Tetracycline antimicrobials are bacteriostatic agents that act by binding to the bacterial 30S ribosomal subunit to inhibit protein synthesis. These agents have broadspectrum activity and have been used for the treatment of various microbial infections including those caused by anaerobic bacteria, Chlamydophila (Chlamydia) spp, and Rickettsia spp. Minocycline is a second-generation antimicrobial of the tetracycline group and has greater lipophilicity and antimicrobial activity than other tetracycline agents.1,2 The excellent lipid solubility of minocycline leads to good distribution in various tissues throughout the body, including penetration of the blood-brain and blood-ocular barriers.3 In dogs, rats, and humans, tissue concentrations of minocycline are generally higher than their serum concentrations.3–5
Minocycline is more active against gram-positive bacteria than gram-negative bacteria.6 Gram-positive bacteria of particular relevance include tetracycline-resistant strains or methicillin-resistant strains of Staphylococcus aureus, and pharmacokineticpharmacodynamic models for activity against S aureus have been reported.2,7 A study5 involving humans revealed minocycline is rapidly and completely absorbed after oral administration and has a prolonged biological half-life of approximately 16 hours. The long half-life allows minocycline to be used effectively to treat infections with administration of 1 or 2 doses/d.
In horses, minocycline may be used when other antimicrobials, such as gentamicin or cephalothin, have failed. This drug is commonly administered at the recommended human dosage (2.2 mg/kg, IV, q 12 h or q 24 h) in horses, but the effect of this dosage has not been evaluated in horses. Tetracyclines are known to cause serious adverse reactions that consist mainly of cardiovascular depression or hypotension in dogs, ruminants, and horses.8–12 For the effective and safe use of antimicrobials, it is important to evaluate their pharmacokinetic and tissue distribution in the target species. Pharmacokinetic data on minocycline are available for dogs,1,8 ruminants,13,14 rabbits,15 and humans.5,16,17 In horses, the pharmacokinetics and tissue distribution of doxycycline, which has a similar structure and bacteriologic properties to minocycline, have been reported18–20 but there is no comparable pharmacokinetic information for minocycline. The purpose of the study reported here was to determine the pharmacokinetics and tissue localization of minocycline in mares after a single IV injection.
Materials and Methods
Horses—Five healthy Thoroughbred mares (age, 3 years; body weight, 476 to 496 kg) kept for research purposes were used in the pharmacokinetic experiment. Six healthy Thoroughbred mares (age, 7 to 13 years; body weight, 476 to 492 kg) that had been withdrawn from racing because of poor performance were used for the tissue distribution experiment. All horses were kept in individual stalls during the experiments and had ad libitum access to grass hay and water. The procedures for both experiments conformed to the guidelines of the Japanese Association for Experimental Animals for the care and use of research animals and was approved by the Animal Care and Use Committee of the Equine Research Institute.
Experimental protocol—Minocycline hydrochloridea was dissolved in saline (0.9% NaCl) solution to achieve a concentration of 2.0 mg of minocycline hydrochloride/mL. A dose of 2.2 mg of minocycline hydrochloride/kg was injected into the left jugular vein of each mare over 5 minutes. Physical examinations, including evaluation of respiratory rate, heart rate, rectal temperature, and the condition of feces and urine, were performed for each mare after drug administration.
Pharmacokinetic experiment
After minocycline was administered, blood samples were obtained at 0 hours (immediately before administration) and at 0.1, 0.5, 1, 2, 3, 6, 9, 12, 24, and 48 hours after administration. Samples were collected via a catheter (1.7 × 133 mm) placed in the right jugular vein. Each blood sample was heparinized, and the plasma was separated and frozen at −40°C pending analysis.
Tissue distribution experiment
The 6 mares were nonrandomly allocated to 1 of 2 groups of 3 horses each. The first group was euthanatized by IV administration of an overdose of thiopentalb and succinylcholinec following sedation with medetomidined at 0.5 hours after minocycline administration. The second group was similarly euthanatized at 3 hours after minocycline administration.
Eighteen tissue and 6 body fluid samples were obtained from each horse immediately after euthanasia, including samples of renal cortex, renal medulla, parotid gland, liver, lung, heart, stomach, uterus, small intestine, trachea, inguinal lymph node, gluteus muscle, diverticulum of the auditory tube (guttural pouch), bone marrow, skin, bulb of heel, flexor tendon, brain, blood (for plasma harvesting), peritoneal fluid, pleural fluid, articular fluid, CSF, and aqueous humor. Tissue samples were blotted dry on filter paper. Fluid samples were centrifuged at 1,800 × g for 5 minutes at 4°C, and the supernatants were collected. All samples were frozen at −40°C pending analysis.
