Concentrations of Trimethoprim and Sulfamethoxazole in Cerebrospinal Fluid and Serum in Mares With and Without a Dimethyl Sulfoxide Pretreatment Sherril L. Green, I.G. Mayhew, Murray P. Brown, Ronald R. Gronwall and Gabrielle Montieth

ABSTRACT Each of seven mares was given an intravenous (IV) injection of 40% dimethyl sulfoxide (DMSO) at a dosage of 1 g/kg, over 35 min, immediately followed by a single IV injection of a trimethoprim (TMP) and sulfamethoxazole (SMZ) combination (SMZ 83%, TMP 17%) at a combined dosage of 44 mg/kg (7.48 mg/kg TMP; 36.52 mg/kg SMZ). Each horse served as its own control and was alternately treated with an identical dose of TMP-SMZ treatment alone at least seven days following or preceding the DMSO and TMP-SMZ treatment. Serum and cerebrospinal fluid (CSF) concentrations of TMP and SMZ were measured over a six hour period. Dimethyl sulfoxide treatment caused no significant difference in the mean serum concentration of SMZ or in the mean CSF concentrations of TMP or SMZ. The mean serum concentration of TMP was significantly (p < 0.05) increased at the two, four and six hour sampling time in the mares receiving pretreatment with DMSO. The clearance of TMP was also significantly (p < 0.05) decreased from 675 mL/h/ kg to 327 mL/h/kg by DMSO administration. Concentrations of TMP and SMZ in the CSF in both treatment groups exceeded the minimum inhibitory concentrations for

many common bacterial pathogens of significatives au niveau des concentraequine origin. In addition, CSF tions seriques en SMZ et dans les concentration of TMP exceeded the concentrations en TMP et SMZ du serum concentrations required for liquide cephalorachidien. Les concen50% inhibition of dihydrofolate trations seriques en TMP furent reductases of protozoan origin. Serum augmentees de fason significative TMP and SMZ concentration were (p < 0,05) apres deux, quatre et six similar to those reported to be heures chez les chevaux ayant resu un effective against Toxoplasma gondii traitement avec du DMSO. La vitesse in in vitro studies on the killing or d'elimination du TMP passa de 675 a inhibition of the organism. 327 mL/h/kg suite a l'administration de DMSO. Les concentrations de

RESUME

TMP et SMZ notees dans le liquide cephalorachidien depassaient les concentrations minemales inhibitrices

Des injections de dimethylsulfoxide 40% (DMSO) furent faites a sept juments par voie intraveineuse, au dosage de 1 g/kg administre sur une periode de 35 minutes. Immediatement apres, on proceda a une injection par voie intraveineuse de trimethoprime (TMP) et de sulfamethoxazole (SMZ) (combines a raison de SMZ a 83% et de TMP a 17%) a la dose de 44 mg/kg (7,48 mg/kg, TMP; 36,52 mg/ kg, SMZ). Chaque cheval etant son propre controle, ils furent traites de faqon alternative avec une dose identique de TMP-SMZ seule au moins sept jours avant ou apres avoir resu une dose combinee de DMSO et TMP-SMZ. Les concentrations de TMP et SMZ furent mesurees sur une periode de six heures dans le liquide cephalorachidien. Le traitement au DMSO n'amena pas de differences

requises pour la plupart des bacteries pathogenes rencontrees chez le cheval. De plus, les concentrations seriques en TMP depassaient les besoins pour obtenir 50% d'inhibition de la reductase dihydrofolique qui provient des protozoaires. Les concentrations seriques de TMP et de SMZ etaient similaires a celles rapportees comme etant efficaces contre Toxoplasma gondii lors d'etudes faites in vitro en regard de la destruction ou la suppression de croissance du

microorganisme. INTRODUCTION The inability of many therapeutic agents to adequately penetrate the blood-brain fluid barrier (BBB) has been a major limiting factor in the

Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610-0126 (Green, Mayhew, Brown, Gronwall) and Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2WI (Montieth). Present address of Dr. S. L. Green: Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N I G 2W 1. Present address of Dr. 1.G. Mayhew: Department of Clinical Studies, Animal Health Trust, Belaton Lodge, P.O. Box 5, Newmarket, Suffolk, CB8 7DW, England. Supported by the American Association of Equine Practitioners, Hoffman-LaRoche Inc., Nutley, New Jersey and Syntex Animal Health Inc., West Des Moines, Iowa. Submitted July 13, 1989.

