Pharmacological Research 49 (2004) 487–491 Pharmacokinetics of doxycycline after administration as a single intravenous bolus and intramuscular doses to non-lactating Egyptian goats A.M. Abd El-Aty , A. Goudah , H.-H. Zhou a Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, 12515-Giza, Egypt b Pharmacogenetics Research Institute, Xiang-Ya School of Medicine, Central South University, 88 Xiang-Ya Road, ChangSha, Hunan 410078, PR China Abstract
The pharmacokinetics of doxycycline hydrochloride (DoxHcl) at a dose of 5 mg kg−1 BW was studied after an intravenous (i.v.) bolus and intramuscular (i.m.) injections in non lactating goats. A microbiological assay employing Bacillus subtilis as the test organism wasused to measure its concentrations in serum and urine. Following a single i.v. injection, the serum concentration-time curves of doxycyclinehydrochloride were best represented by a two-compartment open model. The drug was rapidly distributed and slowly eliminated withhalf-lives of distribution (t1/2␣) and elimination (t1/2␤) of 0.52 and 4.62 h, respectively. After i.m. injection of the same dose, the peakserum concentration Cmax was 1.60 ␮g ml−1 attained at 0.86 h (Tmax). Following i.v. and i.m. injections, the concentrations of doxycyclinein urine were much higher than that in serum. Urinary drug concentrations decreased gradually till reaching its lowest detectable level 12and 24 h post-injections, respectively. The extent of serum protein binding percent was 32.8% and the systemic bioavailability was 99.40%after i.m. injection of 5 mg kg−1 BW 2003 Elsevier Ltd. All rights reserved.
Keywords: Doxycycline hydrochloride; Goats; Pharmacokinetics; Bioavailability 1. Introduction
There has been much recent interest worldwide (includ- ing Egypt) in rearing of goats. This is because goats are Doxycycline, a member of tetracycline’s, is a structural superior in many respects to other farm animals. They isomer of the parent molecule and is synthesized from have modest feeding requirements and provide milk, meat, oxytetracycline or methacycline. It differs from tetracy- and hides of excellent quality. Goats are more hardened to cline in that it is more lipophilic (5–10 times) and has a unfavorable environmental conditions of the surrounding greater plasma protein binding capacity, the overall result- than other farm animals. They are more independent and ing in higher tissue penetration, larger volume of distribu- accustomed to live free. The therapeutic usefulness of doxy- tion, better antimicrobial properties and prolonged half-life cycline in goats relates in part to its disposition, thus the both in humans and animals. The antibacterial effect of objectives of this study were to establish the bioavailability doxycycline is best described as interfering with the bind- and pharmacokinetic constants of doxycycline following ing of aminoacyl-tRNA to the mRNA ribosome complex, intravenous and intramuscular injection of 5 mg kg−1 BW thereby hindering the protein synthesis in growing and/or multiplying organisms Doxycycline is an inexpensive,broad-spectrum antibacterial agent that remains the drug offirst choice for several infections such as atypical pneumo- 2. Materials and methods
nias, skin and soft tissue, genitourinary infection includinggonorrhea, syphilis, non-specific uretheritis, and prostatitis pharmacokinetic aspects of doxycycline have beenfully documented in man and several animal species Five clinically healthy, non-lactating Egyptian goats Only a limited data are available in lactating goats weighing from 30 to 35 kg (2-year-old) were used. The an-imals were determined to be clinically normal on the basis ∗ Corresponding author. Tel.: +20-2-7548926; fax: +20-2-5725240.
of a result of physical examination. Body weights were E-mail address: [email protected] (A.M. Abd El-Aty).
recorded for each animal on the day prior to initiation of 1043-6618/$ – see front matter 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phrs.2003.10.012 A.M. Abd El-Aty et al. / Pharmacological Research 49 (2004) 487–491 the study. Each animal was given one week period to ac- climatize to its environment before being used in the studyand maintained indoors on a diet of hay and pelleted feed The extent of doxycycline binding to serum proteins of concentrate and had the access to water ad libitum. The goats was determined in vitro by ultrafiltration study was conducted using two-way crossover design with antimicrobial-naive goat’s serum fortified with a known con- 15 days washout interval between experiments to ensure centration of doxycycline 1.25, 0.625, and 0.312 ␮g ml−1.
