Distribution, tolerability and tissue compatibility ... - Wiley Online Library

Acta Anaesthesiol Scand 1998; 42: 786-793

Copyricghi0 Acia Annesthesid Scnrid 1998 -

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ACTA ANAESTHESIOLOGICA SCANDINAVICA

ISSN 0001-51 72

Distribution, tolerability and tissue compatibility of intrathecal tizanidine in the sheep G. OCHS,M. LOEW,J. T O N N ~ and K. TOYKA Departments of Neurology and 'Neurosurgery, Julius-Maximilians-UniversitutWiirzburg, Germany

Background: Tizanidine (TZD) is an alpha-2-adrenergic drug with potential spinal analgesic action and could be a substitute or additive for intrathecal (i.t.) opiates in chronic pain treatment. However, long-term tolerability and tissue compatibility are not yet established. Methods: Three sheep were infused intrathecally with TZD up to 4000 pg/d over a time period of up to 440 d using implantable drug administration devices; one control animal was infused with vehicle only; standard values were collected from untreated sheep. CSF samples and blood samples were analyzed to determine pharmacokinetics and systemic redistribution. Behavioral effects were studied. The spinal cord tissue was investigated using standard laboratory histology. Results: Bolus kinetics after i.t. injection of TZD are best described by a two-phase model. Elimination half-lives of TZD in CSF were 15 min and 152 min, respectively. Clearance of TZD from lumbar CSF was 0.48 mlimin. Doses higher than 500 pg i.t. caused dose-dependent motor inactivity and sleepi-

(i.t.) and epidural use of opiates is highly effective and well established for chronic pain treatment (2, 13, 19). In addition to the well-known side-effects, such as pruritus, amenorrhea, bladder / bowel dysfunction, nausea, sympathetic dysfunction, and respiratory depression (1, 31, 37), many patients require escalating doses (6). Some pain states, in particular deafferentation pain, respond only poorly to opioids. Those shortcomings limit the viability of long-term i.t. narcotics, especially for patients with non-malignant or neuropathic pain conditions, even though the additive use of spinal local anesthetics improves efficacy in some cases. Spinally acting non-narcotic analgesics such as alpha-2-adrenergic agonists, used either alone or in conjunction with an opioid, can provide greater efficacy and reduce side-effects. Clonidine exhibits antinociceptive effects (38, 39) and is used in pain syndromes relatively unresponsive to opiates, such as allodynia or hyperalgesia due to deafferentation, with reasonable analgesic effect (5, 8). Tizanidine (TZD) is another alpha-2-adrenergic agonist approved and

T

HE INTRATHECAL

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ness. Continuous i.t. TZD up to 4 mg/d was well tolerated regarding behavior, physical activity, heart rate, muscle strength, and coordination. Minor elevations of CSF cell counts and total protein were detected both in saline and TZD-treated animals, presumably due to local irritation by the catheter and repeated sampling procedures. Histological evaluation of the spinal cord and adjacent tissues showed no abnormalities. Conclusion: The long-term intrathecal infusion of TZD is well tolerated and non-toxic in the sheep. The data favor clinical trials in patients with chronic pain. Received 5 August 1997, accepted for publication 11 February 1998

Key words: Tizanidine; intrathecal application; toxicity; pharmacokinetics; alpha-2-adrenergic. 0 Acta Anaesthesiologica Scandinavica 42 (19Y8)

widely used as an oral antispastic (11, 25). Neurophysiological evidence suggests both a spinal and supraspinal site of action (17) and antinociceptive action has been demonstrated in animal models (16, 22). In humans, analgesic properties have been reported for trigeminal neuralgia (14) and a number of other pain conditions (12, 28, 32). Intrathecal TZD can produce long-lasting antinociception comparable to clonidine and morphine in the rat (24). It blocks the hyperalgesic mechanism underlying neuropathic pain, but has less effect than morphine on pain transmission (22, 23). In dogs, i.t. injection of 1000 pg and 2000 pg caused analgesia in a foot-withdrawal paradigm similar to clonidine (20). CSF-concentrations of most i.t. drugs are 100- to 1000-fold higher than with systemic application, for which most of them are approved. In fact, for some drugs, evidence of neurotoxicity has precluded the advancement to i.t. clinical studies (4, 15, 27, 35, 36). Since the main indication for i.t. delivery is chronic, therapy-resistant pain, tests have to be performed in an animal model allowing long-term drug administra-

Intrathecal tizanidine

tion. We therefore used an implantable drug administration device for the i.t. delivery of TZD in a sheep model to evaluate long-term tolerability and clinical applicability of this potentially powerful spinal analgesic.

change in concentration, i.e. 490+87 pg/ml at day 24 (n=lO) and 477278 pg/ml at day 14-16 (n=8). Consequently, the pump reservoir was refilled every 2 weeks or earlier as needed.