Can J Vet Res 1990; 54: 215-222

215

management of central nervous system (CNS) disease (1-6). Various techniques, such as hypercapnia, intracarotid infusion of hypertonic solutions and systemic hypertension, can partially open the BBB, but the results are not consistent or predictably reversible (7-9). The ability of dimethyl sulfoxide (DMSO), a powerful aprotic, hygroscopic solvent, to penetrate many biological membranes without irreversible damage is well established (10-12). However, the potential therapeutic use of DMSO for the purpose of increasing the deliverance of other substances across the BBB has been the subject of conflicting reports (10-20). There have been reports of the ability of DMSO to reversibly open the BBB to the protein tracer horseradish peroxidase and of its ability to increase serum and brain tissue concentration of ketaconazole and several antineoplastic drugs (13,18,20,21). The exact mechanism by which DMSO may reversibly increase the permeability of the BBB to some substances is not known. Possible explanations include its exchange and interchange for water across biological membranes and the differential effect of DMSO on the compound to be delivered, depending on its lipid solubility, effective diameter, degree of polarity and molecular weight (11,12). It has been suggested that an upper limit of 70,000 daltons exists for the size of substances that can be delivered across the BBB upon exposure to DMSO (18). Also, it has been proposed that, the more water soluble an agent is, the greater the enhancement of DMSO's assisted penetration of it into cerebrospinal fluid (CSF) (10-12,18). Dimethyl sulfoxide may also stimulate pinocytosis and thus increase the transport of substances across the microvascular endothelium or cause a transient opening of the tight junctions between the continuous endothelial cells which compose the normal BBB (18). In addition to its direct effects, DMSO may indirectly alter the BBB by inducing systemic hypertension, hyperosmosis and expansion of the intravascular volume (18).

Trimethoprim (TMP)-sulfamethoxazole (SMZ) a combination antimicrobial, was selected for this

216

nous processes of the sixth lumbar (L6) and the second sacral vertebra (52)(29). Using aseptic technique, 6.0 mL of 2% lidocaine hydrochloride solution was injected subcutaneously above the LS site. A stab incision through the skin was made with a No. 15 scalpel blade. A 17.5 cm x 1.4 mm outer diameter, 17 gauge Huber point (Tuohy) needle with fitted stylet (Becton, Dickenson & New & Co., Rutherford, New Jersey) was inserted perpendicular to the spinal cord, with the needle bevel directed craniad. The spinal needle was then advanced until entry into the SAS was confirmed by aspiration of freely flowing CSF after the stylet was removed. Approximately 0.50 mL of 1.0% lidocaine was injected into the SAS to provide local analgesia (21). Some horses also required light sedation with xylazine (Rompun-Haver, Mobay Corporation, Animal Health Division, Shawnee, Kansas) (0.5 mg/kg) or restraint with a twitch. A 91.5 cm, 19 gauge teflon catheter (Continuous Epidural Tray, American Hospital Supply, McGraw Park, Illinois) reinforced with a stainless steel guide wire, was passed through the needle and threaded cranially in the subarachnoid space for a distance of about 10 cm. The Huber point needle was then MATERIALS AND METHODS withdrawn over the catheter and the Seven healthy adult mares (388-460 guide wire removed, leaving the kg body weight) were used. Four of the catheter in situ. The catheter was seven were randomly selected to first secured at the exit point from the skin receive treatment with DMSO and with an adhesive foam support pad TMP-SMZ and then to be treated (Continuous Epidural Tray, Ameriwith TMP-SMZ alone seven to nine can Hospital Supply, McGraw Park, days later. The remaining three mares Illinois). A catheter adapter (Continuwere treated first with TMP-SMZ ous Epidural Tray, American Hospialone and then were treated with tal Supply, McGraw Park, Illinois) DMSO and TMP/ SMZ seven to nine was placed on the distal end of the LS days later. The experiments followed SAS catheter to facilitate CSF the guidelines of the Guide to the Care collection with a 10 mL syringe. A 16 and Use of Experimental Animals of gauge 13.75 cm teflon catheter (Abbothe Canadian Council on Animal Cath-T, Abbott Hospital Inc., North Care. Chicago, Illinois) was then aseptically Prior to each treatment, an indwel- placed in the jugular vein. A 90% ling catheter was placed in the DMSO preparation (Syntex Animal lumbosacral (LS) subarachnoid space Health Inc., West Des Moines, Iowa) (SAS) of each mare. The procedure was mixed with normal sterile saline was modified from techniques des- ( 2.5 L). In horses receiving DMSO, cribed for regional subarachnoid and a 40% concentration was delivered IV epidural anesthesia (26-28). Each by gravity flow at a total dose of 1 g/ kg mare was restrained in a stock and the given over 35 min. Immediately after LS intervertebral space determined by DMSO/saline infusion, TMP/SMZ palpation of the depression on the (Bactrim IV Infusion, 80 mg TMP and dorsal midline between the supraspi- 400 mg SMZ per 5 mL, Roche

