
N
Mwt=241
H
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time-->
Abundance
Ion 285.00 (284.70 to 285.70): ca-spl3-b.D\data.ms 600000 500000 400000 300000 200000 100000 0 10.00
CH 3 HO O
Mwt=285
CH 3 N
M3
CH 3
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time-->
Fig. 3. (GC/MS) SIM Chromatogram of an enzyme hydrolyzed urine extract camel No a. (basic fraction). (A) Pre-administration; (B) 5 h after ORPH administration. M1:N-desmethylorphenadrine; M2:N,N-didesmethylorphenadrine; M3: hydroxyorphenadrine.
blood level monitoring for orphenadrine levels is not routinely performed. To the best of our knowledge this is the first study that describes the pharmacokinetics of orphenadrine in camels. Our results were characterized by a short elimination half-life 3.57 ± 0.55 h, body clearance
0.60 ± 0.09 l h1 kg1 and relatively large volume of distribution 1.92 ± 0.22 l kg1 the volume of distribution could be due to the alkaline nature of the drug and to that orphenadrine is very tightly bound by the tissues and not by blood, so that the drug appear to be dissolved in large
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that camel’s clear phenylbutazone, flunixin, tolfenamic acid, and caffeine more slowly than in horses (Wasfi et al., 1997, 1998a,b, 2000b). However, we also found that the clearance of antipyrine and triplennamine were similar in these two species Wasfi et al. (1998c), Wasfi et al.. (2000a). The dosage regimens for the drug in camels should be established after pharmacokinetic/pharmacodynamic studies in this species, as otherwise drug toxicity or failure of therapeutic benefit may be expected. The metabolism of orphenadrine in camels is extensive, producing basic metabolites. While different from that reported in humans Moffat et al. (1986), we could not determine acidic metabolites in all urine samples collected
volume like lipid soluble bases, imipramine and chlorpromazine. We could not find any results reporting pharmacokinetic of orphenadrine in horses and cattle with which to compare our results. However, in human adults and children the terminal half-lives have been reported to range from 13 to 25 h (Labout et al., 1982; Rutigliano and Labout, 1982; Lee et al., 2006). Due to the limited number of pharmacokinetic studies in camels, dosage regimens for camels are usually extrapolated from those for horses and cattle, owing to the reason that the pattern of disposition of drugs in camels is not consistent with that in other species. Previously we reported that camel clear diphenhydramine about twice as fast as horse (Wasfi et al., 2003a) and
TIC:ca-blk-b.D\data.ms
A
5000000 4500000 4000000
Abundance
3500000 3000000 2500000 2000000 1500000 1000000 500000 8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
Time-->
B
TIC: ca-spl3-b.D\data.ms 3.2e+07 3e+07
M3
2.8e+07 2.6e+07 2.4e+07
Abundance
2.2e+07 ORPH
2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07
M2
1e+07 8000000 6000000 4000000
M1
2000000 6.00
7.00
8.00
9.00
10.00
11.00 12.00
13.00 14.00
15.00
16.00
17.00 18.00
19.00
Time-->
Fig. 4. Total ion chromatogram of a urine extract (basic fraction) of camel. (A) Pre-administration; (B) 5 h after ORPH administration , M1 Ndesmthylorphenadrine, M2 N,N-didesmethylorphenadrine, M3 hydroxyorphenadrine.
M. Elghazali et al. / Research in Veterinary Science 85 (2008) 563–569
in this study. The major route of ORPH elimination, however, was as free or conjugated orphenadrine in the basic urine fraction that could be glucuronide and/or sulphate, we could not differentiate between these two conjugates. This may be due to the nature of the enzyme we routinely used in post-race sample hydrolysis. The three main basic metabolites found in this study were N-monodesmethylorphenadrine (tofenacin, which was reported as orphenadrine active metabolite), N,N-didesmethylorphenadrine,and hydroxyorphenadrine (Fig. 4). This study showed that the metabolic pathway of orphenadrine followed common phase 1 elimination route in camel. We have consistently determined that N-dealkylation and hydroxylation were major routes of drugs elimination in camels, for example N-dealkylation of etamiphylline Elghazali et al. (2002) and hydroxylation of flunixin, tolfenamic acid and declofenac (Wasfi et al., 1998a,b; Wasfi et al., 2003b). The exact locations of the hydroxyl group in the parent drug molecule can not be ascertained from this study. One of the main objectives of this study was to determine the withdrawal time for orphenadrine in racing camels. Orphenadrine was found detectable in camel for about three days after single i.v. administration. The long detection time of orphenadrine, may be due to several reasons. The alkaline nature of camel urine may be result in hydrolysis of unstable glucuronide conjugate and recycling of the parent compound. Also orphenadrine has a large volume of distribution, lipid soluble drug; because drug in the vascular system can only be available to organs for elimination. Camels have a small urine volume [about 1.0 l d1; Wasfi et al. (1998a)] and a low glomerular filtration rate [0.55–0.65 ml k1 min1; Wilson (1984)], which allows ample time for in vitro hydrolysis and reabsorption. All of these factors taken together will allow detection of orphenadrine for a long period. The camel racing authority in the United Arab Emirates prohibits the presence of any foreign substances in blood or urine samples collected after racing. Detection of prohibited substances and their metabolites usually result in loss of any prize along with suspension sever penalties, or both for the owner, trainer and veterinarians of the camels. For this reason guidance is needed to help them determine when to discontinue the use of legitimate, therapeutic drugs to avoid their accidental presence after racing. Based on the data obtained from this study, we recommend withholding administration of orphenadrine for a minimum period of four days before racing therefore as precautionary measure. Acknowledgments Authors would like to thanks Brigadier Dr. Ahmed Al Awadhi, director of the forensic science laboratory for his full support and fruitful advice. Thanks extended to Dr. Basal, and N. Al braiki for technical assistance. Special thanks go to all staff of the doping laboratory.
