Intrathecal Ketorolac in Dogs and Rats

TOXICOLOGICAL SCIENCES 80, 322–334 (2004)DOI: 10.1093/toxsci/kfh168Advance Access publication May 12, 2004

Intrathecal Ketorolac in Dogs and Rats

Tony L. Yaksh,*,1 Kjersti A. Horais,* Nicolle Tozier,* Michael Rathbun,* Phil Richter,* Steve Rossi,* Marjorie Grafe,†

Chuanyao Tong,‡ Carol Meschter,§ J. Mark Cline,‡ and James Eisenach‡

*University of California, San Diego, Department of Anesthesiology, Research Laboratory-0818, 9500 Gilman Drive, La Jolla, California 92093-0818; †Oregon

Health and Science University, Department of Pathology, Portland, Oregon 97239; ‡Wake Forest University School of Medicine, Department of Anesthesiology,

Winston-Salem, North Carolina 27157; and §Comparative Biosciences, Inc., 2672 Bayshore Parkway, Suite 515, Mountain View, California 94043

Received February 13, 2004; accepted May 2, 2004

This study was conducted to assess spinal safety of the cyclo-

oxygenase inhibitor ketorolac in dogs and rats. Beagle dogs were

preparedwith lumbar intrathecal catheters and received continuous

spinal infusions of 5 mg/ml ketorolac (N 5 6), 0.5 mg/ml ketorolac

(N 5 8), or salinevehicle (N 5 6) at 50ml/h (1.2ml/day) for 28days.

No systematic drug or dose-related changes were observed in motor

function, heart rate, or blood pressure. Histological examination

revealed a mild pericatheter reaction in all groups with no drug

or dose related effect upon spinal pathology at the lumbar site of

highest drug concentration. Cisternal CSF protein was elevated for

all treatment groups at necropsy, and cisternal glucose was within

normal range for all treatment groups, though three dogs displayed

decreases in cisternal glucose. Significant reductions in hematocrit

were noted, and increased incidence of gastric bleeding at necropsy

was observed in animals receiving ketorolac. Intrathecal ketorolac

kinetics revealed a biphasic clearance: t1/2s 5 10.3 and 53 min,

respectively. After initiation of infusion (0.5 mg and 5 mg/ml/

50 ml/h), lumbar CSF concentrations of ketorolac were 3.8 and

52.7 mg/ml, respectively. Bolus and continuous infusion of intra-

thecal ketorolac resulted in significant reduction of lumbar CSF

PGE2 concentrations. In rats, with intrathecal catheters, four

daily bolus deliveries of saline or ketorolac (5 mg/ml/10 ml) had

no effect upon spinal histology or upon spinal cord blood flow.

These data indicate that intrathecal ketorolac in two species at

the dose/concentrations employed does not induce evident spinal

pathology but diminishes spinal prostaglandin release.

Key Words: nonsteroidal anti-inflammatory drug (NSAID);

spinal; cyclo-oxygenase; COX; prostaglandin.

Following tissue injury and inflammation a behaviorally

defined hyperalgesia can be identified in animal models and in

humans (Yaksh et al., 1999a). Current thinking suggests that this

behavioral syndrome results from the chronic activation of pri-

mary afferent input and/or the presence of circulating cytokines

that initiate a spinal cascade that serves to sensitize spinal noci-

ceptive processing. An important component of this cascade

appears to be the synthesis and extracellular movement of

prostaglandins through an activation of spinal cyclooxygenase

(Svensson and Yaksh, 2002, Yaksh et al., 2001). Thus, previous

studies have shown that a variety of COX inhibitors, such as

acetylsalicylate, ibuprofen, and ketorolac given intrathecally in

rat models will diminish hyperalgesic states at intrathecal doses

thatare100to300timessmallerthanthoserequiredaftersystemic

delivery and that have no evident effects after systemic delivery

(MalmbergandYaksh,1992a,b;Yaksh,1982;Yakshetal.,2001).

Ketorolac is a water-soluble nonsteroidal anti-inflammatory

drug (NSAID) with potent inhibitor effects upon both cyclo-

oxygenase isozymes. Administered systemically in humans,

ketorolac has potent antihyperalgesic actions in postoperative

pain (Gillies et al., 1987). Its limiting effects are gastrointestinal

ulceration, effects upon renal blood flow, and depressed platelet

function. These effects are believed to be common to the block-

ade of peripheral systemic cyclooxygenase isozyme activity

(Eisenberg et al., 1994). These potent effects of cyclo-oxygenase

(COX) inhibitors on hyperalgesic states in humans suggest that,

as in preclinical models, the spinal delivery of COX inhibitors

may also display significant efficacy with diminished systemic

side effects.

To consider the possible reaction of intrathecal ketorolac in

humans, it is necessary to establish the pharmacokinetics and

the effects upon spinal cord after intrathecal delivery in

well-defined preclinical models that are believed to robustly

define the safety (or lack thereof) of the test articles in question.

These studies should be undertaken at the maximum usable

formulation of the GMP (Good Manufacturing Practice)

product. The present study was conducted to obtain such data.

The dog and rat are species routinely used to satisfy regulatory

requirements for drug safety evaluation and are chosen because

there is considerable knowledge of these species’ responses to a

wide variety of intrathecal drugs (Yaksh and Malkmus, 1999;

Yaksh et al., 1999b).

MATERIALS AND METHODS

All animal work was carried out according to protocols approved by

the Institutional Animal Care and Use Committee of the University of California,

1 To whom correspondence should be addressed. Fax: (336) 716-3220.

E-mail: [email protected].

Toxicological Sciences vol. 80 no. 2 # Society of Toxicology 2004; all rights reserved.

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San Diego and was in compliance with the USDA Animal Welfare Act (USDA

Title 9 Code of Federal Regulations Part 3, Federal Register 15 February 1991).

Animals

Dogs. Beagle dogs (Covance, Inc., Princeton, NJ and Marshall Farms,

Inc., North Rose, NY), 10 to 24 months in age and weighing 6 to 12 kg, were

individually housed in runs with wood shavings and given ad libitum access to

food and water with a daily 12-h light/dark cycle. Kennels were maintained

within the range of 62–82�F. Animals were adapted for a minimum of 10 days

prior to surgery. A nylon vest (Alice King Chatham Medical Arts, Hawthorne,

CA) was placed on each dog approximately 48 h prior to intrathecal catheter

placement surgery for acclimation.

Rats. Male Sprague-Dawley rats (Harlan-Industries, Indianapolis, IN),

250–370 g, were individually housed and given ad libitum access to food

and water in a regulated temperature (66–76�F) and illumination (12 h light/

12 h dark) environment. Body weight was assessed before implantation of the

intrathecal catheter and daily during study drug treatment.