complete elimination of any residual drug. The Advisory Samples of 5 ml each were placed into conditioned semiper- Committee constituted by the FVM deemed university miable membrane (Centriflow Cones CF-50, Amicon, Cor- approved the experimental protocol used in the present poration, Lexington, MA, USA) resting on a porous coni- cal polyethylene support on the top of centrifuge tubes. Thetubes were centrifuged at room temperature at 1500g for 45 min. Serum samples and their corresponding ultrafiltrateswere assayed by the same method to determine doxycycline Doxycycline (ADWIA, Cairo, Egypt) powder was dis- solved in sterile distilled water and injected intravenously(i.v.) into the right jugular vein and intramuscularly (i.m.) into the lower third of the right cervical musculature at a doseof 5 mg kg−1 BW. Blood samples of 3 ml each were collected A computerized curve-stripping program (R Strip, Mi- in plastic centrifuge tubes from the contralateral vein just cromath Scientific Software, Salt Lake City, UT, USA) was prior to each treatment (zero time) and at 5, 10, 20, 30, and used to analyze the concentration–time curve for each indi- 45 min and 1, 2, 4, 6, 8, 10, 12, and 24 h post-administration vidual goat after the administration of doxycycline by i.v.
of the drug by both routes. The blood was allowed to clot at and i.m. routes. Following intravenous injection, the dispo- room temperature for 2 h and then the serum was harvested sition curves of doxycycline that express the decline in drug by centrifugation at 3000g for 5 min. The urine samples concentration as a function of time was approximated to a were collected via rubber balloon catheter (Foletex Norta, 14 FR—30 cc, Beijing, China). The catheter was fixed inside the bladder and allowed to discharge its urine content at thetime of sampling. The sampling times for collection of urine where Cop is the concentration of drug in serum at time t, A were 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h post-injection.
is intercept of the distribution phase with the concentration All the samples were stored at −20 ◦C pending assay and axis expressed as ␮g ml−1, B is intercept of the elimination were analyzed within one week of obtainment.
phase with the concentration axis expressed as ␮g ml−1, αis distribution rate constant expressed in units of reciprocal time (h−1), β is elimination rate constant expressed in unitsof reciprocal time (h−1), and e is base of natural logarithm.
Serum and urine doxycycline concentrations were deter- Following i.m. administration, each individual curve mined by using modified agar plate diffusion assay method of doxycycline over the time was analyzed to determine with Bacillus subtilis (ATCC 6633) as a test organism the peak drug concentration Cmax (extrapolated from the growing on Mueller-Hinton agar (Mast Group Ltd., Mersey- curve) and the time to peak concentration Tmax. Elimination side, UK). Six wells (8 mm diameter) were punched at half-life (t1/2el) was calculated as ln(2/β). The areas under equal distances in standard Petri dishes containing 25 ml of the concentration-time curves (AUCs) were calculated by seeded agar. The wells were filled with 0.1 ml of either the the trapezoidal rule and further extrapolated to infinity by test samples or doxycycline standards. The plates were kept dividing the last experimental concentration by the terminal at room temperature for 2 h before incubation at 37 ◦C for slope (β). Mean residence time (MRT) was calculated as 18 h. The drug concentrations were determined by compar- AUMC/AUC, where AUMC is the area under the first mo- ing the diameter of zones of inhibition with those of various ment curve AUC is the area under the curve. The dilutions of standard prepared in pooled antibacterial-free absolute bioavailability (F%), is the percentage of the i.m.
goat’s serum by the use of linear regression analysis. Stan- dose absorbed and calculated from AUCi.m./AUCi.v. × 100.