study because of its broad spectrum of activity against numerous strains of pathogenic organisms commonly encountered in veterinary medicine and its potential use in the treatment of CNS diseases in large animals (2225). Specifically, the use of DMSO to enhance penetrability of this antimicrobial agent across the BBB would be a major initial therapeutic advancement in the treatment of protozoal and bacterial diseases of the CNS in the horse (2,22). There is a need for a method of reversibly altering the BBB which would allow predictable increases in the concentration of a drug in the CSF. Only limited pharmacokinetic information regarding the concentration of the TMPSMZ in equine CSF is presently available. The purposes of this study were (i) to compare the pharmacokinetics of TMP and SMZ in the serum and CSF of mares in the immediate posttreatment period with and without concurrent intravenous (IV) administration of 40% DMSO to mares, and (ii) to determine the concentrations of TMP and SMZ in CSF when the drug is given at 44 mg/ kg as a single IV injection.

Laboratories, Nutley, New Jersey) was administered IV as a bolus dose of 44 mg/kg of the combined drug (7.48 mg/kg TMP; 36.52 mg/kg SMZ). When treatment consisted of TMPSMZ alone, the drug was given IV at the same dosage. In pilot studies, by 6 h posttreatment the concentration of TMP had declined to barely detectable levels by our method of assay. A 6 h sample collection period was selected based on those results. Also, we were most interested in the immediate effects of a single DMSO pretreatment on concentrations of TMP-SMZ in the CSF and serum. Blood samples were collected by venipuncture from the opposite jugular vein at 0 (pretreatment), 5,10,20 and 30 min and 1,2,4 and 6 h after TMP-SMZ administration in both groups. Meningeal inflammation and potential disruption of the BBB was monitored by CSF analysis. Cerebrospinal fluid (5 mL) was collected at 0 (pretreatment), 1,2,4 and 6 h after antimicrobial treatment. At pretreatment and at 4 h posttreatment time, CSF analysis (30) was performed and included white (WBC) and red (RBC) blood cell counts and total protein concentration (Spencer Bright-Line hemacytometer, American Optical Co., Buffalo, New York). At the end of each 6 h sampling period the LS SAS catheters were removed. Seven to nine days later, the LS SAS catheter was placed a second time and the horses were treated with the appropriate regimen, either DMSO and TMP-SMZ or TMP-SMZ alone. Cerebrospinal fluid analysis, as described above, was also performed during the second LS SAS catheterization. Blood and CSF samples were again collected as described. Clotted blood samples were centrifuged and serum harvested. Serum and CSF samples were frozen at -20°C until assayed. Trimethoprim and SMZ concentrations in serum and CSF were determined by high performance liquid chromatography (HPLC) according to a previously described method (31). Serum and CSF samples were prepared by adding 200 ,L of 0.1 M tetrabutylammonium hydroxide and 1 mL of 0.05 M buffer solution (pH 10, Fischer Scientific) to 1 mL of thawed sample and then were vortexed for

