Study The First Pass Effect And Mechanism Of Tylosin Tartrate After Oral In Chicken

Tylosin tartrate is widely used in the livestock and poultry farming.It is broad spectum macrolide antibiotic which has high activity and little adverse reaction.The absorption property of it is rapid,the distribution is wide and the bioavailability is very low.However,the studies of the effect factors on the low oral bioavailability of tylosin tartrate are rare at present.So,the experiments were carried out to study the first pass effect and mechanism of tylosin tartrate in broilers.By investigated the mechanism of the first pass effect of tylosin tartrate,studying the methods of improving the oral bioavailability of tylosin tartrate.The details are as follows:1.Analysing the oral bioavailability of tylosin tartrate in chicken The HPLC detection method of tylosin is established.This study preliminary studied the oral bioavailability of tylosin tartrate.Twenty healthy broilers were randomly divided into two groups in this study and each group has 10 chickens.The two groups are tylosin tartrate oral group and intravenous group.All chickens were given via p.o.and iv administration at a single dose of 10 mg/kg of tylosin tartrate.The blood samples were taken from each chicken at time points of 0～24 h post administration.The plasma concentration of tylosin was detected by high performance liquid chromatography.The pharmacokinetic analysis of tylosin was performed using 3P97.The results showed that plasma concentration-time data of tylosin after oral administration in chickens was best described by a one-compartment open model,after iv administration in chickens was best described by a two-compartment open model.After oral administration,the AUC was0.79±0.11μg/mL·h and the Tpeak was 1.33±0.18 h;after iv administration,the AUC was 6.82±0.56 L/kg/h.And the oral bioavailability was 11.58%.The result suggested that the oral bioavailability of tylosin tartrate is low and the absorption is rapidly after oral.2.Relative contribution of the small intestine and liver to the first-pass effect of tylosin tartrate.To differentiate the hepatic and intestinal first-pass effect of tylosin tartrate,four different dosing routes:po,id,ipv and iv were adopted.In brief,the chickens were fasted at the night before the experiment,as well as 4 h after drug administration.Only free access to water was allowed throughout the experiment.The chickens were infused with tylosin tartrate by the four different dosing routes,at a dose of 10 mg/kg after anesthesia.Blood samples(approximately 0.15 mL)were collected from the wing vein into heparinized tubes at predose and at 10,20,30,40,50 min,1,1.5,2,3,4,6,8,12 and 24h postdose.The blood samples were centrifuged immediately at 3000 g for 5 min,and the obtained plasma samples were kept at-80℃until they were analysed.As the result shown,after administration of tylosin tartrate,the AUC(0-∞)values of tylosin tartrate were0.74±0.03μg·mL-1·h-1,0.86±0.05μg·mL-1·h-1,4.77±1.13μg·mL-1·h-1 and 6.57±0.43μg·mL-1·h-1 for po,id,ipv and iv,respectively.The values of terminal half-life and total clearance(CL/F)of tylosin tartrate are similar.A comparison of intestinal and hepatic extraction is calculated.EH and El values calculated using the indirect method are based on the current chicken data using 10 mg/kg doses of po,id,ip and iv administration.The value of EH is estimated to be 27.4%,and the value of El is estimated to be 82.0%.3.Determining the absorb and transport characteristic of tylosin tartrate in intestinal The transport experiments were performed in Caco-2 cell monolayer 21-27 days post seeding in DMEM medium and intestinal perfusion model.In Caco-2 cell monolayer model,to determine whether the transport of tylosin tartrate was time-dependent,the first design focused on time course of tylosin tartrate transport over a 120 min incubation period;to elucidate the mechanism of tylosin tartrate transport,the effect of initial loading concentration of tylosin tartrate on the transport rate was examined after 60 min incubation;the third tested the role of the paracellular pathway in the absorption of tylosin tartrate;the fourth investigate possible efflux transporters over 60 min.In intestinal perfusion model,to determine the main absorbing bowel and the effect of P-gp on tylosin tartrate absorption,duodenum,jejunum and ileum were single-pass perfusion respectively with EGTA and verapamil presence or not.The results showed that there were no significant differences in the efflux ratios throughout the concentration range studied,indicating there was a passive diffusion process for the transport of tylosin tartrate across Caco-2 monolayers.Tylosin tartrate appeared to be transported across the Caco-2 cell monolayers predominantly via the transcellular and paracellular pathway.Tylosin tartrate transport is significantly altered in the presence of verapamil,suggesting that P-glycoprotein is involved in tylosin tartrate transport.And the Papp values indicated that the gut