dards were included in each assay plate to compensate Results are presented as mean ± S.E.M. throughout the for plate-to-plate variation. No recovery was estimated as the serum samples were analyzed without extraction. Theintra-assay CV was 6.66 and inter-assay CV was 11.06. Theresponse of DoxHcl was linear over a range of concentra- 3. Results
tions from 0.20 to 10 ␮g ml−1, with a correlation coefficient(r2) of 0.988 ± 2.6. The mean standard curve coefficient of The present investigation revealed that the serum variance was 10%. The limit of quantification (LOQ) was concentration-time curve of DoxHcl was best fitted to a two- compartment open model following a single i.v. injection of A.M. Abd El-Aty et al. / Pharmacological Research 49 (2004) 487–491 Fig. 1. Mean ± S.E.M. serum doxycycline concentrations following i.v. and i.m. injection of 5 mg kg−1 BW in goats (n = 5).
5 mg kg−1 BW The drug showed a higher serum was 1.60 ␮g ml−1 attained at 0.86 h (Tmax calc). The elimina- p ) 5 min post-injection, subsequently the tion half-life (t1/2el) was 3.65 h and the mean residence time drug concentration decreased gradually over time (MRT) was 2.80 h. The systemic bioavailability of doxycy- Values of pharmacokinetic parameters for DoxHcl after i.v.
cline hydrochloride in goats after i.m. injection of 5 mg kg−1 injection are shown in The drug was rapidly dis- BW was 99.40% and the protein binding percent was 32.8%.
tributed with half-life of distribution (t1/2α) of 0.52 h and Following i.v. and i.m. injections of DoxHcl, the concen- slowly eliminated with half-life of elimination (t1/2β) of trations of DoxHcl in urine were much higher than those 4.62 h. DoxHcl showed a volume of distribution (Vdarea) of in serum. The drug concentrations decreased gradually to 6.48 l kg−1and total body clearance (Cltot) of 0.71 l kg h−1.
reach its lowest level 12 and 24 h post-injections, respec- Following intramuscular injection of DoxHcl at a dose tively, (No concentrations were detected thereafter.
of 5 mg kg−1 BW, the drug was detected in serum 5 minpost-injection. It continued to increase gradually thereafterto reach its maximum concentrations 0.75 h (observed Tmax) post-injection (The serum concentration decreased Mean ± S.E.M. of the pharmacokinetic parameters of doxycycline fol- gradually till reached its minimum level 0.2 ␮g ml−1 8 h lowing i.v. and i.m. injection of 5 mg kg−1 BW in goats (n = 5) post-administration (No doxycycline concentrations were detected thereafter. Values of pharmacokinetic parame- ters for DoxHcl in goats following i.m. injection are shown in calculated peak serum concentration (Cmax calc) t1/2␤ (t1/2el) Mean ± S.E.M. serum doxycycline concentrations following i.v. and i.m.
injection of 5 mg kg−1 BW in goats (n = 5) β: Elimination rate constant; t1/2␣: distribution half-life; t1/2ab: absorption half-life; t1/2␤: elimination half-life; t1/2el: elimination half-life; K12 and K21 first-order rate constants for drug distribution between the central and peripheral compartments; Vdarea: volume of distribution; Cltot: total body clearance; AUC: area under the curve from zero to infinity by the trapezoidal integral; MRT: mean residence time; AUMC: area under the first moment curve; Cmax: maximum serum concentration; Tmax: time to peak concentration; F%: bioavailability.
A.M. Abd El-Aty et al. / Pharmacological Research 49 (2004) 487–491 also lower than that obtained in goats, being 2.29 l kg−1 in Mean ± S.E.M. urine doxycycline concentrations following i.v. and i.m.
cows and ewes 0.93 l kg−1 in dogs The high injection of 5 mg kg−1 BW in goats (n = 5) Vdarea obtained during the present study might be attributed to the wide distribution of the drug coupled with its storage in tissue depots, most likely in fat.
Following intramuscular injection of DoxHcl at a dose of 5 mg kg−1 BW, the serum concentrations of DoxHcl ex- ceeded the MIC90 (<0.25 ␮g ml−1, for most sensitive pathogens for a longer time than after intravenous injection.