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References Armbruster, D.A., Tillman, M.D., Hubs, L.M., 1994. Limit of detection (LOD)/Limit of quantitation (LOQ): Comparison of the empirical and the statistical methods exemplified with GC–MS assays of abuse drugs. Clinical Chemistry 40, 1233–1238. Bast, A., Noordhoek, J., 1982. Spectral interaction of orphenadrine and it metabolites with oxidized and reduced hepatic microsomal cytochrome P-450 in the rat. Biochemical Pharmacology 31, 2745–2753. Elghazali, M., Wasfi, I.A., Al Katheeri, N.A., Hadi, A.A.A., Hussein, M.M., 2002. The pharmacokinetics, metabolism and urinary detection time of etamiphylline in camels after intramuscular administration. Journal of Veterinary Pharmacology and Therapeutics 25, 43–48. Gibaldi, M., Perrier, D., 1982. Pharmacokinetics. Marcel Dekker, NY, pp. 955–971. Labout, J.J., Thijssen, C.T., Keijser, G.G., Hespe, W., 1982. Difference between single and multiple dose pharmacokinetics of orphenadrine hydrochloride in man. European Journal of Clinical Pharmacology 21, 343–350. Lee, S.-Y., Oh, H.J., Kim, J.W., Kim, Y.G., Moon, C.J., Lee, E.H., 2006. Pharmacokinetic study of orphenadrine using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Journal of Chromatography B 839, 118–123. Moffat, A.C., Jackson, J.V., Moss, M.S., Widdop, B., 1986. Clarke’s isolation and identification of drugs, second ed. The pharmaceutical press, London, pp. 833–834. Murray, M., Reidy, G.F., 1990. Selectivity in the inhibition of mammalian cytochromes P-450 by chemical agents. Pharmacological Reviews 42, 85–101. Rutigliano, G., Labout, J.J., 1982. The bioavailability of orphenadrine hydrochloride after intramuscular an oral administration. The Journal of International Medical Research 10, 447–450. Wasfi, I.A., Abdel Hadi, A.A., Zorob, O., Osman, M.E., Boni, N.S.A., 1997. Pharmacokinetics of Phenylbutazone in camels. American Journal of Veterinary Research 58, 636–640. Wasfi, I.A., Boni, N.S., Abel Hadi, A.A., El Ghazali, M., Zorob, O., Alkatheeri, N.A., Barezaiq, I.M., 1998a. Pharmacokinetics, metabolism and urinary detection time of flunixin after intravenous administration in camels. Journal of Veterinary Pharmacology and Therapeutics 21, 203–208. Wasfi, I.A., El Ghazali, M., Abel Hadi, A.A., Zorob, O., Boni, N.S., Alkatheeri, N.A., Barezaiq, I.M., 1998b. Pharmacokinetics of tolfenamic acid and its detection time in urine after intravenous administration of the drug in camels (Camelus dromedarius). American Journal of Veterinary Research 59, 1451–1458. Wasfi, I.A., Zorob, O.M., Boni, N.S., Hadi, A.A.A., Agha, B.A.O., Elghazali, M., 1998c. The activity of mixed function oxidases, estimated by in vitro Antipyrine clearance, is similar in horses and camels. Comparative Biochemistry and Physiology-Part C 2, 139–142. Wasfi, I.A., Abdel Hadi, A.A., Boni, N.S., Alkatheeri, N.A., Barezaig, I.M., Muharami, A.M., Hamid, A.M., 2000a. Comparative disposition of triplennamine in horses and camels after intravenous administration. Journal of Veterinary Pharmacology and Therapeutics 23, 145–152. Wasfi, I.A., Boni, N.S., El Ghazali, M., Abel Hadi, A.A., Barezaiq, I.M., Alkatheeri, N.A., 2000b. The pharmacokinetics, metabolism and urinary detection time of caffeine in camels. Research in Veterinary Science 69, 69–74. Wasfi, I.A., Abel Hadi, A.A., El Ghazali, M., Alkatheeri, N.A., Hussain, M.M., 2003a. Comparative pharmacokinetics of diphenhydramine in camels and horses after intravenous administration. Veterinary Research Communications 27, 463–473. Wasfi, I.A., Hussain, M.M., El Ghazali, M., Alkatheeri, N.A., Abel Hadi, A.A., 2003b. The disposition of declofenac in camels after intravenous administration. The Veterinary Journal 166, 277–283. Wilson, R.T., 1984. The camel. The Print House, Singapore. Yamoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’s information criterion (CIA) in the evaluation of linear pharmacokinetic equations. Journal of Pharmacokinetics and Biopharmaceutics 6, 165–175.
