animals : six crossbred dogs with naturally occurring tvt ( three males and 
 three females , 25 years of age and weighing 1129 kg ) were obtained from a community that 
 promotes the welfare of dogs and dogs family planning ( dog chance , ratchaburi province , 
 thailand ) .
the dogs were individually housed in cages at the laboratory animal facility of 
 the faculty of veterinary medicine at kasetsart university and were acclimatized to the 
 environment for one week .
the animals were allowed access to a standard diet and drinking 
 water ad libitum throughout the experimental period . a cytological 
 examination and a subsequent histological examination of the biopsies
the processes used to monitor the health 
 status of all of the dogs , which included a physical examination , a complete blood cell 
 count ( cbc ) and a serum biochemistry profile related to renal and hepatic function , were 
 performed weekly throughout the studies .
the tumor sizes were also measured using a vernier 
 caliper at the beginning of the experiment and weekly before the administration of 
 vincristine .
this study was performed according to the guidelines for animal experiments and 
 approved by the animal ethics research committee of the faculty of veterinary medicine at 
 kasetsart university .
drug administration and sample collection : the dogs were treated with 
 vincristine sulfate at a dosage of 0.7 mg / m of body surface area through 
 intravenous administration .
blood samples for single - dose pharmacokinetic analysis were 
 collected from the cephalic vein through iv catheter using edta tubes at 0 , 5 , 10 , 15 , 30 
 and 45 min and 1 , 2 , 4 , 6 , 8 , 12 , 24 and 48 hr after drug administration .
the samples were 
 centrifuged , and the plasma was separated and stored at 20c until analysis .
the treatments 
 were performed weekly until the tumor had visibly disappeared up to a maximum of eight 
 treatments . the response to treatment and toxicity
chemicals and reagents : the reference standards of vincristine sulfate and 
 vinblastine sulfate ( internal standard , is ) were purchased from sigma - aldrich ( st . louis , 
 mo , u.s.a . ) .
vincristine sulfate ( v.c.s. ; 1 mg / ml ) for treatment was 
 purchased from boryung pharmaceutical co. , ltd .
sample preparation : the samples were prepared according to a published 
 method   with slight modifications . briefly , 500 
 l of plasma was diluted with 500 l of 4% phosphoric 
 acid , and 20 l of 500 ng / ml is solution 
 was then added to the mixture . after thorough mixing ,
the mixture was applied to an oasis 
 hlb cartridge ( water , milford , ma , u.s.a . ) .
the cartridge was washed with 5% methanol in 
 water and then with 1 ml of methanol : water ( 50:50 , v / v ) .
the eluate was evaporated and 
 reconstituted in 100 l of a mixture of mobile phase a ( 5 mm ammonium 
 acetate in water titrated to ph 3.5 with acetic acid and mobile phase b ( acetonitrile ) 
 ( 50:50 , v / v ) .
the supernatant was 
 transferred to micro insert vials , and 10 l was injected into the lc - ms / ms 
 system for analysis .
chromatographic system : chromatographic separation through an lc system 
 was performed using a poroshell 120 ec - c18 , 3.0  50 mm , 2.7-m 
 column ( agilent technologies , palo alto , ca , u.s.a . ) .
the mobile phase a consisted of 5 mm 
 ammonium acetate in water titrated to ph 3.5 with acetic acid , and mobile phase b was 100% 
 acetonitrile .
the gradient elution was performed as follows : 0 to 0.5 min , isocratic 90% 
 mobile phase a ; 0.5 to 3.0 min , 90% to 5% mobile phase a ; 3.0 to 5.0 min , isocratic 5% 
 mobile phase a. the flow rate was 0.5 ml / min .
the mass spectrometric analysis was performed using a 6460 triple 
 quadrupole mass spectrometer ( agilent technologies ) and programmed using the agilent 
 masshunter b.06.00 software ( agilent technologies ) .