Sample preparation—To measure concentrations of minocycline in the body fluid samples, 0.5 mL of each sample was poured into a glass tube and mixed with 10 μL of phosphoric acid and 2 μg of demeclocyclinee as the internal standard. For analysis of the tissue samples, 0.5 g of the sample fraction (wet weight) was weighed into a glass tube. Six milliliters of 0.1M citrate buffer (pH, 4.0), including 0.2M EDTA disodium salt and 10 μg of the internal standard, was added to the tube, and the tissue fraction was minced and suspended in the solution. The suspended tissue was homogenized with a homogenizerf placed in crushed ice, then centrifuged at 1,800 × g for 30 minutes at 4°C, after which the supernatant solution was collected. When the concentrations of minocycline in the sample exceeded the maximum quantifiable concentration for analysis, the sample solution was diluted with citrate buffer.
Extraction cartridgesg were used to extract minocycline from the body fluid and tissue samples. Each sample solution (1 mL) was applied to a cartridge conditioned with methanol (99.8%) and distilled water. The cartridge was rinsed with a 20% methanol solution (2 mL) and then eluted with 100% methanol (2 mL). The eluate was dried under nitrogen gas at 40°C and resolved to 100 μL of the mobile phase for RP-HPLC; then, 20 μL of the prepared sample was injected onto the RP-HPLC apparatus. The amounts of minocycline recovered from body fluid and tissue samples in this method ranged from 65% to 87%.
RP-HPLC analysis—An RP-HPLC system with UV detectionh was used for the analysis of minocycline. The mobile phase consisted of 0.3% nonafluoropentanoic acidi and acetonitrile (75:25 [vol/vol]), and the flow rate was 1.0 mL/min. A monomeric type of C18 phasej was used.j The column effluent was monitored via UV detection (350 nm),h and areas of peaks were calculated by use of a computer program.h Minocyclinek standards were prepared at concentrations ranging from 0.1 to 16.0 μg/mL for each fluid sample and from 0.25 to 16.0 μg/g for each tissue sample. All analyses of the standards and unknown samples were performed in duplicate. The limit of quantification that was defined as the sample concentration resulting in a peak area of signal-to-noise ratio of 3.0 were 0.1 μg/mL for the fluid samples and 0.25 μg/g for the tissue samples in this analysis. All standard curves were constructed via linear regression analysis in which peak area ratios (peak area of minocycline divided by that of the internal standard) were regressed against the concentrations of standards, and the coefficient of correlation for all measured curves exceeded 0.999. The intraday and interday precision for plasma at a concentration of 0.2 μg/mL were 4.92% and 5.45%, respectively, and those for tissue samples (liver) at a concentration of 0.5 μg/g were 5.91% and 7.59%, respectively.
In vitro plasma protein binding—Binding of minocycline to plasma proteins was determined via equilibrium dialysis. Pooled drug-free plasma samples were obtained from mares that did not receive any medication. The plasma samples were spiked with minocycline at a concentration of 1.0 μg/mL and equilibrated against PBS solution (pH, 7.4) at 37°C for 18 hours. After the equilibrium dialysis procedure, concentrations of minocycline in the plasma and PBS solution were measured by means of RP-HPLC, and the protein binding was calculated by use of the following equation:
in which Cp and Cf are the concentrations of minocycline in plasma and PBS solution after the dialysis, respectively, and V and V0 are the plasma volumes after and before the dialysis, respectively.
Pharmacokinetic analysis—Plasma concentrations of minocycline versus time for each mare were plotted on a semilogarithmic graph, and the plotting curves were analyzed with a computer curve-fitting program.l The best fit of the proposed compartment model to the experimental data was determined on the basis of the Akaike information criterion, and it was obtained by use of a 2-compartment model represented by the following equation:
in which Ct is the concentration of drug at time t; C1 and C2 are the y-axis intercepts for the distribution and elimination phases of the curve, respectively; and α1 and α2 are the slopes of the distribution and elimination phases of the curve, respectively.
The AUC and AUMC from 0 to 24 hours were calculated by use of the trapezoidal method. The AUC0-∞ and AUMC0-∞ were calculated by adding the terminal portion of the curve, estimated from the plasma concentration at 24 hours divided by α2, to the AUC and AUMC from 0 to 24 hours.