Research in Veterinary Science 85 (2008) 563–569 www.elsevier.com/locate/rvsc
Pharmacokinetic, metabolism and withdrawal time of orphenadrine in camels (Camelus dromedarius) after intravenous administration M. Elghazali *, I.A. Wasfi, A.A. Abdel Hadi, A.M. Latum Camel Racing Forensic Laboratory, Directorate for Forensic Sciences, P.O. Box 253, Abu Dhabi, United Arab Emirates Accepted 22 January 2008
Abstract The pharmacokinetics of orphenadrine (ORPH) following a single intravenous (i.v.) dose was investigated in six camels (Camelus dormedarius). Orphenadrine was extracted from the plasma using a simple sensitive liquid–liquid extraction method and determined by gas chromatography/mass spectrometry (GC/MS). Following i.v. administration plasma concentrations of ORPH decline bi-exponentially with distribution half-life (t1/2a) of 0.50 ± 0.07 h, elimination half-life (t1/2b) of 3.57 ± 0.55 h, area under the time concentration curve (AUC) of 1.03 ± 0.10 g/h l1. The volume of distribution at steady state (Vdss) 1.92 ± 0.22 l kg1, volume of the central compartment of the two compartment pharmacokinetic model (Vc) 0.87 ± 0.09 l kg1, and total body clearance (ClT) of 0.60 ± 0.09 l/h kg1. Three orphenadrine metabolites were identified in urine samples of camels. The first metabolite N-desmethyl-orphenadrine resulted from Ndealkylation of ORPH with molecular ion m/z 255. The second N,N-didesmethyl-orphenadrine, resulted from N-didesmethylation with molecular ion m/z 241. The third metabolite, hydroxyl-orphenadrine, resulted from the hydroxylation of ORPH with molecular ion m/z 285. ORPH and its metabolites in camel were extensively eliminated in conjugated form. ORPH remains detectable in camel urine for three days after i.v. administration of a single dose of 350 mg orphenadrine aspartate. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Orphenadrine; Pharmacokinetics; Metabolism; Detection; Camels
1. Introduction Orphenadrine (N,N-dimethyl-2(o-methyl-alpha-phenylbenzyloxy) ethylamine is a close structural analogue of diphenhydramine. It is widely accepted as a skeletal muscle relaxant and is used as a therapeutic agent in the treatment of Parkinson’s disease and the neuroleptic syndrome. Orphenadrine inhibits it own elimination when administered by multiple dose regimen and prolongs the half-life of co-administered haloperidol Labout et al. (1982). ORPH is a relatively non-potent inhibitor of the cytochrome P450 (CYP) monooxygenase system that mediates drug biotransformation and undergoes CYP-dependent biotransformation within the alkylamine side chain to generate a potent inhibitory metabolite Bast and Noordhoek (1982). ORPH metabolite binds tightly to the CYP heam, thus *
Corresponding author. Tel.: +971 50 581 7969; fax: + 971 2 446 3470. E-mail address: [email protected] (M. Elghazali).