Surgical Preparation

Dog. To permit chronic delivery of drug into the lumbar intrathecal

space, a catheter was passed from the cisterna magna to the lumbar intrathecal

space. This placement was accomplished approximately 72 h prior to intrathe-

cal dosing. The antibiotic sulfamethoxazole-trimethoprim (240 mg tablet,

15–25 mg/kg, po, twice daily) was given 48 h prior to surgery and for

48 h after surgery. Dogs received atropine (0.04 mg/kg, im) prior to sedation

with xylazine (1.5 mg/kg, im). Anesthesia was induced with isoflurane, 1–2%

in N2O/oxygen (60/40%) with a mask followed by endotracheal intubation

and spontaneous ventilation. Animals were continuously monitored for

oxygen saturation, inspired and end tidal values of isoflurane, CO2, N2O

and oxygen and heart and respiratory rates.

Surgical areas were shaved and prepared with chlorhexadine scrub and

solution. Using sterile technique, the cisterna magna was exposed and a

small incision (1–2 mm) was made in the dura. APE-10 l catheter (fabricated

with polyethylene tubing and sterilized by electron beam irradiation) was

inserted and passed caudally approximately 40 cm to a level corresponding

to the L2–3 segment. Dexamethasone sodium phosphate (0.25 mg/kg, im) was

administered just after catheter placement. The external catheter was then tun-

neled subcutaneously to exit at the left scapular region. Upon closure of the

incision, isoflurane was discontinued and butorphanol tartrate (Torbugesic; 0.04

mg/kg, im) was administered to relieve postoperative pain as necessary.

For pharmacokinetic studies, dogs were prepared as described above, but a

second catheter (polyethylene, PE50) was also placed in addition to the

infusion catheter for purposes of lumbar cerebrospinal fluid (CSF) sampling.

The sampling catheter was passed such that the tip was approximately 2 cm

cephalad to the infusion site. The catheter was externalized similarly on the

contralateral side.

Following recovery, the nylon vest was again placed on the animal (ani-

mals having been previously acclimated to the vest prior to surgery), and an

infusion pump (PANOMAT C-10; Disetronic Medical Systems, Saint Paul,

MN) secured in the vest pocket where it was connected to the externalized

end of the PE-10 intrathecal catheter. An infusion of 0.9% (w/v) Sodium

Chloride for Injection, USP at 50 ml/h was initiated to ensure catheter patency

prior to infusion of Test Article.

Rats. For neurotoxicologic assessment, rats were anesthetized with 2–

4% halothane in oxygen/air. Polyethylene catheters were inserted through a

small incision in the atlanto-occipital membrane and passed 8 cm caudally

to the level of the lumbar enlargement, as previously described (Yaksh and

Rudy, 1976). To confirm correct placement of the catheters, 10 ml of 2%

lidocaine was injected, followed by a 10 ml 0.9% saline flush the day after

surgery. All animals developing a bilateral motor block of the hind limbs

within 30 s were included in the study. After surgery and testing with

lidocaine, the animals recovered for at least five days. Animals with any

evident neurologic deficit were excluded from the study.

Test Article

Ketorolac (5 mg/ml; Acular PFTM; Allergan, Irvine, CA) was supplied as

a sterile additive-free saline solution. Sterile saline (0.9% (w/v) Sodium

Chloride for Injection, USP, Abbott Laboratories, North Chicago, IL; 10 ml/vial)

was used as the control solution and as flush for the catheter. From each cartridge

change of the test article treatment group, drug samples were randomly

collected during the filling of the pump cartridges and analyzed for test article

concentration.

Dog Procedures

These studies had three phases: dose ranging, safety, and pharmacokinetics.

Table 1 indicates the dog treatment assignments.

Dose-ranging phase. Two dogs were assigned to receive the maximum

concentrations (5 mg/ml) at the maximum intrathecal infusion rate routinely

employed for spinal delivery in this model (100 ml/h). In the event of deleterious

side effects, the animal was terminally anesthetized and underwent necropsy and

histopathology.

Safety phase. Dogs were randomly assigned to receive the lumbar intrathe-

cal infusion of the maximum available concentration of ketorolac (5 mg/ml of

TABLE 1

Summary of Dog Group Assignment and Treatment

Dog ID Gender Weight

(kg)

Treatment

group

3376087 Male 7.2 5 mg/ml Ketorolac

test article at 100 ml/h

(2.4 ml/day)

3381293 Male 9.4

385 8979 Male 13.0 0.5 mg/ml Ketorolac


(1.2 ml/day)

389 3243 Male 11.8

389 4631 Male 12.8

OFR-1 Male 9.0

YZR-1 Male 9.2

OER-1 Male 9.6

389 2328 Male 12.4

388 4376 Male 12.2

CPH AVL Male 11.0 5 mg/ml Ketorolac


(1.2 ml/day)

CPH AYU Male 10.4

CPH AUI Male 10.6

CPH AZX Female 9.0

CPH ATY Female 7.6

CPH AER Female 6.0

CPH AWK Male 7.8 Saline control article

at 50 ml/h (1.2 ml/day)CPH AVH Male 11.4

CPH AXA Male 10.2

CPH ASL Female 7.8

CPH ATW Female 9.8

CPH APB Female 7.6

CRDAIC Male 11.8 Pharmacokinetics-prostanoids

samplingCRDATI Male 12.3

3359565 Male 8.6

3377130 Male 8.6

INTRATHECAL KETOROLAC IN DOGS 323

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ketorolac), 0.5 mg/ml ketorolac, or an equal volume of saline delivered at 50ml/h

for a minimum of 28 days, unless euthanasia was deemed necessary.

Pharmacokinetics phase. Serum and lumbar intrathecal CSF samples (0.5

and 0.3 ml, respectively) were taken before and at intervals after the intrathecal

injection of ketorolac (5 mg/ml) as a 1 ml bolus and assayed for ketorolac. After a

3–5 day interval, intrathecal infusion of ketorolac (5 mg/ml) at a rate of 50 ml/h

was initiated. Before and at 6–12 h after the initiation of infusion, serum, and

lumbar CSF samples were taken and assayed for ketorolac. In addition, CSF

samples were split and assayed for PGE2.

Dog Clinical Observations

A physical examination of each dog was conducted prior to its inclusion into

the study. Animals were observed for health status and clinical signs at least twice

daily. Catheters and pumps were checked at least once daily.

Neurological examination. For safety phase animals, a neurological exam-

ination was undertaken prior to the initiation of drug delivery and within three

days prior to sacrifice (e.g., on or about study Day 25–28). Examinations were

performed without knowledge of treatment group.

Periodic observation. After surgery, each dog’s rectal temperature was

recorded daily. Specific behavioral indices chosen to assess each animal’s

state of arousal, muscle tone, and motor coordination were undertaken twice

daily throughout the in-life phase of the study, as described in detail elsewhere

(Yaksh et al., 1997). Body weights, heart rate, and blood pressure measurements

were recorded on Days �5, �3, prior to initiation of test or control article, at

regular 4-day intervals, and on the day of necropsy.