10 s. After addition of 4 mL of methylene chloride, the mixture was agitated on an Eberbach horizontal shaker for 2 min. Each sample was centrifuged at 3322 x g for 15 min at 100C. The aqueous layer was aspirated and discarded and 500 ,uL of the organic phase were injected into the HPLC. The HPLC consisted of a dual piston reciprocating pump, variable wave length detector operated at 285 nm and a silica column (Resolves; Fischer Scientific, Orlando, Florida). The mobile phase was a mixture of 976 mL of chloroform, 50 mL of methanol, 2 mL of water (H20) and 0.6 mL of ammonium hydroxide (NH40H). The mobile phase flow rate was 2 mL/ min. Under these conditions the retention times for TMP and SMZ were 7 and 14 min, respectively. Standards were prepared by adding TMP and SMZ to horse serum; standards were extracted in the same way as were the unknown samples. Standard curves were constructed by linear regression analysis, using the area under the absorbance-vs-time curve against TMP and SMZ concentrations. The lowest limit of the assay was 0.02 ,ug of TMP/ mL and 0.05 ,ug of SMZ/ mL. Prior to the experiment, it was verified that the presence of DMSO did not affect the TMP and SMZ assay. DATA ANALYSIS

The serum TMP and SMZ concentration versus time data were fitted to the following mathematical model using a digital computer program that minimized the sum of squared deviations (32) Ct

=

Cl.e-xl.t + C2.e-X2.t

where Ct is the serum concentration of SMZ or TMP, t is the time from administration of SMZ/TMP, e is the base of the Napierian logarithms, and Cl, C2, XI, and X2 are the fitting components of the equation. Total systemic clearance was calculated as the dose divided by the area under the serum concentration versus time curve (AUC) value. The apparent volume of distribution at steady-state (Vss)

Vss = (Dose)(AUMC)

(AUC)2

was calculated as the dose times the area under the moment curve (AUMC) divided by the square of the AUC. AUC was calculated from the

following equation: AUC = C1/X1 + C2/X2 The AUMC was calculated, using the following equation: AUMC = CI/XA/Xl + C2/X2/X2 The mean CSF concentration from the time 0 to 6 h was calculated as the area under the CSF TMP or SMZ concentration versus time data curve, determined using spline interpolation to follow a smooth curve between data points, divided by the 6 h duration of sampling (33). The overall elimination rate constant was equated to X2. The elimination half-life (t/2) was calculated as the ratio of the natural logarithm of 2X (0.693) to X2. Serum and CSF concentrations, AUC, mean residence time (MRT), MRT=AUMC AUC and clearances for TMP and SMZ were analyzed for differences between treatments by the Student's paired t test. P values less than 0.05 (t = 1.934) were considered significant. Due to the low number of samples, and the variability of the trauma produced at each LS catheterization, the CSF analyses were not analyzed statistically for differences between treatment with and without DMSO. The range of values for the CSF RBC, and WBC counts and total protein concentrations were reported as a reference for the expected results from this catheterization technique. RESULTS There were no signs of toxicity associated with the administration of the 40% DMSO solution given over 35 min. One horse did develop muscle tremor after rapid injection of the TMP-SMZ IV infusion. No complication was observed as a result of LS SAS catheterization. Pretreatment CSF analysis at the initial LS SAS catheterization reflected blood contamination of the CSF from the catheterization (Table I). The mean WBC and RBC counts were lower in all horses at the 4 h posttreatment 217

TABLE I. The effect of LS SAS catheter placement on CSF analysis in seven mares 1st catheterization pretreatment 4h 13.00 2.25 11.40 1.93 340.00 327.25 318.46 131.66

CSF parameter WBCa (cells/! L) Mean ± SEM RBCb (cells/,uL) Mean ± SEM Protein mg/dL) Mean ± SEM aWhite blood cell counts bRed blood cell counts

26.42 3.40

31.66 3.33

analysis. At the time of the second catheterization seven to nine days later, CSF WBC, RBC counts and TP concentrations had increased although cell counts were again lower at 4 h posttreatment. Catheters were easily maintained over the 6 h sampling periods and no horse developed signs of infection postcatheterization. Both TMP and SMZ were detectable in the CSF. Dimethyl sulfoxide treatment did not significantly alter the CSF concentration of either drug. The overall mean concentration of SMZ in the CSF when the horses were

18 -

16

g

14 12

~

Clo8

LL.