The MRT was calculated as the AUMC0-∞ divided by the AUC0-∞. The Vdarea was calculated as follows:
The Vdss was calculated as follows:
The t1/2α and t1/2β were calculated as the natural logarithm of 2 divided by α1 and α2, respectively.
Results
Animals—No signs of unusual physical conditions were detected in any mare after minocycline administration.
Pharmacokinetic experiment—The mean plasma concentration-time curve for minocycline and the theoretical curve calculated from the biexponential equation after IV administration were constructed (Figure 1). The clearance of minocycline from plasma can be described as having rapid and slow disposition phases and as following the biexponential equation. Drug was detected in plasma samples from all mares at 24 hours after minocycline administration but was not detected in any mares at 48 hours. Values of pharmacokinetic parameters were summarized (Table 1). The percentage of binding of plasma protein to minocycline was 68.1 ± 2.6%. Plasma concentrations of free minocycline were calculated as the percentage of protein binding. The concentration was 0.12 μg/mL at 12 hours, and the AUC from 0 to 24 hours in a dose of 2.2 mg/kg, IV, every 12 hours was estimated as 10.3 μg × h/mL.
Mean ± SD values of pharmacokinetic variables associated with minocycline hydrochloride (2.2 mg/kg, IV, once) administration in 5 mares.
| Variable | Value |
|---|---|
| Body weight (kg) | 488 ± 8 |
| C1 (μg/mL) | 3.56 ± 0.66 |
| C2 (μg/mL) | 1.18 ± 0.08 |
| λ1 (h−1) | 2.66 ± 0.45 |
| λ2 (h−1) | 0.09 ± 0.02 |
| AUC0-∞ (μg × h/mL) | 14.5 ± 4.0 |
| AUMC0-∞ (μg × h2/mL) | 156 ± 93 |
| CL (L/h/kg) | 0.16 ± 0.04 |
| MRT (h) | 10.10 ± 2.86 |
| Vdarea (L/kg) | 1.69 ± 0.07 |
| Vdss (L/kg) | 1.53 ± 0.09 |
| t1/2α (h) | 0.27 ± 0.04 |
| t1/2β (h) | 7.70 ± 1.91 |
C1 and C2 are the y-axis intercepts for the distribution and elimination phases of the curve, respectively, and λ1 and λ2 are the slopes of the distribution and elimination phases of the curve, respectively.
CL = Total body clearance.
Mean ± SD plasma concentrations (solid line) of minocycline in 5 Thoroughbred mares after a single IV injection (2.2 mg of minocycline hydrochloride/kg). The theoretical curve calculated from a biexponential equation is represented by a dotted line.
Citation: American Journal of Veterinary Research 71, 9; 10.2460/ajvr.71.9.1062
Tissue distribution study—At 0.5 hours after drug administration, minocycline was detectable in 17 tissue samples and 4 fluid samples (Table 2). The highest minocycline concentration was in renal tissue, in which concentrations in tissues from the renal cortex and medulla were 12.6 and 6.0 times as high as those in the plasma, respectively. The minocycline concentrations in parotid gland, liver, and lung tissues were also 3.5 to 4.6 times as high as those in the plasma. In contrast, minocycline concentrations in peritoneal fluid, pleural fluid, bulb of heel, tendon, and articular fluid were only 10% to 30% of those in the plasma. Minocycline was not detected in brain tissues, CSF, or aqueous humor.
Mean ± SD concentrations of minocycline in tissue (μ/g) and body fluid (μ/mL) samples, and the mean ± SD ratio of sample to plasma concentration in 3 mares at 0.5 and 3 hours after a single IV injection of minocycline hydrochloride (2.2 mg/kg).