the ionization source parameters were optimized as follows : 
 capillary voltage , 3500 v ; gas temperature , 330c ; gas flow rate , 8 l / min ; 
 nebulizer , 50 psi .
the fragment energy was set to 230 v. the multiple reaction monitoring 
 ( mrm ) transitions were selected to be m / z 825.4 precursor ion to 
 m / z 765.4 production ion for vincristine and m / z 811.4 
 precursor ion to m / z 751.3 product ion for vinblastine .
validation procedures : in this study , the lc - ms / ms method was developed 
 and validated for the identification and quantification of vincristine in the plasma of dogs 
 with tvt .
the calibration standard concentrations were prepared by spiking the working 
 standard solution into blank plasma to yield final concentrations of 0.5 , 1 , 2.5 , 5 , 10 , 50 , 
 100 and 150 ng / ml .
five duplicates of the quality control 
 ( qc ) sample at concentrations of 2 , 50 and 100 ng / ml were 
 prepared and used to determine the recoveries , intra - day and inter - day precision , and 
 accuracy of the method .
the procedure was repeated five times within the same day to gain an 
 intra - day run precision and accuracy and five times of each concentration over five 
 different days to obtain an inter - day run precision and accuracy .
the precision of the assay 
 was assessed by calculating the relative standard deviation ( r.s.d . ) for each concentration 
 level .
the accuracy was calculated by comparing the average measurements with the nominal 
 values and is expressed as a percentage . a blank plasma sample ,
eight calibrated standard 
 concentrations and five duplicates of the qc samples were included in each run .
the 
 quantification was obtained using the multiple reaction monitoring ( mrm ) transitions for 
 vincristine ( m / z 825.4  765.4 ) and is ( m / z 811.4  
 751.3 ) .
the 
 calibration curve exhibited linearity ( r=0.990 ) over the 
 concentration range of 0.5 to 150 ng / ml . the lower limit 
 of quantification ( lloq ) , defined as the lowest concentration yielding a signal - to - noise 
 ratio ( s / n ) higher than 10 , was 0.5 ng / ml . pharmacokinetic study : the determination of the pharmacokinetic parameters 
 of vincristine was performed using a two - compartmental pharmacokinetic model with pk 
 solution 2.0(summit research services , montrose , co , u.s.a . ) .
the 
 cp term was the extrapolated plasma concentration time curve at zero time , 
 k12 and k21 were micro - rate constants , auc was the area under the 
 curve , t1/2 was the distribution half - life , 
 t1/2 was the elimination half - life , 
 vdarea was the volume of distribution , cl was the plasma clearance , and mrt was 
 the mean residence time .
method validation : the results of intra - day and inter - day precision and 
 accuracy of the assay are presented in table 
 1table 1.intra-day and inter - day accuracy and precision of vincristine sulfate in dog 
 plasma ( n=5)qc sample concentration 
 ( ng / ml)mean  sd(ng / ml)accuracy(%)precision(% r.s.d)intra - day ( 5 days)2.02.05  0.201026.4650.049.9  0.8199.71.59100.099.9  0.9999.90.92inter - day ( 5 days)2.02.22 
the intra - day precision and accuracy ranged from 0.92% to 6.46% and from 
 99.7% to 102% , respectively .
the inter - day precision and accuracy ranged from 0.902% to 
 4.39% and from 98.9% to 108% , respectively .
the extraction recoveries were 86.3  10.54 , 
 77.3  9.88 and 88.9  5.49% for low , medium and high qc levels , respectively ( table 2table 2.recovery of vincristine following extraction process ( n=5)qc sample concentration(ng / ml)mean extraction recovery(%)coefficient of variation(%)2.0 86.3  10.5412.350.0 77.3  9.8811.8100.0 88.9  5.496.18 ) .
pharmacokinetic analysis : the level of vincristine sulfate in plasma was 
 detectable up to 120 min after drug administration .