C

2nd catheterization pretreatment 4h 53.85 35.00 19.87 12.24 5183.95 1772.16 2945.86 1058.37

6 4 2

uI

62.85 10.62

63.00 12.5

treated with DMSO was 5.78 ± 0.647 ,ug/mL compared with 5.64 ± 1.01 ,ug/mL in the trials without DMSO (Fig. 1, Table II). Sulfamethoxazole concentration in the CSF was still increasing at 6 hours posttreatment in both groups. The overall mean CSF concentration of TMP was 0.463 ± 0.68 ,ug/ mL in horses with DMSO and 0.337 ± 0.54 ,ug/ mL in trials without DMSO. Although concentrations of TMP in the CSF of the DMSO treated group were greater, the increase was not statistically significant (Fig. 1, Table III). Treatment with DMSO had no significant effect on the mean concentration of SMZ in the serum but did significantly increase the mean concentration of serum TMP at the 2,4 and 6 h sampling time (Fig. 2, Tables IV and V). The overall mean serum concentrations of SMZ with and without DMSO were 47.3 ± 1.70 ,g/ mL and 46.8 ± 3.90 ,ug/ mL respectively. The overall mean serum concentration of TMP with DMSO was 1.58 ± 0.240 ,ug/ mL and

2.45 ± 0.18 ,ug/ mL without. Dimethyl sulfoxide did not significantly alter the Vss of either TMP or SMZ (p > 0.05). The mean Vss of TMP was 2042 mL/ kg in horses with DMSO and 2794 mL/kg in trials without DMSO. The mean Vss of SMZ was 442 mL/kg with DMSO and 504 mL/ kg in trials without DMSO. Dimethyl sulfoxide had no statistically significant effect on the clearance, MRT or AUC of SMZ (Table VI). The clearance of SMZ was 92.9 mL/ h/ kg and 82.6 mL/ h/kg with and without DMSO respectively. Dimethyl sulfoxide pretreatment did not alter the MRT or the AUC of TMP (Table VII). The only tested result that was significantly changed (p < 0.05) was the clearance of TMP which was decreased from 675 mL/ h/ kg to 327 mL/ h/ kg by DMSO administration (Table VII). Treatment with DMSO did not alter the t/2 values for TMP and SMZ. The t/2 values for TMP were 5.27 ± 2.18 and 3.45 ± 1.78 h with and without DMSO respectively. The t/2 values for SMZ were 3.45 ± 0.48 and 4.78 ± 1.54 h with and without DMSO respectively.

DISCUSSION This study provided evidence that DMSO did not enhance the penetration of TMP-SMZ across the BBB. Neither did it alter the concentration of SMZ in the serum. Mean serum concentrations, Vss, t½2, and clearance values for TMP and SMZ with or without DMSO were similar to those of previous reports (34,35), the

0.9

0.8 CA 0.7

TABLE II. Mean cerebrospinal fluid concentrations of sulfamethoxazole over six hours without and with pretreatment with DMSO

X 0.6

2 0.5 .L 0.4

DMSO

Q 0.3

Hour 0

0

0.2 0.1 0.0

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0

1

2

3

4

5

6

Time (hours)

Fig. 1. Mean cerebrospinal fluid (± SEM) concentrations of trimethoprim (TMP) and sulfamethoxazole (SMZ) in horses given 7.48 mg TMP/kg IV and 36.52 mg SMZ/kg IV with (--) and without (--- ) concurrent dimethyl sulfoxide at 1 g/kg IV. The lines represent the mathematical model fit to the mean concentration.