| Sample | Concentration | Concentration ratio (sample vs plasma) | ||
|---|---|---|---|---|
| 0.5 hours | 3 hours | 0.5 hours | 3 hours | |
| Tissue | ||||
| Brain | − | 0.77 ± 0.45 | − | 0.48 ± 0.24 |
| Guttural pouch | 1.84 ± 0.25 | 2.62 ± 0.63 | 0.58 ± 0.08 | 1.65 ± 0.28 |
| Parotid gland | 14.7 ± 0.38 | 7.68 ± 0.76 | 4.62 ± 0.38 | 4.88 ± 0.31 |
| Trachea | 3.16 ± 0.36 | 4.62 ± 0.18 | 1.00 ± 0.19 | 2.94 ± 0.19 |
| Lung | 11.2 ± 1.15 | 4.70 ± 1.12 | 3.52 ± 0.37 | 2.99 ± 0.69 |
| Heart | 6.18 ± 1.19 | 3.35 ± 0.48 | 1.93 ± 0.24 | 2.14 ± 0.38 |
| Stomach | 5.26 ± 0.59 | 3.38 ± 0.63 | 1.65 ± 0.20 | 2.14 ± 0.23 |
| Small intestine | 3.68 ± 0.87 | 2.26 ± 0.19 | 1.15 ± 0.23 | 1.44 ± 0.10 |
| Liver | 11.5 ± 3.45 | 7.22 ± 1.63 | 3.56 ± 0.72 | 4.59 ± 0.93 |
| Renal cortex | 40.3 ± 7.59 | 12.1 ± 1.50 | 12.7 ± 2.78 | 7.74 ± 1.21 |
| Renal medulla | 19.3 ± 8.20 | 10.4 ± 2.10 | 6.07 ± 2.76 | 6.70 ± 1.95 |
| Uterus | 4.20 ± 0.38 | 3.24 ± 0.55 | 1.32 ± 0.04 | 2.08 ± 0.45 |
| Inguinal lymph node | 3.16 ± 0.90 | 2.29 ± 0.04 | 0.98 ± 0.18 | 1.46 ± 0.11 |
| Bone marrow | 1.57 ± 0.63 | 0.98 ± 0.09 | 0.49 ± 0.19 | 0.62 ± 0.01 |
| Bulb of heel | 0.65 ± 0.09 | 1.14 ± 0.28 | 0.20 ± 0.04 | 0.72 ± 0.12 |
| Gluteus muscle | 3.00 ± 0.99 | 2.68 ± 0.16 | 0.95 ± 0.35 | 1.71 ± 0.20 |
| Skin | 1.47 ± 0.44 | 2.72 ± 0.91 | 0.46 ± 0.16 | 1.70 ± 0.44 |
| Flexor tendon | 0.43 ± 0.05 | 0.58 ± 0.18 | 0.14 ± 0.03 | 0.37 ± 0.11 |
| Fluid | ||||
| Articular fluid | 0.32 ± 0.11 | 0.45 ± 0.06 | 0.10 ± 0.04 | 0.29 ± 0.02 |
| Aqueous humor | − | − | − | − |
| CSF | − | 0.30 ± 0.13 | − | 0.19 ± 0.07 |
| Peritoneal fluid | 0.85 ± 0.19 | 0.97 ± 0.13 | 0.27 ± 0.06 | 0.61 ± 0.03 |
| Plasma | 3.19 ± 0.29 | 1.57 ± 0.13 | 1.00 ± 0.00 | 1.00 ± 0.00 |
| Pleural fluid | 0.80 ± 0.06 | 0.82 ± 0.05 | 0.25 ± 0.03 | 0.52 ± 0.04 |
— = Not detected.
Gutteral pouch is the diverticulum of the auditory tube.
At 3 hours after drug administration, minocycline was detectable in all tissue and fluid samples, with the exception of aqueous humor. Plasma minocycline concentration at 3 hours decreased to half of the concentration at 0.5 hours; however, concentrations in 9 types of tissue samples were higher than at 0.5 hours. The ratios of tissue concentrations to plasma concentrations were larger in 20 tissue samples at 3 hours than at 0.5 hours. None of the aforementioned differences were statistically tested.
Discussion
In the study reported here, the pharmacokinetics and tissue distribution of minocycline were evaluated in healthy mares. With regard to the adverse effects of tetracycline antimicrobials in horses, IV administration of oxytetracycline and doxycycline can induce cardiovascular collapse.9,10 Therefore, it has been suggested that the IV administration of doxycycline should be discouraged in horses.11 Similarly, rapid IV injection of minocycline can cause cardiovascular depression in dogs.8 In our study, we did not notice any clinical signs of adverse effects in any of the mares after a single IV injection of minocycline hydrochloride at a dose of 2.2 mg/kg. On the basis of this result, we cannot determine whether this route of minocycline administration is safe in horses. However, this treatment has been used in racehorse clinics in our country, and serious adverse effects are rarely reported.