Measurement of cardiovascular parameters. Measurements were made

using a tail cuff manometer (Dinamap 8100, Criticon). Respiration rates were

measured by observation of chest expansion and contraction.

Nociception response. The thermally evoked skin twitch response was

measured using a probe with approximately 1 cm surface area maintained at

approximately 62.5 6 0.5�C with a feedback controller. The probe was applied

to two shavedareas of the back (right and left lateral) at the thoracolumbar level of

the spine. Typically, this results in a brisk contraction of the local musculature

within 1–3 s of probe placement. Upon the appearance of this response, the probe

was removed and the latency to response recorded. Failure to respond within 6 s

was cause to remove the probe and assign that value (6 s) as the latency.

For analysis purposes, the nociceptive response was presented as the mean of

the two latencies.

Dog Laboratory Measurements

Blood analyses. Blood was collected from each dog by cephalic venipunc-

ture, after an overnight fast of approximately 16 h (no food was allowed but free

access to water was provided) for a complete blood count and blood chemistry

panel.

Complete blood counts included the following analyses: leukocyte, erythro-

cyte, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular

hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differ-

ential leukocyte count (absolute and relative), reticulocyte count, and blood cell

morphology evaluation.

Blood chemistry panels included the following analyses: sodium, cholesterol,

potassium, aspartate aminotransferase, chloride, alanine aminotransferase, total

protein, alkaline phosphatase, albumin, triglycerides, calcium, globulin (calcu-

lated), phosphorous, albumin/globulin ratio, creatinine, total and direct bilirubin,

glucose, and urea nitrogen. After initiation of test or control article, weekly

hematocrit values were determined on site over the duration of the safety

phase of the study.

Drug samples. To determine drug concentration in the blood, duplicate

cephalic blood samples (41 ml) were collected on wet ice and prepared as

serum, then transferred to Eppendorf tubes and stored at �20 6 10�C until

analysis. To determine test article concentration in the cerebrospinal fluid

(CSF), samples of CSF (approximately 0.3 ml) were collected on ice then

transferred to Eppendorf tubes and stored at �20 6 10�C until analysis.

Ketorolac analysis. Serum and CSF samples were quantitatively extracted

by C-18 reverse phase cartridge chromatography and eluted with acetonitrile.

Concentrated eluates were injected onto an HPLC equipped with a Phenomenex

‘‘Prodigy’’ C-18 reverse phase column (250 mm34.6 mm). Peaks were detected

withanAgilentModel1100UVdetectorsetatawavelengthof313.Allunknowns,

standards, and controls contained an equal quantity of internal standard, 200 ng of

indoprofen. With this assay, a single peak for ketorolac is found at the retention

timeof10.68min,theinternalstandardindoprofenelutesat12.09min.Plasmaand

CSF extracts showed no interfering chromatography throughout the integration

time period. The absolute sensitivity of the ketorolac assay was 5 ng/ml, and the

coefficient of variation was 510% within the concentration range 5–500 ng/ml.

Dog Necropsy

Allanimalswereanesthetizedwithanivdoseofsodiumpentobarbital(35mg/kg

or dose to effect). Blood and cisternal CSF (by percutaneous puncture with a 22 ga

spinal needle) were then sampled. The aorta was then catheterized though a thor-

acotomy and the blood cleared by perfusion with approximately four liters of 0.9%

(w/v)salinefollowedbyapproximatelyfourlitersof10%neutralbufferedformalin.

Spinal dissection. After perfusion, the dura of the spinal cord was exposed

by laminectomy. Methylene blue dye was injected through the intrathecal cathe-

ter to determine the catheter patency, the location of the tip, and dye spread

around the tip. The condition of the spinal cord was noted and a photographic

(digital) record of the spinal cord/catheter relationship was made. The cord was

cut (taking care to keep the dura intact) into four sections: cervical (section A),

thoracic (section B), high lumbar (catheter tip region, section C), and lower

lumbar (just caudal to catheter tip section D), for histopathological analysis.

In addition to the spinal cord, portions of gastrointestinal tract, kidney, and

bladder were also retained

Tissue preparation. Formalin-fixed tissues were trimmed into cassettes,

processed and embedded in paraffin, sectioned at approximately 3–5 mm and

stained with hematoxylin and eosin. In addition, special stains were performed on

the spinal cord in the block at the catheter tip region (block C). These sections

were immunohistochemically stained for glial fibrillary acidic protein (GFAP)

(DAKO, Carpenteria, CA; Kit 20334, Lot # CH096302), Gram stain

(McDonald’s Gram Stain, American Master, Lodi, CA; Kit # KTMGS) for

bacteria and Grolcott’s Methenamine Silver (GMS), (Grolcott’s Methenamine

Silver Stain Kit # KTMGSMIC, American Master, Lodi, CA) for fungi. Spinal

cord sections were evaluated by light microscopy independently by two

pathologists (C.M. and M.G.) without knowledge as to treatment. A text descrip-

tion was written for each section. The severity of the inflammation was graded on

a scale of 0–3 for increasing severity, and the animals were then forced ranked in

order of increasing severity. To more precisely rank the pathology scores, all

slides were re-reviewed, still without knowledge of treatment groups, and within

each score category an additional increment of 60.25 could be given if

the reaction was slightly more or less severe than that of animals with whole

number scores.

Rat Procedures

There were two phases to the rodent studies: intrathecal toxicity and spinal

cord blood flow.

Intrathecal toxicity. To assess the response of the intrathecal space to

repeated bolus delivery, rats were randomized to receive 50 mg ketorolac or

an equal volume (10 ml) 0.9% saline daily by intrathecal bolus for four days

(n 5 10 per group). Arousal, motor coordination, paw withdrawal latency to

radiant heat, and general behavior were assessed daily after each injection, as

previously described (Yaksh et al., 1995). On day 6, animals were deeply

324 YAKSH ET AL.

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anesthetized with pentobarbital, perfused transcardially with saline followed by

paraformaldehyde, the spinal cords removed, fixed, then sectioned and stained

with hematoxylin-eosin. Sections of spinal cord near the catheter tip in the lumbar

region, and distant from the tip in the thoracic region and cervical region were

examined by a pathologist unaware of treatment group.

Spinal cord blood flow. Rats were anesthetized with sevoflurane. Femoral

venous and arterial catheters were inserted; then a tracheotomy was performed

for controlled ventilation. A small laminectomy was performed at the atlanto-

occipital membrane, the dura excised, and a tissue well created. A PE 50 catheter

was tunneled under the skin of the back, exiting above the spinal cord surface, and

the well perfused with saline solution through this catheter. Laser-doppler flow

was performed by placement of a flowmeter probe directly over the spinal cord

surface. Following baseline measurements, the spinal cord was continuously

perfused with saline, followedby ketorolac, 2.5 and then 5 mg/ml. Blood pressure

and heart rate were monitored throughout the experiment.