218

treatment without with

CSF/SMZ (ALg/ mL) 0 0 2.98 3.25 4.89 4.96 7.26 7.42

n 7 7 7 7 7 7 7 7 7 7

SEM

Variance

3.28 without 0.694 7.55 with 1.038 2 6.32 without 0.950 7.74 with 1.051 4 11.57 1.295 without with 0.600 2.52 6.08 6 without 8.41 1.511 0.56 2.20 with 8.44 DMSO: Dimethyl sulfoxide CSF SMZ: Cerebrospinal fluid concentration of sulfamethoxazole *paired t-test (p = 0.05; t = 1.943) 1

t*

p

0.18

0.861

0.04

0.968

0.08

0.936

0.02

0.985

TABLE III. Mean cerebrospinal fluid concentration of trimethoprim over time with and without DMSO

DMSO CSF/TMP Hour treatment n SEM Variance (lg/ mL) 0 without 0 7 with 0 7 I without 7 0.415 0.099 0.069 with 7 0.467 0.071 0.036 2 without 0.443 7 0.078 0.041 with 0.576 7 0.074 0.038 4 without 0.328 7 0.062 0.027 with 7 0.506 0.063 0.028 6 without 0.222 7 0.052 0.019 with 0.342 7 0.027 0.005 DMSO: Dimethyl sulfoxide CSF TMP: Cerebrospinal fluid concentration of trimethoprim *paired t-test (p = 0.05; t = 1.943)

200

,9100 t*

p

50

N

2

CO)

0.34

0.748

E 10

0.97

0.372

0

1.98

0.095

2.24

0.066

co

101 %-

N.=_

2 1

E

differences likely reflecting the higher combined IV dose (nearly three times those used in earlier studies) used in our study. Failure of DMSO to enhance the penetration of TMPSMZ into the CSF may have been due in part to the moderate osmolarity of our 40% DMSO solution or due to the low water solubility of the TMP-SMZ agents; however, the molecular weights of the TMP-SMZ preparation were 290.3 and 253.28 daltons respectively and well within the weight range (up to 70,000 daltons) proposed for the enhancement of penetration of substances across the BBB by DMSO (18). Delayed clearance of TMP from

serum and the increase of TMP concentrations in the serum of horses treated with DMSO have not been reported previously. The mechanism or the biological significance of this interaction of DMSO with TMP is not clear. Despite this finding, the results of this study do not support the use of DMSO to increase the penetration of TMP-SMZ in the CSF in the immediate posttreatment period (within 6 h). Serial measurements of TMP and SMZ in the CSF after a single IV injection have not been previously reported. Although pretreatment with DMSO failed to increase the penetra-

a') i

.

.

.

1

2

3

4

5

6

Time (hours)

Fig. 2. Mean serum concentrations (± SEM) of trimethoprim (TMP) and sulfamethoxazole (SMZ) in horses given 7.48 mg TMP/kg IV and 36.52 mg SMZ/kg IV with ( ) and without (--- ) concurrent dimethyl sulfoxide at 1 g/kg, IV. The lines represent the mathematical model fit to the mean concentration.

tion of TMP-SMZ across the BBB, the results of our study document good penetration of the agent into the CSF. In our study, CSF concentrations of TMP-SMZ after a single IV injection at the combined dose of 44 mg/kg TABLE IV. Mean serum concentration of sulfamethoxazole over time with and without DMSO body weight exceeded the minimal pretreatment inhibitory concentration (MIC) of 0.25 ,ug TMP/ mL-4.75 ,ug sulfadiaDMSO Serum SMZ zine (SDZ)/mL for 90% of the t* Hour treatment (,ug/ mL) n SEM Variance p isolates of equine origin: following 0 without 0 7 Corynebacterium pseudotuberculosis, with 0 7 Staphylococcus sp. and Streptococcus 0.083 without 108.69 7 11.139 868.601 0.68 0.524 with 119.27 7 14.998 1574.627 zooepidemicus (36). In vitro antimi0.166 without 93.80 7 8.163 466.432 0.56 0.597 crobial susceptibility testing for TMPwith 7 100.01 7.297 372.690 SDZ has yielded results comparable to 0.333 without 7 77.83 6.007 252.647 0.94 0.385 those for TMP-SMZ (25,37). In with 85.89 7 4.965 172.542 0.5 without addition, in a study of 94 isolates of 74.68 7 7.227 365.654 0.58 0.586 with 79.65 7 4.119 118.773 obligate anaerobes, including 31 of 1 without 63.16 7 5.220 190.776 0.55 0.604 equine origin, 90% were inhibited by with 66.54 7 2.034 28.974 minimum concentrations of 0.25 MAg 2 without 52.72 7 4.166 121.510 -0.012 0.985 TMP/mL-4.75 MAg SMZ/mL (38). In with 52.64 7 1.908 25.478 3 without 45.83 7 4.172 121.820 -1.12 0.307 our study, the CSF concentrations of with 41.03 7 1.788 22.387 TMP-SMZ exceeded the MIC for 4 without 36.37 7 3.578 89.625 -0.23 0.826 those anaerobes. Although the with 35.34 7 1.468 15.082 reported MIC for TMP-SDZ for 6 without 28.62 7 3.348 78.452 -1.35 0.225 Escherichia coli and Streptococcus with 23.18 7 1.264 11.193 0.5 MAg TMP/mL-9.5 MAg SDZ/ equi, DMSO: Dimethyl sulfoxide mL (36) was slightly higher than Serum SMZ: Serum concentration of sulfamethoxazole *Paired t-test (p = 0.05; t = 1.943) concentrations of TMP or SMZ 219