In our pharmacokinetic analysis, the clearance of minocycline in the mares after a single IV administration was adequately described by a 2-compartment biexponential equation. In studies13,15,16 involving other animal species, the disposition of minocycline after IV injection was also characterized by a 2-compartment model. The mean total body clearance of minocycline in the study mares was similar to that in dogs8; however, it was approximately half that in sheep14 and double that in humans.17 On the other hand, the t1/2β of minocycline in the study horses was shorter than that in humans.17 Minocycline is reportedly metabolized in the liver, and the parent compound and metabolites are eliminated in the urine and feces.3 Species differences in rates of hepatic biotransformation and renal elimination may explain the variation evident in these kinetic values, suggesting that the therapeutic effects of minocycline may vary among species. The Vdss of minocycline in the study mares was similar to that in dogs, sheep, and humans,8,14,17 whereas the percentage binding of minocycline to plasma proteins was lower in the mares than in other species.1,5,13 The values of Vdss and protein binding obtained in our study suggest that minocycline has good penetration in equine tissues.
In the tissue distribution experiment, minocycline was detected in almost all types of tissues and body fluids evaluated at 0.5 hours after IV injection. These data suggested that this antimicrobial can penetrate rapidly into the potential sites of infection in horses. Plasma minocycline concentration at 0.5 hours was approximately twice that at 3 hours; however, the concentrations in some tissue samples were higher at 3 hours than at 0.5 hours. This finding indicated that distribution equilibrium of minocycline was not attained in those tissues at 0.5 hours after administration. High minocycline concentrations, > 3 times the plasma concentrations, were detected in renal, parotid gland, liver, and lung tissues, and the highest concentration was detected in renal cortical tissue. In a study3 of tissue distribution of minocycline in dogs, high concentrations were detected in renal, liver, and lung tissue but not in parotid gland tissue and the highest concentration was detected in liver tissue. In our study, minocycline was not detected in aqueous humor samples but it was detected in the aqueous humor samples in the canine study. These discrepancies may have arisen from differences in the injection dose and collection time of the tissue samples. In the canine study, a higher dose (10 mg/kg, IV) of minocycline was administered, with tissue samples collected 4.5 hours afterward. Doxycycline was also detected in the aqueous humor of horses in 1 study,18 but it was not detected in another study.20 That discrepancy was explained by the difference in dose.18 The minocycline dose used in the present study may be inadequate for diffusion into healthy eyes at 3 hours after administration.
The pharmacodynamics of minocycline activity against S aureus strains in an in vitro pharmacokinetic model have been reported.6 The AUC:MIC ratio based on free-drug serum concentrations was best explained by the antimicrobial effect, and the effective ratio was ≥ 33.9. For bacteria with an MIC ≤ 0.3 μg/mL, a dose of 2.2 mg of minocycline/kg, IV, every 12 hours will achieve this ratio in horses. This would include Staphylococcus spp isolated from horses.6 The plasma concentration of free minocycline at 12 hours after drug administration in that study was equal to the MIC of Streptococcus spp; therefore, an IV administered dose of 2.2 mg/kg every 12 hours would maintain trough plasma concentrations higher than the MIC for Streptococcus spp and should be efficacious against infections with these bacteria in horses. For less susceptible bacteria, use of a higher dose or more frequent administration of minocycline may be required. The pharmacokinetics and tissue distribution data in the present study will contribute to appropriate use of minocycline in horses.
ABBREVIATIONS
| AUC0-∞ | Area under the plasma concentration vs time curve from 0 to infinity |
| AUMC0-∞ | Area under the first moment curve from 0 to infinity |
| MIC | Minimal inhibitory concentration |
| MRT | Mean residence time |
| RP-HPLC | Reverse-phase high-performance liquid chromatography |
| t1/2α | Distribution half-life |
| tl/2β | Elimination half-life |
| Vdarea | Apparent volume of distribution based on area under the curve |
| Vdss | Apparent volume of distribution at steady state |
Minopen, Sawai Pharmaceutical Co, Yodogawa, Osaka, Japan.
Ravonal, Mitsubishi Tanabe Pharma Co, Osaka, Japan.
Relaxin injection, Kyorin Pharmaceutical Co, Tokyo, Japan.
Domitor, Nippon Zenyaku Kogyo Co, Fukushima, Japan.
Demeclocycline hydrochloride, Sigma-Aldrich Co, St Louis, Mo.
Polytron-aggregate, Kinematica Inc, Littau-Lucerne, Switzerland.
Oasis HLB extraction cartridge, Waters Co, Milford, Mass.