Material and methods

The animals were infused continuously over 180 to 441 d with cumulative TZD doses of 126 to 330 mg (see Table 1). In one animal, 16 mg/d was applied for a single day and 8 mg/d for several days after stepwise dose escalation.

D r u g administration and dosing Experimental animals Four female Merino sheep (body weight: 6527 kg, 12 years of age) were sedated by i.m. 0.7 to 1.1 mg/kg xylazin and anesthetized with pentobarbital 0.23 mg kg-' min-l. A silicon rubber spinal catheter (model 8703, Medtronic) was introduced with a Touhy needle at L5/L6 or L6/S1 into the subarachnoidal space and advanced to vertebral level L3. An infusion pump (SynchroMed, Medtronic) was inserted subcutaneously at the lateral aspect of the abdominal wall and connected to the catheter. An Ommaya reservoir was inserted through a bore hole into the lateral ventricle. Both the device and the catheter are standard equipment also used in patients for i.t. therapy of pain or spasticity (30). The infusion device contains a drug reservoir of 20 ml and can be filled through a rubber septum percutaneously. The infusion rate can be telemetrically adjusted. Bolus injections and CSF sampling can be performed through an inborn access port. Pain medication and prophylactic antibiotics were given postoperatively. The treatment with TZD commenced 2 to 3 weeks after surgery. The animals could move freely during the observation period of 7 to 14 months (see: Table 1). They were sacrificed after sedation with 10 ml T61 (HoechstPharma, Frankfurt, Germany). All surgical procedures and animal care were performed strictly according to the national guidelines for animal experimentation. The recommendations of the Committee for Research and Ethical Issues of the IASP (40) have been followed. The protocol was approved by the local Animal Practices Committee and the Bavarian State Authorities.

Drug preparation and application TZD (Sandoz Pharma, Basel, Switzerland), a watersoluble white powder, was reconstituted in saline, sterile filtered and aliquots of 500 pg/ml and 1000 mg/ml were prepared and stored at 5-7°C in lightprotected vials. Injections into the port or the pump reservoir were performed through an external, sterile, 0.22-pm bacterial filter. For the bolus injections, 750 pg TZD, i.e. 1.5 ml of solution, was used. The pump reservoir was filled with TZD solution of 500 yg/ml. Samples taken over 2 weeks showed no

Blood and CSF sample Blood samples were obtained either from the ear vein or the forelimb veins. Standard serum chemistry, i.e. electrolytes, BUN, creatinin, liver enzymes, bilirubine, creatine kinase, total protein, and glucose was performed within 1 h of sampling. Standard values were determined from six untreated animals. TZD steadystate levels in plasma were measured 3 d or more after dose changes. For CSF sampling, the pump was halted and a dead volume of 1 ml was discarded. For bolus kinetics, test samples of 1 ml were taken at 0.5, 1, 2, 3, 6, 9, 12 h after the TZD injection. In addition, 1 ml of spinal and ventricular CSF was taken for standard CSF chemistry and cell counts. CSF analysis, including bacteriological tests, was performed within 1 h of sampling. For the toxicity study, samples were collected 2 to 4 times per month during the entire follow-up period. CSF and blood samples to evaluate TZD concentration were centrifuged for 5 min at 5000 RPM and immediately frozen at - 18°C until later analysis. Drug solution taken from the pump reservoir to verify stability was also frozen for subsequent analysis.

Analysis of T Z D levels TZD plasma and CSF levels were determined by a radio-immuno-assay (RIA).RIA-kits (Sandoz Pharma,

Table 1 ~

____

~

Application characteristics.

Animal #1 (TZD) #2 (TZD) #3 (TZD) #4 (NaCI)

Total time of application Id1

Daily TZD dose Imddl

Daily fluid volume Im13

Cumulative TZD dose [mgl

44 1 312 180 389

0.1-3 0.1-8* 0.1-4

0.2-3 0.2-8 0.2-4 0.2-1

330 317 126

-

-

Application characteristics for the i.t. delivery of TZD. Animal #4 was treated with an average flow rate of 0.5 ml/d of saline. *dose applied for last week only.

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Basel, Switzerland) contained tritium-labeled TZD, TZD standard, standard human plasma samples, the polyclonal antiserum and charcoal (Merck Nr. 2186) for separation. The standard reference material is tizanidine hydrochloride and the quality control sample was analyzed in duplicate each time. Samples from blood and ventricular CSF were used undiluted, lumbar CSF samples were diluted by 500 or 5000, and the TZD solution from the reservoir was diluted by lo5. Plasma samples were deproteinized before measuring. Samples were incubated for 4 h at 4°C before separating by charcoal for 10 min in ice water. Subsequently, they were centrifuged in a refrigerated centrifuge at 1400 g and the supernatant was decanted into vials containing scintillation gel. The limit of quantification is approximately 0.1 ng/ml in sheep CSF. The coefficient of variation (CV) within assays is 14.8 and between assays 19.2 for means of duplicates. Six assays were included in the calculation of the CV. The linear range for the assay system is between 0.5 and 10 ng/ml. According to the manufacturer, the RIA is highly specific and has no interference with the five major known metabolites of TZD.

in 4% paraformaldehyde with phosphate buffer at pH 7.2, rinsed in buffer, and embedded in paraffin. H&E, Nissl, Kluever-Barrera, Masson-Goldner, and Bielschowski stainings were performed on serial 15-ym sections. Nerve roots and dorsal root ganglia were embedded in plastic and semi-thin 1-ym sections were stained with toluidine blue.