TABLE V. Mean serum concentration of trimethoprim over six hours, with and without DMSO pretreatment Serum TMP DMSO n SEM (,ug/ mL) Hour treatment without 7 0 0 with 7 0 7 0.894 0.83 without 6.128 7 1.275 with 67.504 7 0.634 0.166 without 4.845 with 7 0.771 5.969 7 0.570 0.333 without 3.804 7 0.533 with 4.833 7 0.789 0.5 without 3.877 with 7 0.430 4.091 7 0.473 1 2.723 without with 7 0.328 3.393 7 0.242 2 1.557 without 7 0.298 2.695 with without 7 0.229 1.268 3 with 7 0.191 1.857 4 without 7 0.166 0.927 with 7 0.206 1.716 7 0.133 6 without 0.597 7 0.176 1.343 with DMSO: Dimethyl sulfoxide Serum TMP: Serum concentration of trimethoprim *Paired t-test (p = 0.05; t = 1.943)

achieved in the CSF in our study, SMZ CSF concentration had not yet peaked at the end of our short 6 h sampling period. In a study of human patients with noninflamed meninges, the tendency for SMZ to accumulate in the CSF was observed (39). Multiple treatments with TMP-SMZ may have a cumulative effect (in addition to maintaining effective serum and CSF concentrations), thereby increasing its usefulness against susceptible strains of E. coli and S. equi, two of the most common pathogens associated with purulent disease of the CNS in the equine species. At present, the treatment of choice for equine protozoal disease of the CNS is the combination of pyrime-

Variance

t*

p

5.596 11.390 2.817 4.157 2.273 1.987 4.360 1.296 1.566 0.752 0.411 0.620 0.367 0.255

0.88

0.411

0.99

0.361

1.16

0.290

0.21

0.837

0.92

0.393

2.51

0.046

2.88

0.028

3.42

0.014

3.72

0.009

0.296 0.123 0.216

thamine and a trimethoprimpotentiated sulfonamide (2). These drugs act synergistically as inhibitors of protozoan dihydrofolate reductase. Information regarding the inhibition of dihydrofolate reductase of equine protozoal CNS pathogens is not currently available. However, serum concentration of TMP required for 50% inhibition of dihydrofolate reductase of the protozoan Plasmodium berghei in experimentally infected mice was 70 nM, (< 0.02 Mg/ mL), and was exceeded by CSF concentrations of TMP in our study (40). In addition, in vitro studies (using mouse cell cultures) on the killing and inhibition of replication of Toxoplasma gondii demonstrated irreversible inhibition and death of the

intracellular organism with serum concentrations of 2 ,ug TMP/ mL and 50 ug SMZ/ mL after 18 h of treatment (41). Serum concentrations of TMPSMZ of this magnitude were achieved in the mares used in this study. Due to the ethical considerations which have limited the study of tissue concentrations of drugs within the CNS and due to the difficulty in obtaining repeated, serial CSF samples from the horse, there has been limited information regarding drug penetration across the BBB, especially in horses. Functional and anatomical differences between BBB and the blood cerebrospinal fluid barrier are recognized; however, they are not completely separate and their differences with respect to their pharmacokinetic compartments is of uncertain clinical significance (1-4). Although ventricular CSF may have higher drug concentrations than in CSF collected from the LS SAS (3,4,5) in the authors' (MPB, IGM) experience ventricular catheterization (42) has been a somewhat difficult procedure and was complicated by difficulties maintaining catheter patency and by postcatheterization infection. Repeated sampling of CSF from a LS SAS catheter proved a workable alternative. Although an area of mild, focal, nonseptic meningitis did develop around the LS SAS catheter, as evidenced by the elevated CSF cell counts and TP concentration, any disruption of the BBB at that site did not statistically alter the pharmacokinetic data between these two catheterizations of horses. Despite the mild inflammatory response around the meninges at each LS SAS catheterization site, the CSF concentrations of TMP and SMZ remained relatively consistent over time. Meningeal