Statistical Analysis

Data of the respective observations (e.g., mmHg, beat/min, mg/ml) are

expressed as means and SEs, or as medians and quartiles. Continuous normal

data were compared using repeated measures and one-way ANOVA with post

hoc Bonferroni tests for multigroup comparisons. Distribution-free testing of

data, such as ranked spinal cord pathology, was performed using the Jonckheere

test for comparison of three or more groups with post hoc Mann-Whitney U

tests for multigroup comparisons. All statistical comparisons were made at the

p 5 0.05 level of significance. Pharmacokinetic data were analyzed using PK

Solutions 2.0 (#1999, Summitt Research Services, Ashland, OH) statistical

software for noncompartmental pharmacokinetic data analysis. Analysis

included half-lives, descriptive curve parameters, curve area, statistical moment,

and volume of distribution calculations.

RESULTS

Dog Investigations

All animals survived the surgical preparation without evi-

dence of significant neurological deficit due to the implantation

of the catheters.

Dose-Ranging Phase

There were no significant changes in motor function, cardio-

vascular status, or body temperature in either dog receiving 5 mg/

ml, 100 ml/h. However, due to prominent tarry stools, a signifi-

cant lowering of hemtocrit (pre vs. post: 47% to 27% and 41% to

25%) and deteriorating physical condition, ketorolac infusion (5

mg/ml, 100 ml/h) of dog 3376087 was terminated on day 13, and

this animal was euthanized on day 18. Dog 3381293 displayed

blood in stools and some deterioration of physical status, but was

infuseduntil euthanasiaon infusionday34. Itwasconsidered that

these outcomes reflected the systemic effects arising from the

daily dose of ketorolac of 2 mg and 1.3 mg/kg/day, respectively,

based on body weight at the time of termination of infusion.

Serum ketorolac concentrations at sacrifice were 7.83 and

3.36 mg/ml, respectively. Inspection of the spinal cord revealed

no pathology (see Table 4). Based upon these results, the ketor-

olac concentration of 5 mg/ml was retained, but the infusion rate

for the safety phase was reduced to 50 ml/h.

Safety Phase

All vehicle dogs, six of eight 0.5 mg/ml test article dogs and

five of the six 5 mg/ml test article dogs completed the 28-day

infusion sequence. The two 0.5 mg/ml dogs (3892328 male,

3884376 male) were euthanized at day 13 and 23, respectively,

and the single 5 mg/ml test article dog (CPH AER, female) was

sacrificed at day 10 of infusion.

Behavioral and Physiological Observations

Neurological examination. Baseline behavioral scores for

arousal, muscle, and motor coordination were scored as ‘0’ or ‘1’

at the start of infusion. In vehicle animals, all neurological signs

were judged minor and limited to mild increases in hind limb

motor tone and minimal gait abnormalities. Behavioral mani-

festations, i.e., arousal, muscle tone, and motor coordination,

were typically scored as ‘0’ or ‘1’ throughout the interval of

infusion. In the 0.5 mg/ml treatment group, six of the eight

displayed minimal neurologic changes comparable to the vehi-

cle group, with behavioral scores typically ‘0’ or ‘1’. However,

two animals, 389 2328 and 388 4376, were euthanized at day 13

and 23, respectively, due to progressive hypertonicity of the neck

and hind limbs, and pain behavior. These conditions developed

between days 9 and 13 for dog 389 2328 and days 14 to 20 in dog

388 4376. In the 5 mg/ml treatment group, as in the vehicle-

treated group, all abnormalities identified were minimal and

typically reflected mild hind limb and minimal gait impairment,

with behavioral scores for arousal, muscle tone, and motor

coordination typically scored as ‘0’ or ‘1’ throughout the interval

of infusion. A single 5 mg/kg dog (CPH AER, female) was

sacrificed at day 10 of infusion due to the acute development

of a lateral recumbent posture and whole body stiffness.

Physiological measures. Pre-infusion values for tempera-

ture (101.86 0.6�C), respiration (306 4 b/m), heart rate (1256

21 b/m), and blood pressure (diastolic: 79 6 16; mean: 996 17;

systolic: 136 6 17 mmHg) were comparable for each group.

Over the interval of the infusion, no treatment group displayed

any clinically significant trend in these indices in any animal,

even in those undergoing early sacrifice.

Body weight. Mean body weight upon initiation of infusion

was 9.7 6 0.5 kg and did not differ between treatment groups.

Neither the test nor control article treatments had a significant

effect upon mean body weight. CPH AER displaying significant

falls in hematocrit and euthanized at 10 days showed a 13%

decrease in body weight over the interval of test article admin-

istration (see below).

Nociceptive response. Baseline thermal escape latencies

were 3.6 6 0.4 s. There were no significant changes in this

thermal escape latency over time in any treatment group.

Clinical Chemistry

Blood chemistry values were not different among the

three groups at surgery, and the medians for selected


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values are presented in Table 2. Examination and statistical

comparison of these parameters at the time of sacrifice reveals

no systematic dose-dependent effects. As noted, several animals

underwent early sacrifice or showed distinct reactions, and their-

data are presented separately to permit independent assessment.

Hematocrit/RBC. As indicated in Table 2, there were no

systematic changes between surgery and sacrifice in animals

receiving vehicle. Dogs receiving 0.5 mg/ml ketorolac typically

displayed a slight (< 15–20%) reduction in hematocrit and red

blood cell count. With the 5 mg/ml infusion (50 ml) treatment,

one of six dogs displayed a prominent drop in hematocrit

and RBC count. Red cell count paralleled the changes noted

in the respective hematocrit. As noted above, both animals

receiving the higher infusion dose showed prominent drops in

hemtocrit values.

WBC/neutrophils. Animals in vehicle and ketorolac treat-

ments typically displayed little or no change in WBC and neu-

trophil counts (Table 2). Treatment animals undergoing early

sacrifice or showing prominent dysfunction typically showed an

increase in both WBC and neutrophil count.

CSF analysis. Summaries of cisternal CSF clinical ana-

lysis results are presented in Table 2. Mean CSF protein and

glucose were not different among treatment groups at the time of

surgery.

Vehicle and both test article groups displayed increases in

protein at sacrifice. Cisternal glucose levels were within

expected ranges before and at the time of necropsy, except

for animal 385 8979 in the 0.5 mg/ml ketorolac group. White

blood cell counts showed an increase over the 28-day period

that was similar in both vehicle and test article groups.

Differential cell counts showed a similar profile for both

vehicle and test article groups. Statistical comparison of

these parameters at the time of sacrifice reveals no dose-

dependent differences among treatment groups ( p 4 0.10).

In the animals undergoing early sacrifice, cisternal CSF pro-

tein concentrations were notably elevated (see Table 2). CSF

glucose levels were comparable at sacrifice in all animals

with the exception of low glucose levels in 3858979, 388437,

and 3376087, the latter two of which underwent early

sacrifice.