0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.01.006
generating a metabolite intermediate (M1)-complex similar to those produced by other alkylamines, such as the macrolide antibiotics Murray and Reidy (1990). In adults, orphenadrine is readily absorbed after oral administration and rapidly distributed. Up to 60% of an oral dose is excreted in the urine in three days. In the 24 h following dosage, under uncontrolled conditions, 4% of a dose is excreted as unchanged drug, 5% as N-monodesmethylorphenadrine (tofenacin), 3% as N,N-didesmethylorphenadrine, 3% as orphenadrine N-oxide, 8% as a conjugated of 2-methylbenzhdoxyacetic acid, and 6% as conjugate of 2-methylbenzhydrol. The urinary excretion appears to be dependent on urinary pH Moffat et al. (1986). Data concerning ORPH disposition, metabolism and the minimum effective plasma concentration in camels are not available. The purpose of this work was to: (1) determine the disposition of orphenadrine in camels following intravenous administration; (2) investigate the metabolism
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of orphenadrine in camels; and (3) determine the withdrawal time of orphenadrine in camels. 2. Materials and methods 2.1. Experimental animals Six healthy male camels (Camelus dormedarius), 7–14 years old, approximately 400 kg in body weight were used. The camels were kept in open pens. The camels were determined to be clinically normal before the experiment by physical examination and confirmed by serum biochemical analysis. Good quality hay and Lucerne (alfalfa) were fed to the camels once daily and water allowed ad labitum. The Ministry of Agriculture, Veterinary Department approved this study. 2.2. Experimental design Orphenadrine citrate (401.3 mg equivalent to 350 mg orphenadrine aspartate, Gulf Pharmaceutical Industries, Julphar) dissolved into 15 ml sterile sodium chloride 0.9% ‘‘normal saline”. 15 ml was administered to each camel as a single intravenous bolus into the jugular vein. Lack of pharmacokinetic data for orphenadrine in camels prompted us to use the manufacturer (USO veterinario, industria) clinically recommended dose of orphenadrine in horses. Blood samples (15 ml) were collected in heperinized tubes from the opposite jugular vein at zero (preinjection) and at 5, 10, 15, 30, and 45 min and 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and 24 h after drug administration. The blood samples were centrifuged at 4000g for 10 min. Separated plasma was kept at 20 °C. Samples were assayed within 10 days. Urinary detection time and metabolites were determined in two camels by collecting their urine at zero-time, (blank sample) and at 1, 2, 5, and 8 h (the first day) and at 8:00 a.m. daily for 7 days. Urine collection was done as per Wasfi et al. (1997) briefly, urine samples collected by the use of locally made plastic bags sewn to cotton cloth to give them extra strength. The bag was fastened to the camel via three straps. For male the bag was fitted directly onto the penis. For female camels, a flexible metallic bar was added to the bag. And configure to fit under the tail, separating the anus from the vagina, thus avoiding contamination of urine samples with fecal matter. When bags were half full, they were replaced by new bags. Voided urine transferred to plastic containers and kept in a freezer bending analysis. 2.3. Assay procedures The concentration of the drug in camel plasma was determined by GC/MS. Briefly, ORPH was extracted from 0.5 ml plasma by the addition of 100 ll chlorpromazine, internal standard (10 lg/ml in methanol). Then 100 ll of 1 N sodium hydroxide was added followed by 2.5 ml ethyl
acetate. Samples were agitated on horizontal mixer for 10 min and centrifuged for 10 min at 3500g. Two milliliters of the organic supernatant was removed and transferred to a clean glass tube. The organic layer was evaporated to dryness at 40 °C under a low stream of nitrogen. The residue reconstituted in 100 ll ethyl acetate. Two microliters were immediately analyzed by GC/MS. 2.4. GC/MS analysis GC/MS was carried out using an Agilent Technologies 6890N, net work GC system linked with 5973Network Mass selective detector with 7683B series injector and sample tray. Injections were made in the splitless mode onto a 30 m 0.25 mm i.d. HP-5MS column (Agilent Technologies). The initial oven temperature was 100 °C programmed to rise to 300 at 30 °C min1. Helium was used as the carrier gas. Data were acquired in the selective ion mode (SIM) monitoring the ions m/z 58, 165 and 272, 318 for orphenadrine and chlorpromazine, respectively. The interference of endogenous compounds and drugs was assessed by analyzing standard orphenadrine and chlorpromazine collect drug free plasma from the camels under investigation (six camels) before the start of experiment, plasma spiked with orphenadrine and chlorpromazine and plasma obtained from subjects given orphenadrine. Method proved to be selective for orphenadrine and the internal standard chlorpromazine. The linearity of the method was from 10 to 5000 ng/ml of ORPH in spiked plasma (r2 > 0.995). Maximum recovery was reported in this method. The intra- and inter-assay relative standard deviation and standard errors of mean were used to validate the precision and accuracy of the assay by determining standard samples of orphenadrine in camel plasma. For intra-day validation, six sets of controls at two ORPH concentrations 10 and 1000 ng/ml were assayed with one standard curve on the same run. The range of the relative standard deviation was reported. For inter-day validation six sets of two concentration of the drug in camel plasma were evaluated on three different days. The range of the relative standard deviation was also reported. The intra-coefficient of variation (CV%) for 10 and 1000 ng/ml orphenadrine spiked plasma was 7.33% and 6.59%, respectively. The inter-coefficient of variation (CV%) for 10 and 1000 ng/ml orphenadrine plasma spiked was 10.82% and 5.63%, respectively. The accuracy was determined by comparing the calculated concentration using calibration curve to known concentration. Limit of detection (LOD) and lower limit of quantitation (LLOQ) were determined according to the procedure described by Armbruster et al. (1994). The LOD, defined as the concentration at which all routine GC/MS acceptance criteria (retention time within 2% of calibrator, ion ratios within 20% of calibrator) are met at least 90% of the time, was 2.5 ng/ml. The LLOQ, defined as the lowest concentration above which quantitation can be carried out with adequate accuracy and precision, was 5 ng/ml.