Prominence, Shimadzu Co, Tokyo, Japan.
Nonafluoropentanoic acid, Sigma-Aldrich Co, St Louis, Mo.
COSMOSIL 5C18-MS-2, Nacalai Tesque Inc, Kyoto, Japan.
Minocycline hydrochloride, Sigma-Aldrich Co, St Louis, Mo.
SAAM- II program, University of Washington, Seattle, Wash.
References
- 1.
Barza M, Brown RB, Shanks C, et al. Relation between lipophilicity and pharmacological behavior of minocycline, doxycycline, tetracycline, and oxytetracycline in dogs. Antimicrob Agents Chemother 1975; 8:713–720.
- 2.↑
Steigbigel NH, Reed CW, Finland M. Susceptibility of common pathogenic bacteria to seven tetracycline antibiotics in vitro. Am J Med Sci 1968; 255:179–195.
- 3.↑
Kelly RG, Kanegis LA. Metabolism and tissue distribution of radioisotopically labeled minocycline. Toxicol Appl Pharmacol 1967;11:171–183.
- 4.
Colovic M, Caccia S. Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat. J Chromatogr B Analyt Technol Biomed Life Sci 2003; 791:337–343.
- 5.↑
Macdonald H, Kelly RG, Allen ES, et al. Pharmacokinetic studies on minocycline in man. Clin Pharmacol Ther 1973; 14:852–861.
- 6.↑
Ensink JM, van Klingeren B, Houwers DJ, et al. In-vitro susceptibility to antimicrobial drugs of bacterial isolates from horses in The Netherlands. Equine Vet J 1993;25:309–313.
- 7.
Karen EB, Alan RN, Alasdair PM. Pharmacodynamics of minocycline against Staphylococcus aureus in an in vitro pharmacokinetic model. Antimicrob Agents Chemother 2008; 52:4370–4373.
- 8.↑
Wilson RC, Kitzman JV, Kemp DT, et al. Compartmental and noncompartmental pharmacokinetic analyses of minocycline hydrochloride in the dog. Am J Vet Res 1985; 46:1316–1318.
- 9.
Gyrd-Hansen N, Rasmussen F, Smith M. Cardiovascular effects of intravenous administration of tetracycline in cattle. J Vet Pharmacol Ther 1981; 4:15–25.
- 10.
Potter WL. Collapse following intravenous administration of oxytetracycline in two horses. Aust Vet J 1973; 49:547–548.
- 11.↑
Riond JL, Riviere JE, Duckett WM, et al. Cardiovascular effects and fatalities associated with intravenous administration of doxycycline to horses and ponies. Equine Vet J 1992; 24:41–45.
- 12.
Riond JL, Duckett WM, Riviere JE, et al. Concerned about intravenous use of doxycycline in horses (lett). J Am Vet Med Assoc 1989; 195:846–848.
- 13.
Ziv G, Sulman FG. Analysis of pharmacokinetic properties of nine tetracycline analogues in dairy cows and ewes. Am J Vet Res 1974; 35:1197–1201.
- 14.↑
Wilson RC, Green NK. Pharmacokinetics of minocycline hydrochloride in clinically normal and hypoproteinemic sheep. Am J Vet Res 1986; 47:650–652.
- 15.↑
Nicolau DP, Freeman CD, Nightingale CH, et al. Pharmacokinetics of minocycline and vancomycin in rabbits. Lab Anim Sci 1993; 43:222–225.
- 16.
Yamamoto T, Takano K, Matsuyama N, et al. Pharmacokinetic characteristics of minocycline in debilitated elderly patients. Am J Ther 1999; 6:157–160.
- 17.↑
Savin S, Houin G. Clinical pharmacokinetics of doxycycline and minocycline. Clin Pharmacokinet 1988; 15:355–366.
- 18.↑
Davis JL, Salmon JH, Papich MG. Pharmacokinetics and tissue distribution of doxycycline after oral administration of single and multiple doses in horses. Am J Vet Res 2006; 67:310–316.
- 19.
Womble A, Giguère S, Lee EA. Pharmacokinetics of oral doxycycline and concentrations in body fluids and bronchoalveolar cells of foals. J Vet Pharmacol Ther 2007; 30:187–193.
- 20.↑
Gilmour MA, Clarke CR, Macallister CG, et al. Ocular penetration of oral doxycycline in the horse. Vet Ophthalmol 2005; 8:331–335.