Necropsy Observations

Catheter. Examination of the external catheter tracts typi-

cally revealed a well-healed sheath encasing the catheter as it

exited from the cisternal membrane. The presence of serous fluid

was noted along the sc catheter track in two 0.5 mg/kg dogs

(3884376; 3858979) and one 5.0 mg/kg dog (CPH AER) (see

Table 2). Intrathecal injection of 0.2 ml of methylene blue dye

uniformly revealed coloration at the tip indicating patency and

continuity.

Spinal cord. No visually evident abnormalities were typi-

cally present in the spinal canal at the catheter sites at necropsy.

TABLE 2

Group Summary of Blood/CSF Clinical Chemistry Taken at Surgery and at Sacrifice and Individual Data for Animals Showing

Pathology or Early Sacrifice

Blood CSF

WBC

(#/ml)

Neutrophil

(#/ml)

RBC

(#/ml)

Hematocrit

(%)

Protein

(mg/dl)

Glucose

(mg/dl)

WBC

(#/ml)

Observations

Pre-treatmenta

All animals 10 (9–11) 6 (5–7) 7 (6–8) 47 (45–50) 11 (10–12) 69 (66–72) 4 (1–36)

Post-treatment groupsa

Vehicle (n 5 6) 9 (8–100) 6 (5–7) 8 (7–8) 49 (47–49) 41 (25–151) 66 (63–71) 115 (10–1015)

0.5 mg/ml (n 5 5) 8 (5–10) 5 (3–8) 6 (6–7) 43 (39–45) 178 (36–1050) 65 (56– 67) 38 (19–292)

5.0 mg/ml (n 5 6) 14 (10–16) 10 (6–12) 5 (4–7) 36 (25–43) 63 (5–186) 59 (40–63) 560 (184, 1741)

Post-treatment individual dogs showing notable pathology or early sacrifice

3892328 (13 d) (0.5 mg/ml)b 12 11 7 50 3800 66 c CB. WBH

388 4376 (23d) (0.5 mg/ml) 7 7 7 49 300 44 c SF, WGH

385 8979 (28d) (5 mg/ml) 18 17 6 40 1200 18 1320 CB, SF

CPHAER (10d) (5 mg/ml) 24 22 4 29 c c c CB, GI, SF, WBH

337 6087 (18d) (5 mg/ml)d 15 11 4 25 592 40 800 GI

338 1293 (28 d) (5 mg/ml)d 12 9 4 27 36 63 40 GI

Note.Rank-order comparison across treatment groups (Jonckheere) showed no statistically significant dose-dependent trends for blood or CSF clinical chemistry.

p values ranged from p 4 0.10 to p 4 0.50. Abbreviations: CB: cisternal blood; SF: serous fluid along external catheter track; GI: gastrointestinal bleeding and

ulceration; WBH: whole body hypertonicity.aMedian with 25%–75% quartiles.bSacrifice day.cInsufficient sample.dInfusion rate at 100 ml/h as compared to 50 ml/h for all other animals.

326 YAKSH ET AL.

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However, in two 0.5 mg/kg dogs (3892328; 3858979) and one

5.0 mg/kg dog (CPH-AER), unclotted blood was noted in the

cisterna and upper cervical spinal cord.

Other tissues examined. Administration of 5 mg/ml ketor-

olac (50 ml/h) was associated with gastrointestinal ulceration in

two of six animals (CPH AYU and CPH AZX) and, as noted

above, in both animals receiving 5 mg/ml at 100 ml/h. Small

intestinal mucosal infiltration with lymphoid and monocytic

cells, and edema was present in dog CPH AER. Mild pulmonary

congestion and thrombosis were noted in Dog CPH AER. Tissue

from other organs was not examined by the histopathologist,

however, no other gross abnormalities were noted at necro-

psy. With the low (0.5 mg/ ml) infusion concentration no evi-

dence of gastric bleeding or other untoward systemic effects

were noted.

Spinal Histopathology

Table 3 presents the summary of the incidence of pathology

observations and scores for the four treatment groups. Table 4

presents the individual pathology score presented by animal and

spinal level. In general, observations may be considered in terms

of local catheter compression of the spinal cord, the presence of

dural and arachnoid infiltrates, and root and spinal infiltrates at

the three levels of the spinal cord that were systematically exam-

ined. Several points summarize the overall assessment.

Spinal cord compression. A modest spinal cord compres-

sion adjacent to the catheter was noted with an equal incidence in

all four treatment groups, along the axis of the catheter but not

below the catheter tip (Table 3; see Fig. 1).

Local inflammatory reaction. The incidence of inflamma-

tory infiltrates in dura, nerve root, and spinal cord was noted with

approximately equal incidence observed at all levels in all four

treatment groups (Table 3). At the catheter tip site (section C),

where drug concentrations are highest, the local reaction was

judged to be minimal to mild (score of 1.25 or less) in four of six

control animals, five of eight 0.5 mg/ml treated animals, and five

of six animals in the 5 mg/ml treated group. In these animals, a

small fibrous track inside the dural sac represented the catheter

track (see Figure 1A). Minimal to mild chronic inflammation

was associated with the tracks, consisting of small numbers of

lymphocytes, plasma cell, and macrophages around the catheter

site and in the adjacent dura. In the remaining two animals in the

control group (CPHATW, CPH AVH), three animals in the

0.5 mg/ml treated group (3892328, 3884376, 3858979) and

one animal in the 5 mg/ml (CPH AER) treated group, moderate

to severe inflammation (score of 1.5 or greater) was present in

block C (catheter tip). One dog had white matter vacuolization

TABLE 3

Summary of Histological Observations by Group

Histopathology observations (incidence by group)

Treatment

Spinal

compression by

catheter

Dura infiltrate

(fb/lym/pmn)

Arachnoid infiltrate

(lym/pmn)

Nerve root

infiltrate

(lym/pmn)

Spinal cord

infiltrate

(lym/pmn)

Pathology

score

median

Spinal level

Cervical

Saline 50 ml/h 2/6 5/6 3/6 1/6 0/6 0.75

0.5 mg/ml 50 ml/h 2/7 4/7 6/7 2/7 2/7 1.0

5 mg/ml-50 ml/h 3/6 5/6 6/6 3/6 2/6 1.5

5 mg/ml-100 ml/h 1/2 2/2 1/2 2/2 0/2 (1.5)

Thoracic

Saline 50 ml/h 1/6 6/6 3/6 1/6 2/6 0.87

0.5 mg/ml 50 ml/h 1/7 7/7 7/7 1/7 2/7 1.0

5 mg/ml-50 ml/h 2/6 6/6 6/6 1/6 2/6 1.5

5 mg/ml-100 ml/h 2/2 2/2 2/2 (1.9)

Lumbar (tip)

Saline 50 ml/h 1/6 5/6 2/6 1/6 1/6 1.0

0.5 mg/ml 50 ml/h 4/7 7/7 7/7 2/7 1/7 1.0

5 mg/ml-50 ml/h 1/6 6/6 6/6 3/6 2/6 1.0

5 mg/ml-100 ml/h 0/2 2/2 1/2 1/2 1/2 1.5 (?)