1.time profile of vincristine plasma concentration after its intravenous administration 
 to dogs with tvt at a dose of 0.7 mg / m .
shows the mean plasma concentration of vincristine as a function of time after a 
 single intravenous administration at a dose of 0.7 mg / m to dogs with tvt .
the 
 pharmacokinetic parameters for vincristine in dogs with tvt are presented in table 3table 3.pharmacokinetic parameters ( mean  sd ) of vincristine sulfate after its 
 intravenous administration at a dose of 0.7 mg / mto dogs with tvt 
 ( n=6)pharmacokinetic parameters ( units)average  sdk12 ( min)0.094  0.035k21 ( min)0.029  0.012cp 
 ( ng / ml)119  18.0t ( min)21.5  6.90t ( min)47.6  14.2cl ( l / min / kg)0.010  0.001vd(area ) ( l / kg)0.660  0.210mrt ( min)55.9  19.3auc ( ngmin / ml)2,349  317k12 , k21= micro - rate constants ; cp= plasma 
 concentration at initial time ; t1/2= distribution half - life ; 
 t1/2 = elimination half - life ; cl = clearance ; vd(area ) = 
 volume of distribution ; mrt = mean residence time ; auc = area under the plasma 
 concentration - time curve .. the plasma vincristine concentration immediately after administration 
 ( cp ) was 119  18.0 ng / ml .
the apparent 
 volume of distribution ( vdarea ) and mean residence time ( mrt ) were 0.660  0.210 
 l / kg and 55.9  19.3 min , respectively .
the distribution half - life 
 ( t1/2 ) , elimination half - life ( t1/2 ) and plasma clearance were 
 21.5  6.90 min , 47.6  14.2 min and 0.010  0.001 l / min / kg , 
 respectively .
time profile of vincristine plasma concentration after its intravenous administration 
 to dogs with tvt at a dose of 0.7 mg / m .
k12 , k21= micro - rate constants ; cp= plasma 
 concentration at initial time ; t1/2= distribution half - life ; 
 t1/2 = elimination half - life ; cl = clearance ; vd(area ) = 
 volume of distribution ; mrt = mean residence time ; auc = area under the plasma 
 concentration - time curve .
clinical responses : tumor regression was determined at weekly intervals by 
 a physical examination and histopathological analysis .
complete remission or complete response 
 means that all of the tumors completely disappeared , and partial remission means that the 
 tumors regressed more than 50% , but less than 100% . in our study , three to eight 
 administrations of vincristine at a dose of 0.7 mg / m were able to induce 
 complete remission in five dogs ( 83.33% ) .
in addition , these five dogs exhibited more than 
 50% remission after the first week of drug administration .
in contrast , one dog exhibited 
 only a partially response after the drug was administered eight times .
the tumor mass 
 ( approximately 1 cm in diameter ) in this dog continued to appear on the posterior portion of 
 the gland penis .
gastro - intestinal side effects , such as vomiting , anorexia and diarrhea , 
 were observed after the first drug administration in three dogs .
hematological side effects , 
 such as leucopenia , neutropenia and thrombocytopenia , were observed in two dogs after the 
 third drug administration , and anemia was observed in one dog after the fourth drug 
 administration .
a high 
 incidence of tvt is associated with a large population of free - roaming dogs with 
 uncontrolled sexual activity .
chemotherapy , particularly vincristine sulfate , has been shown 
 to be the most effective treatment and is thus frequently used .
several analytical methods 
 for the determination of vincristine sulfate in biological samples derived from both humans 
 and animals have been reported . currently , lc - ms / ms is the method that provides the most 
 sensitive and reliable qualitative and quantitative analyses .
the present study was 
 conducted to evaluate the pharmacokinetic parameters of vincristine sulfate in dogs with tvt 
 and to determine their correlation with the clinical effects of the drug using the 
 well - suited lc - ms / ms method to determine the concentration of vincristine sulfate in dog 
 plasma .
the results from our study show that three to eight weekly intravenous 
 administrations of vincristine at a dose of 0.7 mg / m produced a good response . 