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2.5. Pharmacokinetic analysis
2.7. Extraction and identification of the major metabolites of orphenadrine
Pharmacokinetic analysis was performed, using standard methods and equations Gibaldi and Perrier (1982) and was aided by use of a computer program (PC Modfit, Cambridgeshire, UK). Plasma concentrations were first plotted against time on a semilogarithmic graph to select initial models. Plasma concentrations over time after i.v. administration were analyzed via compartmental pharmacokinetic method. Examination of the semilogarithmic graphs suggested that a one- or two-compartment model, with first order output and bolus input, would be appropriate. All data were fit to these two models. Fitting parameters selected were weighted least squares method using 1/conc weighting scheme, measurement of Akaikes’s information criterion (AIC) Yamoka et al. (1978) was used to determined the model that was best for the data from each specific camel. For the two-compartment open model a biexponential equation was used: Ct ¼ Aeat þ Bebt
ð1Þ
where C is the drug plasma concentration at time equal t, e is the base of the natural logarithm, A is the intercept, and a is the rate constant for the initial steep phase (distribution) portion of the curve; B is the intercept, and a is the rate constant of the terminal (elimination) portion of the curve. For one-compartment open model, a mono-exponential equation was used: Ct ¼ C 0 ekt
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ð2Þ
where C is the plasma concentration at time t, C0 is the initial plasma concentration at time 0, and k is the elimination rate constant (equivalent to kel). After performing this analysis it was discovered that data for all camels could best fit with 2-compartment model. Also, for data fit to a two-compartment model, there were some instances in which the data defined the distribution phase showed problem with few data points on the distribution portion of the curve. Because of these problems, noncompartmental analysis was used in all camels to minimize the need for compartmental assumptions. The primary pharmacokinetics parameters estimated were intercepts A and B, and the rate constants a and b. Secondary variables were calculated by standard equations Gibaldi and Perrier (1982). Area under the plasma concentration versus time curve (AUC) was calculated using the trapezoidal method, with area from the last time point to infinity estimated from the terminal elimination rate constant. 2.6. Determination of drug withdrawal time The detection time for orphenadrine in racing camels was determined by collecting daily urine samples from the treated camels at about 8:00 a.m. for seven days. These urine samples were then subjected to a routine method for screening drugs in post-race urine samples Wasfi et al. (1998a).
The extraction and identification of the major metabolites of orphenadrine was performed on urine samples collected at 0-time (pre-injection sample; blank), 1, 5, and 8 h after drug administration by the method reported elsewhere Wasfi et al. (2000a). 2.8. Statistical analysis of data The value of the pharmacokinetic parameters is expressed as mean ± SEM. 3. Results The GC chromatographic method used in this study showed good separation between the drug and internal standard. Retention times were 7.1 and 9.2 min for orphenadrine and the internal standard chloropromazine respectively (Fig. 1). The total analysis time for each run was 10 min. We were able to analyze more than 60 samples per day. The best recovery of orphenadrine was obtained when the pH of the sample was adjusted to above 10 and this was achieved by addition of 100 ll of 1 N sodium hydroxide solution. The plasma profile of ORPH following i.v. administration is presented in Fig. 2. Pharmacokinetics parameters (mean ± SEM) are presented in Table 1. Pharmacokinetic results for camel six are not consistence with the other five. Camel six showed large distribution phase, with large distribution half-life (t1/2a) 1.5 h, terminal elimination half-life (t1/2b) > 3 d, Vdss was 4.29 l kg1 and total body clearance ClT 9.5604 l h1 kg1. Although, all camels were treated the same and kept under the same conditions. This may be attributed to misestimating in age and weight. A bi-exponential model best fits the plasma concentration after intravenous administration of orphenadrine in camels. The curve (Fig. 2) of the mean ± SEM plasma concentrations after i.v. orphenadrine administration in camels indicated an initial rapid phase, corresponding to the distribution of the drug with distribution half-life (t1/2a) 0.50 ± 0.07 h. The distribution phase of orphenadrine was followed by a slower elimination phase with elimination half-life (t1/2b) of 3.57 ± 0.55 h. Identification of orphenadrine metabolites was based on the presence of chromatographic peaks in the urine samples collected after drug administration in comparison with urine samples obtained before drug administration. According to our procedure, the acidic–neutral fractions, with or without derivatization, showed no signs of metabolites. The enzymatically hydrolyzed sample, basic fraction of camel’s urine showed three major metabolites in addition to orphenadrine. In this study it appears that the drug and its metabolites were excreted mainly as conjugates, glucournide and/or sulphate: the GC/MS peaks of the drug and its metabolites in the non-hydrolyzed urine samples
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TIC: ca-spl3a.D\data.ms
400000
350000
Abundance
300000
250000
Chlorpromazine
200000
150000
100000
50000
6.60
6.80
7.00
7.20
7.40
7.60
7.80
8.00
8.20
8.40
8.60
8.80
9.00
9.20
9.40
9.60
9.80
Time-->
Orphenadrine Concentration μg ml-1
Fig. 1. Reconstructed selected ion chromatogram obtained from extracted camel plasma 15 min after intravenous administration of orphenadrine. Ions m/z 58, 165 were monitored for orphenadrine and ions m/z 272, 318 were monitored for chlorpromazine (internal standard).