Lumbar (distal)

Saline 50 ml/h 0/6 5/6 5/6 4/6 2/6 1.0

0.5 mg/ml 50 ml/h 0/7 1/7 7/7 0/7 1/7 0.75

5 mg/ml-50 ml/h 0/6 5/6 5/6 2/6 2/6 1.0

5 mg/ml-100 ml/h 0/2 2/2 2/2 1/2 0/2 1.0(?)

Note.Chi square analysis across treatments (vehicle, 0.5 mg/ml, and 5.0 mg/ml) for each class of histological observation vs. treatment at each spinal level revealed

no statistical significance at a critical value corresponding to p 5 0.10. Abbreviations: fb: fibroblasts; lym: lymphocytes; pmn: polymorhonuclear leucocytes.


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TABLE 4

Summary of Spinal Histopathology

Spinal pathology score

Group Dog Sex Cervical Thoracic Lumbar (Tip) Lumbar (Distal)

Control CPH APB F 0.75 0.75 0.75 1

Control CPH ASL F 0.75 0.75 0.75 0.75

Control CPH ATW F 1.25 1 2.25 3

Control CPH AVH M 1.75 2 2.25 2.25

Control CPH AWK M 0.75 1 1 0.75

Control CPH AXA M 0.75 0.75 1 1

0.5 mg/ml OFR-1 M 0.75 1 0.75 1

0.5 mg/ml OER-1 M 1 1 1 1

0.5 mg/ml YZR-1 M 1 1 1.25 0.75

0.5 mg/ml 389 2343 M 1 1.25 1 0.75

0.5 mg/ml 389 4631 M 1.25 1 1 0.75

0.5 mg/ml 385 8979 M 3 3 3 3

0.5 mg/ml 389 2328a M 2 1.25 2 1

0.5 mg/ml 388 4376a M 2 2 2 0.75

5 mg/ml CPH AERa F 3 3 3 3

5 mg/ml CPH ATY F 2 1.25 1 1

5 mg/ml CPH AUI M 1 3 1 2.25

5 mg/ml CPH AVL M 2 1.75 1 1

5 mg/ml CPH AYU M 0.75 1 1 1

5 mg/ml CPH AZX F 1 1 1 0

Note. Inflammationseveritykey:0 5 noabnormalfindings;1 5 mild;2 5 moderate;3 5 severe;incrementsof60.25inscoreindicatedslightlylessormoresevere

than whole number score. Control 5 saline at a volume of 1.2 ml/day, 0.5 and 5 mg/ml ketorolac intrathecally at a volume of 1.2 ml/day. Rank order comparison across

treatmentgroupsateachspinallevelwascarriedoutusingtheJonckheererankedtestfororderedmeansateachspinallevel.Comparisonofgrouppathologyscoresacross

treatment at lumbar distal, lumbar tip and cervical were not statistically significant (p50.10), while comparison across dose at the thoracic level was (p5 0.05).aAnimals euthanized early (day 13, 23, and 10 of infusion respectively).

FIG. 1. Representative histology for dogs receiving 50 ml/h saline (A and D) or 5 mg/ml ketorolac (B and C) for 28 days. Abbreviations: C: catheter, SAS:

subarachnoid space. Arrows indicate the dura. Top left: Dog CPH ATW Saline, spinal cord section D (lumbar). Top right: CPHAVL Ketorolac, spinal cord

section D (lumbar). Bottom left: Dog CPH AYU Ketorolac, spinal cord section D (lumbar). Bottom right: Dog CPH AXA Saline, spinal cord section D (lumbar).

328 YAKSH ET AL.

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(without inflammation); this was focal and not associated with

the catheter site. Consistent with this summary, the median

pathology scores for sections C and D were not different across

treatment group (Jonckheere testp40.10). Consideration of the

individual animals presented in Table 3 emphasizes that the most

severe histopathology reactions were observed in animals with

low CSF glucose at sacrifice (3884376; 3858979, 3376087).

At sites cephalad to the catheter tip (section A: cervical and B:

thoracic) a continuation of the reaction observed distally

was typically noted (see Figs. 1B and 1D). The sections with

inflammation showed variable proportions of lymphocytes,

neutrophils, plasma cells, and macrophages. In some animals,

the inflammation involved spinal nerve roots and epi/perin-

eurium. A variable degree of fibrosis was noted at the catheter

site and in adjacent dura, sometimes with neovascularization.

Two animals (389 2328 and CPH ATW) had focal lymphocytic

infiltration of the white matter adjacent to the catheter site and

one (CPH AVL) had a focal lymphocytic infiltrate along the

Virchow-Robin spaces. Two animals (385 8979, 0.5 mg/ml

ketorolac and CPH AER, 5 mg/ml ketorolac) had extensive

infiltration of the spinal cord parenchyma by predominantly

neutrophils, with focal necrosis and abscess formation. Statis-

tical comparison across groups revealed a statistical significance

by dose for section B (thoracic: p50.05) but not section

A (cervical: p40.10).

In all treated and control groups, spinal GFAP staining was

within normal limits, even at sites adjacent to the catheter site

indicating alack of reactive gliosis.

Gram and GMS stains undertaken in block C (catheter tip)

revealed no signs of bacteria or of fungal elements.

Toxicokinetics of Safety Animals

Infusate. Analysis of injectate (ketorolac 5 mg/ml) indi-

cated that representative aliquots contained 5375 6 58 mg/ml

of ketorolac (N 5 5).

Serum. No animal showed ketorolac levels in their serum

prior to initiation of infusion. The vehicle treatment group did

not show detectable drug levels in serum after 28 days of infu-

sion. In animals receiving ketorolac infusion, the mean serum

ketorolac concentrations on day 4, the first time point after infu-

sion initiation, were 0.18 6 0.05 mg/ml and 1.14 6 0.25 mg/ml

for 0.5 mg/ml and 5 mg/ml ketorolac infusions, respectively.

Serum concentrations measured at the time of sacrifice did not

differ for these respective treatments. In the one animal (CPH

AER, 6.0 kg) in the 5 mg/ml test article treatment group that was

euthanized on day 10 of infusion because of gastric ulceration,

maximum serum ketorolac levels were 2.08mg/ml. As expected,

small animals (such as CPH AER) displayed the highest serum

ketorolac concentrations.

Cisternal CSF. No animals showed any drug levels in

pretreatment cisternal CSF. The vehicle group did not show

drug in cisternal CSF after 28 days of infusion. After 28 days

ofcontinuous infusion, the test article treatment groups hadmean

cisternal CSF drug levels of 0.06 6 0.03 mg/ml and 0.16 6 0.09

mg/ml (0.5 mg/ml and 5 mg/ml groups, respectively) in the

cisternal CSF. The mean cisternal CSF to serum ratio measured

at the time of necropsy was 0.32 6 0.15 and 0.10 6 0.07 for the

0.5 mg/ml and 5 mg/ml treatments, respectively.