The peak serum concentration (Cmax) was 1.60 ␮g ml−1 at- max). This value differs from that recorded after i.m. administration of doxycycline at 20 mg kg−1 BW for pneumonic and healthy goats, 3.87 and 5.56 ␮g ml−1, respectively, differ from that obtained after single intragastric dose to mare, 0.22 ␮g ml−1 1 h post administra-tion similar value was reported in east African goatsafter intramuscular injection of 20 mg kg−1 (1.87 ␮g ml−1 4. Discussion
at 0.85 h, This difference might be attributed to drugformulation, dosage variation and/or the health status of the This study used the bioassay technique to determine the pharmacokinetics of doxycycline in goats. The bioassay The urine concentrations of DoxHcl after i.v. and i.m. in- did not distinguish the active metabolite from the parent jections were much higher than those of serum concentra- compounds. The choice of bioassay method was due to tions and exceeding MIC90 (<0.25 ␮g ml−1, for most cost constraints. By using Akaike’s information criterion sensitive urinary tract pathogens. As the drug maintained at the changes in serum concentrations of the drug af- a very high concentration in urine, it may be of particular ter an i.v. dose of 5 mg kg−1 BW were best fitted to a value in treating urinary tract infections caused by sensitive two-compartment open model. Distribution half-life (t1/2α) values of 0.56, 0.87, and 0.73 h were reported in cows and The systemic bioavailability of DoxHcl in goats after i.m.
ewes, human, and lactating goats respectively.
injection of 5 mg kg−1 BW was 99.4%. This value indicates These values are of similar order to that represented in an excellent absorption of the drug from the site of injection.
this work (0.52 h), suggesting a similar rate of distribu- This finding is in contrast with those reported in calves (70%, tion of doxycycline in goats, cows and ewes, and human.
The value of elimination half-life (t1/2β) reported here In vitro protein binding percent of doxycycline in serum was 4.62 h, the same data was reported both in dogs and of goats was 32.8%, indicating that the drug has a moderate cats 6.99, 4.56 h and pigs 4.04 h differ from tendency to bind to the serum protein. This finding differ that reported in lactating goats (16.63 h, cows and from that recorded both in cats (98.35%) and dogs (91.40%) ewes (24.75 h, and calves (9.5 h, This differ- ence might be attributed to species, interspecies and/or As pharmacokinetic-pharmacodynamic (PK/PD) surro- formulation used. The calculated value for the tissue:serum gate indices (AUIC, AUC/MIC, and Cmax/MIC) for mea- concentration during the elimination phase [k12/(k21 − β)] suring the antibiotic efficacy are only well established for of 2.10 indicate a good distribution of the drug in the pe- ␤-lactams, quinolones and aminoglycosides an addi- ripheral compartments, this fact was supported by the result tional work is needed for others antibiotics including doxy- obtained in lactating goats (4.86, DoxHcl is highly cycline. In addition, owing to the high variations in MIC lipophilic in nature and would be expected to be distributed of sensitive veterinary pathogens, it is more important that more in fatty tissue depots Thus the higher tissue: doxycycline dosage regimens be calculated according to serum concentration obtained in goats compared with that the sensitivity of the individual pathogen, site of infection in cattle may be due to higher muscle fat content of goats and clinical response, than by following a percent dosage The volume of distribution (Vd) of a drug can be calcu- lated by different methods. Vd calculated by the area method(Vdarea) only correctly predicts the distribution of the drug 5. Ultimately
in the body during the longer elimination phase TheVdarea of 6.48 l kg−1 obtained in these goats was near to Since doxycycline is eliminated at very high concentra- that obtained in lactating goats (9.78 l kg−1, and higher tions in urine, this drug should be extremely useful for than those obtained in children’s (0.9 0.75 l kg−1 treating suitable urinary tract infections caused by sensitive in adult humans In other animals, values of Vdarea were A.M. Abd El-Aty et al. / Pharmacological Research 49 (2004) 487–491 References
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