were very small compared with enzymatically hydrolyzed urine samples. The main orphenadrine metabolites detected in this study had molecular ion fragment at m/z 255 (M1, Fig. 3B) and m/z 241 (M2, Fig. 3B) suggestive N-desmethylation, and N,N-didesmethyl-orphenadrine, respectively. N-desmethylorphenadrine known as tofenacin is an active orphenadrine metabolite and it acts as antidepressant. The third metabolite with molecular ion fragment m/z 285 (M3, Fig. 3B) suggests hydroxy-orphenadrine. The present of the hydroxyl metabolite was confirmed by LC/ MS/MS chromatograph, method not validated. Using our routine method for screening post-race urine samples, we were able to detect orphenadrine for up to three days.
1.00
0.10
0.01
0
5
10
15
4. Discussion
Time (h)
Fig. 2. Orphenadrine concentration (mean ± SEM) in plasma after administration of 350 mg orphenadrine aspartate/camel intravenously (n = 6).
Orphenadrine is a widely used anticholinergic drug in the treatment of Parkinson’s disease. However, to date,
Table 1 Orphenadrine PK parameters (mean ± SEM) following administration of a single i.v. dose of 350 mg orphenadrine aspartate to five male camels Camel No. PK Parameter
Camel 1
Camel 2
Camel 3
Camel 4
Camel 5
Mean ± SEM
t1/2a (h) t1/2b (h) ClT (l h1 kg1) Vdss (l kg1) AUC (g h1 l1) VC (l kg1)
0.38 2.24 0.50 1.29 1.17 0.73
0.58 4.33 0.52 1.75 1.12 0.73
0.62 4.79 0.52 2.54 1.11 1.10
0.30 2.26 0.95 2.31 0.61 1.07
0.60 4.21 0.51 1.70 1.13 0.74
0.50 ± 0.07 3.57 ± 0.55 0.60 ± 0.09 1.92 ± 0.22 1.03 ± 0.10 0.87 ± 0.09
Where t1/2a and t1/2b, half-lives of the distribution and elimination phase, respectively; ClT, total body clearance; Vdss, volume of distribution at steady state; AUC, area under the curve from time 0 to infinity; Vc, volume of central compartment.
Abundance
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Ion 255.00 (254.70 to 255.70): ca-blk-b.D\data.ms
14000 12000 10000 8000 6000 4000 2000 0
A
10.00
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
11.60
11.80
Abundance
Time--> 14000 12000 10000 8000 6000 4000 2000 0
Ion 241.00 (240.70 to 241.70): ca-blk-b.D\data.ms
10.00
10.20
10.40
10.60
10.80
11.00
11.20
11.40
Abundance
Time--> 14000 12000 10000 8000 6000 4000 2000 0 10.00
Ion 285.00 (284.70 to 285.70): ca-blk-b.D\data.ms
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
Time-->
Abundance
Ion 255.00 (254.70 to 255.70): ca-spl3-b.D\data.ms 600000 500000 400000 300000 200000 100000 0 10.00
CH 3 O
M1
Mwt=255
B
H N CH 3
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time-->
Abundance
Ion 241.00 (240.70 to 241.70): ca-spl3-b.D\data.ms 600000 500000 400000 300000 200000 100000 0 10.00
CH 3
H
O
M2
N
Mwt=241
H
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time-->
Abundance
Ion 285.00 (284.70 to 285.70): ca-spl3-b.D\data.ms 600000 500000 400000 300000 200000 100000 0 10.00
CH 3 HO O
Mwt=285
CH 3 N
M3
CH 3
10.20
10.40
10.60
10.80
11.00
11.20
11.40
11.60
11.80
12.00
Time-->
Fig. 3. (GC/MS) SIM Chromatogram of an enzyme hydrolyzed urine extract camel No a. (basic fraction). (A) Pre-administration; (B) 5 h after ORPH administration. M1:N-desmethylorphenadrine; M2:N,N-didesmethylorphenadrine; M3: hydroxyorphenadrine.