Intrathecal Pharmacokinetics

All pK animals survived the experiments without disability.

One animal CRD ATI failed to sample after the initial studies and

was removed from further analysis. At necropsy, the intrathecal

infusion catheters were typically located at L1/2 and the sampling

catheter was approximately 2 cm proximal to that of the infusion

catheter. Examination of the spinal cords revealed no evidence of

gross pathology or any sign of blood in the intrathecal space.

Bolus intrathecal injections and ketorolac concentrations.

The bolus intrathecal delivery of ketorolac (5 mg/ml/1 ml) in

three dogs resulted in an initial peak concentrations in lumbar

FIG. 2. Ketorolac (mg/ml) in lumbar CSF and serum of three dogs after

bolus lumbar intrathecal injection (T 5 0) of ketorolac (5 mg/1 ml). (Kinetic

data for these experiments are presented in Table 5.)


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CSF and plasma, and this declined in a biphasic fashion (Fig. 2).

Calculations of lumbar CSF kinetic parameters, assuming a one-

compartment model, are presented in Table 5. At the time of

peak serum concentration, the CSF/serum ratios were approxi-

mately 30:1.

Continuous infusion and ketorolac concentrations. To

determine steady state levels of ketorolac in CSF, lumbar

CSF samples were harvested before and at 24–48 h after

initiation of ketorolac infusion in three animals. As indicated

in Figure 3, ketorolac concentrations at 0.5 and 5.0 mg/ml at 100

ml/h were on the order of 3.8 and 53 mg/ml, respectively.

Effects on Lumbar CSF PGE2 Levels

Mean lumbar CSF PGE2 concentrations measured in six dogs

prior to the delivery of ketorolac were 1224626 pg/ml. After the

bolus delivery of ketorolac (5 mg/ml; 1 ml), there was a

significant reduction (48%) in the concentrations of lumbar

PGE2 at 30 min with significant recovery being observed

by 4 h (see Fig. 4). After the initiation of intrathecal ketorolac

infusion (0.5 mg/ml) in three dogs, lumbar PGE2 concentrations

were significantly depressed from baseline 1271 6 18 to

87 6 22 at 24 h.

Rat Investigations

Safety Phase

All animals completed the series of injections, and there were

no alterations in behavior. Ketorolac-injected animals did not

differ from saline-injected animals in assessment of motor tone,

coordination, and arousal (data not shown). Similarly, weight

did not differ before intrathecal drug treatment (288 6 19 g in

saline; 304 6 21 g in ketorolac) at the end of drug treatment

(289 6 22 g in saline; 305 6 18 g in ketorolac), or on any day

during treatment (data not shown). Latency to paw withdrawal

did not differ prior to or following intrathecal drug delivery and

baseline withdrawal latencies did not change over the time

course of drug treatment (data not shown).

Necropsy. At post mortem, spinal cords, meninges, and

nerve roots appeared grossly normal in all animals. Perfusion

was inadequate in one animal (in the saline group).

Histopathology. Histological examination showed a rim of

fibrin, fibroblasts, and inflammatory cells around the catheter

site. Histological changes were observed frequently in both

groups, but with no difference in incidence between groups.

At the lumbar level, the number of animals with no significant

TABLE 5

Summary of Calculated Lumbar CSF PK Parameters for Bolus

Dose (5 mg/ml/1 ml) of Lumbar Intrathecal Ketorolac in Dogs

Parameters

Dog

CRDAIC

Dog

3359565

Dog

3377130

Grouped

dataa

D half life (min) 17.9 7.6 5.5 10.3

E half life (min) 81.2 71 38 52.7

C initial (mg/ml) 1381 6304 4443 2663

MRT-area (min) 46.2 23.4 11.7 34.5

AUC obs (mg 3 min/ml) 39620 99278 43402 54119

Vd obs (ml) 14.8 5.2 6.3 7

Note. D, distribution; E, elimination; C, concentration; MRT, mean residence

time; AUC area under the curve; obs, observed; Vd, volume of distribution.aRegression analysis run on grouped data.

FIG. 3. Left: Lumbar CSF Ketorolac concentrations (mean and individual

data) at T 5 �15 min (Pre) and 24–48 h after the initiation of intrathecal

infusion of ketorolac (0.5 and 5 mg/ml/100 ml/h) in three dogs. Right: Ratio

of lumbar CSF ketorolac measured versus that measured during infusion with

0.5 mg/ml at 100 ml/h.

FIG. 4. PGE2 (% of control concentration) and Ketorolac (mg/ml)

(Mean 6 SD; N 5 3) after bolus intrathecal injection (T 5 0) of ketorolac

(5 mg/1 ml) in lumbar CSF sampled at T 5 �15, 30, and 240 min **p 50.05,

paired Student’s t-test).

330 YAKSH ET AL.

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lesions, and minimal multifocal white matter vacuolation was

three, five, and one in saline-treated animals and one, seven, and

two in ketorolac-treated animals. The white matter vacuolation

was swelling of myelin sheaths, some of which contained myelin

fragments and cellular debris. These changes, when present,

were in all funiculi and at all dermatomal levels, not consistent

with a focal lesion of the cord.

Spinal cord blood flow. Laser-Doppler flow remained stable

following establishment of the experimental model, and was not

altered over 30 min of saline perfusion. Ketorolac, either at the

2.5 or 5.0 mg/ml concentration, had no effect on SCBF, as

measured by relative change in blood flow velocity (Fig. 5).

DISCUSSION

Spinal Cyclo-oxgenase

Cyclo-oxygenase (COX) inhibitors suppress hyperalgesia

that arises secondary to tissue injury. In contrast to the broadly

held belief that NSAID have a peripheral action, these agents

exert a potent antihyperalgesic action following spinal delivery

(Malmberg and Yaksh, 1992a,b; Yaksh, 1982; Yaksh et al.,

2001). This spinal effect reflects three observations: 1) cyclo-

oxygenasesareconstitutivelyexpressedinspinalparenchyma;2)

afferent input evokes spinal prostaglandins release; 3) prosta-

noids exert a facilitatory effect on spinal nociceptive neurons

(Svensson and Yaksh, 2002; Yaksh et al., 2001). While it is clear

that systemically delivered NSAIDs can exert central effects, the

relative central bioavailability of agents such as ketorolac is poor

(Rice et al., 1993). Given the peripheral side effect (e.g., anti-

platelet action), the central effects of a systemic agent may be

limited by the peripheral dose effects. This raises the possibility

of considering spinal delivery. Cancer patients received pain

relief from intrathecal lysine acetylsaliscylate, suggesting a

spinal action in humans (Devoghel, 1983, 1993; Pellerin et al.,

1987). Nevertheless, prior to human intrathecal delivery,

preclinical evaluation of ketorolac safety by this route must be

ascertained. Ketorolac (Acular PF) is a sterile preservative-free

formulation provided in concentrations of 5 mg/ml. We exam-

ined continuous 28-day intrathecal infusion of the highest avail-

able concentration of drug at a dose that could be tolerated by the

dog and 1/10 this highest tolerated concentration. In the rat,

the standard bolus injection volume is 10 ml and, accordingly,

the highest concentration was delivered in that volume, and the

effects of the injections on spinal blood flow were examined.