TABLE VI. Mean sulfamethoxazole measurements made with and without DMSO/saline for mean residence time, area under the curve and clearance

DMSO/ saline treatment

SMZ Mean residence time min Mean residence time min Area under curve CF/D x mL/min Area under curve CF/ D x mL/min Clearance mL/min Clearance mL/ min

without with without with without with DMSO: Dimethyl sulfoxide SMZ: Sulfamethoxazole CF/D: Conversion factor/ dose *Paired t-test (p = 0.05; t = 1.943)

220

n 7 7 7 7 7 7

Mean 388.593 288.412 28907.572 24064.404 595.467 664.063

Stderr 52.660 15.514 3519.853 1204.734 76.494 35.161

Variance 19412.198 1684.851 86725594.386 10159701.729 40959.719 8654.189

t*

-1.68

p 0.144

-1.13

0.303

0.80

0.454

TABLE VII. Mean trimethoprim measurements made with DMSO and without DMSO for mean residence time, area under the curve and clearance DMSO/ saline TMP treatment without Mean residence time with Mean residence time without Area under curve CF/D x mL/min with Area under curve CF/D x mL/min without Clearance mL/min with Clearance mL/min DMSO: Dimethyl sulfoxide TMP: Trimethoprim CF/ D: Conversion factor/ dose *Paired f-test (p = 0.05; t = 1.943)

inflammation did not appear to significantly alter the CSF concentrations of TMP-SMZ in studies in humans (39,43). The known diuretic effects of DMSO could be considered and may have possibly masked the effect of pretreatment with the drug. Diuresis was not observed in our DMSO treated animals; however, the analysis of the diuretic effect of DMSO on the CSF and serum concentrations of TMP and SMZ would have required osmolar and hematological data and 24 hour urine collection. Such investigation was beyond the scope of this study. Although the DMSO treated group received DMSO mixed in 2.5 L of saline, it is doubtful that such a volume would cause significant compartmental fluid shifts in a 500 kg animal and significantly alter the plasma and CSF fluid kinetics. Treatment with various DMSO solutions had no effect on the tissue distribution of gentamicin in rats or on the plasma or CSF kinetics of cyclophosphamide in human patients (14,19). Infections of the CNS, whether bacterial or protozoal, constitute a medical emergency. Initially, the highest dosage of the antimicrobial that can be tolerated by the patient should be used. Liver and kidney function must be monitored. Trimethoprim-sulfadiazine have been associated with gastrointestinal disorders and blood dyscrasias in the horse; however, no clinical sign of toxicity was observed in our research horses. Although the optimum dosage of TMP-SMZ is not known, the results of this study show that after a single IV injection of TMP-SMZ, at a dose of 44 mg/kg of body weight,

n 7 7 7 7 7 7

Mean 254.726 421.704 767.583 519.525 4866.110 2003.966

Stderr 47.346 75.533 120.334 224.760 893.199 437.842

adequate concentrations of the drugs are achieved in the CSF for susceptible equine pathogens. Further pharmacokinetic studies are required before recommendations regarding the clinical use of multiple treatments with TMP-SMZ at a dosage of 44 mg/kg body weight can be made.

9.

10.

ACKNOWLEDGMENTS

11.

The authors wish to acknowledge the technical assistance of Betsy Houston and Linda Siedzik.

12.

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Variance 15692.039 37850.310 101362.021 353620.229 5584635.655 1341939.941

t* 1.65

p 0.151

3.37

0.015

-3.46

0.014

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