blood level monitoring for orphenadrine levels is not routinely performed. To the best of our knowledge this is the first study that describes the pharmacokinetics of orphenadrine in camels. Our results were characterized by a short elimination half-life 3.57 ± 0.55 h, body clearance
0.60 ± 0.09 l h1 kg1 and relatively large volume of distribution 1.92 ± 0.22 l kg1 the volume of distribution could be due to the alkaline nature of the drug and to that orphenadrine is very tightly bound by the tissues and not by blood, so that the drug appear to be dissolved in large
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that camel’s clear phenylbutazone, flunixin, tolfenamic acid, and caffeine more slowly than in horses (Wasfi et al., 1997, 1998a,b, 2000b). However, we also found that the clearance of antipyrine and triplennamine were similar in these two species Wasfi et al. (1998c), Wasfi et al.. (2000a). The dosage regimens for the drug in camels should be established after pharmacokinetic/pharmacodynamic studies in this species, as otherwise drug toxicity or failure of therapeutic benefit may be expected. The metabolism of orphenadrine in camels is extensive, producing basic metabolites. While different from that reported in humans Moffat et al. (1986), we could not determine acidic metabolites in all urine samples collected
volume like lipid soluble bases, imipramine and chlorpromazine. We could not find any results reporting pharmacokinetic of orphenadrine in horses and cattle with which to compare our results. However, in human adults and children the terminal half-lives have been reported to range from 13 to 25 h (Labout et al., 1982; Rutigliano and Labout, 1982; Lee et al., 2006). Due to the limited number of pharmacokinetic studies in camels, dosage regimens for camels are usually extrapolated from those for horses and cattle, owing to the reason that the pattern of disposition of drugs in camels is not consistent with that in other species. Previously we reported that camel clear diphenhydramine about twice as fast as horse (Wasfi et al., 2003a) and
TIC:ca-blk-b.D\data.ms
A
5000000 4500000 4000000
Abundance
3500000 3000000 2500000 2000000 1500000 1000000 500000 8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
Time-->
B
TIC: ca-spl3-b.D\data.ms 3.2e+07 3e+07
M3
2.8e+07 2.6e+07 2.4e+07
Abundance
2.2e+07 ORPH
2e+07 1.8e+07 1.6e+07 1.4e+07 1.2e+07
M2
1e+07 8000000 6000000 4000000
M1
2000000 6.00
7.00
8.00
9.00
10.00
11.00 12.00
13.00 14.00
15.00
16.00
17.00 18.00
19.00
Time-->
Fig. 4. Total ion chromatogram of a urine extract (basic fraction) of camel. (A) Pre-administration; (B) 5 h after ORPH administration , M1 Ndesmthylorphenadrine, M2 N,N-didesmethylorphenadrine, M3 hydroxyorphenadrine.
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in this study. The major route of ORPH elimination, however, was as free or conjugated orphenadrine in the basic urine fraction that could be glucuronide and/or sulphate, we could not differentiate between these two conjugates. This may be due to the nature of the enzyme we routinely used in post-race sample hydrolysis. The three main basic metabolites found in this study were N-monodesmethylorphenadrine (tofenacin, which was reported as orphenadrine active metabolite), N,N-didesmethylorphenadrine,and hydroxyorphenadrine (Fig. 4). This study showed that the metabolic pathway of orphenadrine followed common phase 1 elimination route in camel. We have consistently determined that N-dealkylation and hydroxylation were major routes of drugs elimination in camels, for example N-dealkylation of etamiphylline Elghazali et al. (2002) and hydroxylation of flunixin, tolfenamic acid and declofenac (Wasfi et al., 1998a,b; Wasfi et al., 2003b). The exact locations of the hydroxyl group in the parent drug molecule can not be ascertained from this study. One of the main objectives of this study was to determine the withdrawal time for orphenadrine in racing camels. Orphenadrine was found detectable in camel for about three days after single i.v. administration. The long detection time of orphenadrine, may be due to several reasons. The alkaline nature of camel urine may be result in hydrolysis of unstable glucuronide conjugate and recycling of the parent compound. Also orphenadrine has a large volume of distribution, lipid soluble drug; because drug in the vascular system can only be available to organs for elimination. Camels have a small urine volume [about 1.0 l d1; Wasfi et al. (1998a)] and a low glomerular filtration rate [0.55–0.65 ml k1 min1; Wilson (1984)], which allows ample time for in vitro hydrolysis and reabsorption. All of these factors taken together will allow detection of orphenadrine for a long period. The camel racing authority in the United Arab Emirates prohibits the presence of any foreign substances in blood or urine samples collected after racing. Detection of prohibited substances and their metabolites usually result in loss of any prize along with suspension sever penalties, or both for the owner, trainer and veterinarians of the camels. For this reason guidance is needed to help them determine when to discontinue the use of legitimate, therapeutic drugs to avoid their accidental presence after racing. Based on the data obtained from this study, we recommend withholding administration of orphenadrine for a minimum period of four days before racing therefore as precautionary measure. Acknowledgments Authors would like to thanks Brigadier Dr. Ahmed Al Awadhi, director of the forensic science laboratory for his full support and fruitful advice. Thanks extended to Dr. Basal, and N. Al braiki for technical assistance. Special thanks go to all staff of the doping laboratory.