this response 
 confirmed the efficacy of vincristine sulfate as a single agent for tvt treatment , which is 
 in agreement with previous reports [ 1 , 16 , 20 ] .
for the pharmacokinetic evaluation , the plasma concentration as a function of time was 
 described by a two - compartment model .
the t1/2 indicates the overall 
 rate of elimination and allows the prediction of vincristine accumulation with a value for 
 vincristine of 47.6 min after i.v .
administration in dogs with tvt , whereas the value of 
 t1/2 was 22.8 hr in healthy dogs . 

thus , about 99% of the vincristine from plasma was cleared within 5 hr 
 after i.v .
the distribution half - life ( t1/2 , 21.5 min ) of 
 vincristine obtained in dogs with tvt was longer than in healthy dogs ( 0.14 hr ) .
this may indicate that vincristine is distributed to 
 the peripheral tissues of dogs with tvt , and this finding is further supported by the high 
 uptake of the drug by tubulin - rich tissues [ 3 , 21 ] .
the above - described characteristics of the 
 penetration of vincristine sulfate exhibited a good correlation with the clinical tumor 
 response , which showed that more than 50% remission was obtained after the first drug 
 injection .
the volume 
 of distribution obtained in this study is in agreement with the results from previous 
 investigations in mice , tasmanian devils , children humans   and adult humans . 
 in previous studies , the accumulative concentration of the drug
was found to be high in many 
 organs , including the pancreas , spleen , thyroid , adrenal , intestinal mucosa , lung , liver , 
 kidney and bone marrow , 
 and the spleen was found to be the major organ in which the drug was accumulated . to the best of our knowledge
, this 
 study provides the first demonstration of the clinical pharmacokinetics of vincristine 
 sulfate in dogs with tvt using the lc - ms / ms method . in conclusion , although pharmacokinetic parameters of vincristine sulfate in dogs with tvt 
 showed rapid elimination and therefore no accumulation , it resulted in good clinical effects 
 by dosing once a week .
in addition , it is well recognized that auc is one of the most important pk parameters that 
 have a relationship with an efficacy of antitumor drugs .
based on the value of auc of 
 vincristine in this study , it might indicate the penetration of the drug into the tumor 
 mass , which increases the efficacy and antitumor activity of the drug .
however , further 
 investigation is needed to clarify the pharmacodynamics of the drug in tvt .
 this study was conducted to evaluate the pharmacokinetic characteristics of vincristine 
 and their correlation with its clinical effects in dogs with transmissible venereal tumor 
 ( tvt ) . 
 dogs with tvt were intravenously administered vincristine sulfate at a dose of 0.7 
 mg / m2 of body surface area . 
 blood samples were collected starting from 5 min 
 to 48 hr after drug administration . 
 the plasma concentration of vincristine was determined 
 using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) . 
 the pharmacokinetic 
 parameters of vincristine were characterized using a two - compartmental pharmacokinetic 
 model . 
 the volume of distribution , distribution half - life , elimination half - life and 
 plasma clearance were 0.660  0.210 l / kg , 21.5  6.90 min , 47.6  14.2 
 min and 0.010  0.001 l / min / kg , respectively . 
 tumor regression was 
 determined at weekly interval by a physical examination and histopathological analysis . in 
 our study , three to eight administrations of vincristine at a dose of 0.7 mg / m2 
 were able to induce a complete tumor regression without any evidence of gross lesion of 
 disease . 
 therefore , this investigation provides the pharmacokinetic characteristics of 
 vincristine in dogs with tvt , which may be used as an integration tool to gain a better 
 understanding of the disposition properties of the drug and the correlation of these 
 properties with the drug s clinical effects . 
 in addition , we validated the lc - ms / ms method 
 and found that it is suitable for the pharmacokinetic study of vincristine in dog 
 plasma . 