absorption efficiency is 20%to 70%.In the small intestine perfusion process,tylosin tartrate absorbed rapidly in duodenum,indicated that duodenum is the major absorption bowel.Tylosin tartrate appeared to be transported across the intestinal predominantly via the transcellular pathway.The P-gp mediated efflux was significantly reduced with the presence of Verapamil,the Papps of bowel reveal tylosin tartrate is well absorbed in the duodenum,and the absorption is general in jejunum and ileum.4.Determining the metabolism characteristic of tylosin tartrate in chicken hepatocytes Hepatocytes were isolated from adult male Ross chicken.This study investigated the metabolic properties of tylosin tartrate in hepatocytes.By optimization the incubation time,the hepatocytes seeding density,the concentration of tylosin tartrate and inhibited CYP1A and 3 A,investigated the effects of those factors on the metabolic elimination of tylosin tartrate.The concentration of tylosin tartrate in the culture medium was determined by HPLC.We found that the tylosin tartrate was metabolized 20%in primary cultured hepatocytes over 60 min.But the metabolism was significantly decreased to10%with ketoconazole present.The results indicate that CYP3A is involved in the metabolism of tylosin tartrate.5.The effects of tylosin tartrate on the expression of CYP1A,CYP3A and P-gp in liver and intestine cells Study the effects of tylosin tartrate on the expression of CYP1A,3A and P-gp in vitro and in vivo by using the primary hepatocytes,Caco-2 cells and broiler chickens.In vitro study,there are four groups about primary hepatocytes and Caco-2 cells,they are high dose group,middle dose group,low dose group and control group,respectively.In vivo study,there are two groups,tylosin tartrate group(test group)and control group(blank group).The mRNA levels were detected by real-time reverse transcriptase polymerase chain reaction(Real-time qPCR).The protein levels were detected by western-blot The results showed that CYP1A4,CYP1A5 and CYP3A37 were inhibited significantly in hepatocytes when the doses of tylosin tartrate are 10μg/mL and 30μg/mL.The effects of tylosin tartrate on the Mrna expression of P-gp were inhibited in Caco-2 cells.And the results about the effects of tylosin tartrate on the expression of CYP1A and 3 A in chickens were correlated with the results in hepatocytes.The effects of tylosin tartrate on mRNA expression of P-gp in chickens were correlated with the results in Caco-2 cells.The results suggest that there are inhibition of tylosin tartrate on the expression of CYP1A,3A and P-gp,thereby affecting its pharmacokinetic characteristics or cause drug-drug interaction.6,Preliminary studying the methods of improving the oral bioavailability of tylosin One day old AA broiler chickens were fed drug free feeds until they were 42 days old.One hundred and forty healthy male broilers were randomly divided into seven groups in this study and each group has 20 chickens to investigate the effects on the oral bioavailability of tylosin,from the salt form,structure optimization and inhibiting first-pass effect three facts,respectively.In salt form test,the oral bioavailability of tylosin tartrate and tylosin phosphate were investigated in broiler chickens.In structure optimization test,the oral bioavailability of tylosin tartrate and acetylisovaleryltylosin tartrate were investigated.In inhibiting first-pass effect test,the oral bioavailability of tylosin tartrate was investigated with ketoconazole and verapamil presence or not.The results showed that:in salt form test,after intravenous administration there were no significant differences between tylosin tartrate and tylosin phosphate.After oral administration,there were significant differences in the Cmax(0.09±0.01,0.22±0.09)and AUC(0.56±0.082,0.89±0.12)between tylosin phosphate and tylosin tartrate.The oral bioavailability of tylosin tartrate is higher than tylosin phosphate by improved oral absorption after oral administration;in structure optimization test,after intravenous administration there were significant differences in the Vd(0.61±0.07,0.29±0.04 L/kg)and T1/2ke(1.98±0.21,1.50±0.21 h)between acetylisovaleryltylosin tartrate and tylosin tartrate.After oral administration,there were significant differences in the Tpeak(0.76±0.38,1.87±0.38 h),AUC(1.15±0.12,0.82±0.16),T1/2ke(1.86±0.44,1.46±0.44 h)and F(12.6%.16.7%)between acetylisovaleryltylosin tartrate and tylosin tartrate.The oral bioavailability of acetylisovaleryltylosin tartrate is higher than tylosin tartrate with rapid absorption,wide distribution,slowed metabolism after oral administration;in inhibiting first-pass effect test,there was significant differences in the F(12.1%,14.4%and 14.5%)between control,verapamil and ketoconazole group,the oral bioavailability of tylosin tartrate is improved with ketoconazole presence by inhibiting CYP3A mediated elimination in liver,the oral bioavailability of tylosin tartrate is improved with verapamil presence through inhibiting P-gp mediated efflux.