Behavioral observations and general aspect of the animals The general appearance, feeding behavior, motor activity, limb strength, and the state of arousal were rat-

Bolus kinetics The time course of the TZD concentration in CSF, CCSF(t),was fitted by a two-phase model according to

1 0

I . . ,

2

I

4

’

I

6

’

I

8

’

’

I

10

I

12

time after bolus injection [hours]

CCSF(t)=Al ’ exp(-t/Tl)+Az eXp(-t/T,). The two time constants T~ and z2 in the exponentials and the amplitudes Al and A2 were calculated by an iterative, non-linear least-square fitting algorithm (software: Origin, MicroCal). The mean values of the data points with weights of l / y were used for the approximation procedure. The half-times tl / 2 were calculated according to tl/,(fast)=ln(2) . z1 and tl/,(slow)=ln(2)~ T ~

’

Fig. 1. Time course of spinal T Z D concentration after i t . bolns injection of 750 pg in 1.5 ml. Data from 3 experiments. T Z D concentrations determined by RIA. Curve obtained from least-square fit of the equation CcsF(t)=A, . exp(-t/r,)+A2 . exp(-tlr2). See methods for details.

7

.

The hypothetical drug concentration Co at time t=O is then defined by Co =Al+Az and the initial volume of distribution Vd can be calculated from the injected dose Vd=dose/Cowhere clearance of TZD from lumbar CSF, CLCSF, was calculated by dividing the dose by the area under the concentration-vs-time curve (AUC)

1.4

=

1,2

Y

B0

5

CLcsF=dose/AUC.

His to1ogy Brain, spinal cord and nerve roots were rapidly removed. Catheter location and size of the spinal canal and cord were measured and the tissue samples were fast frozen at -70°C. Tissue samples were also fixed

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0

I

500

I

1000

I

1500

I

2000

daily intrathecal TZD dose [pglday] Fig. 2. Blood levels under continuous i.t. infusion of T Z D , data pooled from 3 animals, T Z D levels determined by RIA. Linear least-square fit to obtain line according to y=0.031+0.0058. x.

Intrathecal tizanidine

Table 2 CSF findings and physiological parameters.

No. of samples Cell counts [per yl] Total protein [mg/dl] Heart rate [per min] Respiratory rate [per min]

Naive controls

Saline controls

6 3.3t2.5 36.7t13.4 n.d. n.d.

7 9.126.1* 20.8233.8 90211 89238

TZD daily dose [pg] 25-450

500-950

23 16.3214.9* 61.1 235 91213 89230

10 19.6t14* 46.5229 922 12 85227

1000-1450

1500-1950

2000-4000

13 17.2221.4* 52.6236 89213 90?33

8 22.1 ?21.8* 50.4 ? 32 8529 71 243

6 6.353.2 35.4222.8 87218 82241

CSF findings, heart rate and respiratory rate measured at different dose levels. Significant differences from the findings in the untreated animals are marked by *, k 0 . 5 , Student’s t-test. All other data did not differ significantly from the control. n.d.=not done.

ed on a global assessment score reading normal (0), moderately affected (1) or substantially impaired (2). The frequency of the score values ’0’ to ’2’ at a particular dose level was correlated with the dose and the non-parametric correlation coefficient, i.e. Spearman r, was calculated to determine significant effects of treatment (Software: GraphPad Prism). The respiratory rate was determined as breaths per minute. Heart rate and blood pressure were measured by a cuff according to Riva-Rocci. A Student’s t-test was performed to detect significant treatment effects. At bolus injections, ratings and measurements were performed at 30 min, 60 min, 2 h, and 3 h, and in long-term experiments 3 d after dose changes. The animals were weighed once a week.

(fast) redistribution half-life and A2=2.91 pg/ml, tlIZ (slow)=152 min for the (slow) elimination half-life. The hypothetical drug concentration Co in CSF at time t=O was calculated Co=44.26 pg/ ml and the initial volume of distribution Vd=16.94 ml. The lumbar CSF clearance was calculated CLcsF=28.9 ml/h=0.48 ml/ min with AUC=25.95 pg . h/ml. Steady state distribution: Plasma TZD levels at steady state were between 0.1 and 1.3 ng/ml with a linear dose dependence (see Fig. 2). The steady-state ventricular TZD concentration at infusion rates under 750 pg/d was below the detection limit of the assay, i.e.