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References Armbruster, D.A., Tillman, M.D., Hubs, L.M., 1994. Limit of detection (LOD)/Limit of quantitation (LOQ): Comparison of the empirical and the statistical methods exemplified with GC–MS assays of abuse drugs. Clinical Chemistry 40, 1233–1238. Bast, A., Noordhoek, J., 1982. Spectral interaction of orphenadrine and it metabolites with oxidized and reduced hepatic microsomal cytochrome P-450 in the rat. Biochemical Pharmacology 31, 2745–2753. Elghazali, M., Wasfi, I.A., Al Katheeri, N.A., Hadi, A.A.A., Hussein, M.M., 2002. The pharmacokinetics, metabolism and urinary detection time of etamiphylline in camels after intramuscular administration. Journal of Veterinary Pharmacology and Therapeutics 25, 43–48. Gibaldi, M., Perrier, D., 1982. Pharmacokinetics. Marcel Dekker, NY, pp. 955–971. Labout, J.J., Thijssen, C.T., Keijser, G.G., Hespe, W., 1982. Difference between single and multiple dose pharmacokinetics of orphenadrine hydrochloride in man. European Journal of Clinical Pharmacology 21, 343–350. Lee, S.-Y., Oh, H.J., Kim, J.W., Kim, Y.G., Moon, C.J., Lee, E.H., 2006. Pharmacokinetic study of orphenadrine using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). Journal of Chromatography B 839, 118–123. Moffat, A.C., Jackson, J.V., Moss, M.S., Widdop, B., 1986. Clarke’s isolation and identification of drugs, second ed. The pharmaceutical press, London, pp. 833–834. Murray, M., Reidy, G.F., 1990. Selectivity in the inhibition of mammalian cytochromes P-450 by chemical agents. Pharmacological Reviews 42, 85–101. Rutigliano, G., Labout, J.J., 1982. The bioavailability of orphenadrine hydrochloride after intramuscular an oral administration. The Journal of International Medical Research 10, 447–450. Wasfi, I.A., Abdel Hadi, A.A., Zorob, O., Osman, M.E., Boni, N.S.A., 1997. Pharmacokinetics of Phenylbutazone in camels. American Journal of Veterinary Research 58, 636–640. Wasfi, I.A., Boni, N.S., Abel Hadi, A.A., El Ghazali, M., Zorob, O., Alkatheeri, N.A., Barezaiq, I.M., 1998a. Pharmacokinetics, metabolism and urinary detection time of flunixin after intravenous administration in camels. Journal of Veterinary Pharmacology and Therapeutics 21, 203–208. Wasfi, I.A., El Ghazali, M., Abel Hadi, A.A., Zorob, O., Boni, N.S., Alkatheeri, N.A., Barezaiq, I.M., 1998b. Pharmacokinetics of tolfenamic acid and its detection time in urine after intravenous administration of the drug in camels (Camelus dromedarius). American Journal of Veterinary Research 59, 1451–1458. Wasfi, I.A., Zorob, O.M., Boni, N.S., Hadi, A.A.A., Agha, B.A.O., Elghazali, M., 1998c. The activity of mixed function oxidases, estimated by in vitro Antipyrine clearance, is similar in horses and camels. Comparative Biochemistry and Physiology-Part C 2, 139–142. Wasfi, I.A., Abdel Hadi, A.A., Boni, N.S., Alkatheeri, N.A., Barezaig, I.M., Muharami, A.M., Hamid, A.M., 2000a. Comparative disposition of triplennamine in horses and camels after intravenous administration. Journal of Veterinary Pharmacology and Therapeutics 23, 145–152. Wasfi, I.A., Boni, N.S., El Ghazali, M., Abel Hadi, A.A., Barezaiq, I.M., Alkatheeri, N.A., 2000b. The pharmacokinetics, metabolism and urinary detection time of caffeine in camels. Research in Veterinary Science 69, 69–74. Wasfi, I.A., Abel Hadi, A.A., El Ghazali, M., Alkatheeri, N.A., Hussain, M.M., 2003a. Comparative pharmacokinetics of diphenhydramine in camels and horses after intravenous administration. Veterinary Research Communications 27, 463–473. Wasfi, I.A., Hussain, M.M., El Ghazali, M., Alkatheeri, N.A., Abel Hadi, A.A., 2003b. The disposition of declofenac in camels after intravenous administration. The Veterinary Journal 166, 277–283. Wilson, R.T., 1984. The camel. The Print House, Singapore. Yamoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’s information criterion (CIA) in the evaluation of linear pharmacokinetic equations. Journal of Pharmacokinetics and Biopharmaceutics 6, 165–175.