Intrathecal Safety Assessment

Spinal drug safety has two components: untoward physiolo-

gical effects mediated by spinal and nonspinal systems, and local

tissue toxicity. In the first case, the effects are proportional to the

total delivered dose. Such outcomes reflect a direct effect on

spinal or extra-spinal mechanisms and often define the maxi-

mum dose of drug to which the dog can be exposed. The limiting

response for opiates is defined by respiratory depression

(Sabbe et al., 1994); for clonidine it is hypotension and sedation.

(Yaksh et al., 1994) Conversely, tissue toxicity is dependent

upon the concentration to which the local tissue proximal to

the delivery site is exposed. Accordingly, the most robust test

of safety will be consideration of the tissue reaction to the highest

concentration that can be delivered. In this case, the available

GMP, preservative-free formulation of ketorolac is 5 mg/ml.

Dose/Concentration Selection

In the dose-ranging series, we observed that 5 mg/ml, 100ml/h

(total dose of 12 mg/day, approximately 1–1.5 mg/kg) resulted in

a reduced hematocrit and gastrointestinal bleeding. Accord-

ingly, the infusion rate in was lowered to 50 ml/h to reduce

the total systemic dose by half. This permitted us to retain the

highest local spinal exposure and reduce, but not abolish,

systemic toxicity.

Intrathecal Ketorolac Effects

Under the present treatment paradigm, all animals including

the saline-control dogs displayed a mild local meningeal inflam-

matory reaction and elevated cisternal CSF protein at the time

of sacrifice. Inflammation at the catheter tip site was noted with

approximately equal incidence in control, 0.5 mg/ml, and

5 mg/ml treated group. At the site of highest drug concentration

(the catheter tip), there were only modest effects in all groups and

none were statistically drug/dose related. Interestingly, group

scores revealed an elevation of pathology score only for thoracic

sections. No difference was noted in cervical sections. This lack

of difference in pathology scores for lumbar sections proximal to

the catheter tip where drug level is highest makes it difficult to

interpret the significance of the thoracic response. Finally,

although the higher dose of ketorolac (5.0 mg/ml/100 ml/h)

was not systematically examined, the incidence and magnitude

FIG. 5. Effect of intrathecal space perfusion of saline (solid squares),

ketorolac, 2.5 mg/ml (open circles), or ketorolac, 5 mg/ml (solid triangles) on

change in spinal cord blood flow velocity (SCBFV) as measured by laser-

Doppler flowmetry, in anesthetized rats (N 5 5 per group). No significant

change over time was observed with any treatment.


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of the spinal pathology was not different from that observed at

the lower doses. These incidental observations over a 10-fold

range of concentration and a 20-fold range of dosing confirm

the lack of an effect of ketorolac at these concentrations on

spinal tissue. These results are in accord with those of Gallivan

et al. (2000) who studied repeated epidural bolus delivery of

ketorolac in dogs and observed no untoward effects on

spinal structure.

While there were no group differences, three animals had

severe acute inflammation in the spinal cord parenchyma, two

(3858979 at all spinal levels and 3884376 at thoracic level) in the

0.5 mg/ml group and one (CPH AER at all levels) in the 5 mg/ml

group. These observations reflect several variables. First, these

animals displayed systemic neutropenia and reduced cisternal

CSF glucose (when determined), a profile suggestive of a bac-

terial infection. The severity of the effect upon CSF could not be

determined at necropsy from the third animal with severe spinal

cord inflammation. This animal was the smallest animal and had

the most severe gastrointestinal and adverse systemic responses.

Gram stains undertaken at the catheter tip were negative in all

animals. This, however, does not exclude that a peri-catheter

infection at other loci contributed to this picture. This suggestion

is supported by the observation of serous fluid along the external

subcutaneous catheter tract at necropsy in these animals.

Neuraxial bleeding. Blood was noted in the cisterna and

upper cervical cord at sacrifice in two 0.5 mg/kg and one 5.0

mg/kg dog. No bleeding was observed in other animals or vehi-

cle-treated dogs. As there was a percutaneous cisternal tap

approximately 30 min prior to death, it is possible that the obser-

vation of local cisternal blood reflects an event secondary to a

traumatic puncture. The presence of persistent blood ketorolac

might facilitate such bleeding. The fact that bleeding was not

observed in any animal intraparenchymally, or at the lumbar

level where drug concentration was highest supports a local

event at the cisterna. The importance of persistent anticoagula-

tion in this outcome is suggested by the absence of cisternal

bleeding in vehicle animals and in pharmacokinetic dogs receiv-

ing intrathecal ketorolac by bolus and by infusion for two-day

intervals.

Rat Studies

The absence of specific intrathecal drug related toxicity in the

dog was mirrored by the studies carried out in the rat. Here

moderate catheter-related pathological changes were noted.

These effects were not altered by ketorolac. Further, consistent

with previous work suggesting that prostaglandins have minimal

effects on central perfusion (Siesjo and Nilsson, 1982), local

ketorolac had no effect upon spinal blood flow.

Model Reliability

Spinal agents may lead to local reactions including aseptic

inflammation, neuronal death, and demyelination (Weller,

1999), which are driven by local drug concentration and duration

of exposure. (Yaksh et al., 1999b) The most robust assessment of

toxicity is provided by continuous delivery of the drug into the

local intrathecal space at the highest available concentration.

(Yaksh et al., 1999b) Accordingly, we employed the highest

concentration and dose consistent with survival and applied

this by continuous infusions for an interval of 28 days. Given

an absence of tissue effect, an important question is how sensi-

tive is the test model in displaying toxicity. To date, a variety of

agents have examined in this dog model, including baclofen

(Sabbe et al., 1993); clonidine (Yaksh et al., 1994); adenosine

(Chiari et al., 1999); neostigmine (Yaksh et al., 1995); and brain-

derived nerve growth factor (Yaksh et al., 1997). Morphine

given for 28 days as a bolus was also without effect (Sabbe

et al., 1994). In contrast, the opioid peptide DPDPE (Horais

et al., 2003) and high concentrations of morphine (Yaksh et al.,

2003) are associated with toxicity after 28-day continuous infu-

sion. The canine model is thus both robust and sensitive.

Intrathecal Ketorolac Kinetics

Intrathecal ketorolac showed a biphasic clearance after bolus

delivery. This clearance is consistent with a small moderately

lipid soluble molecule that is cleared from the CSF compartment

first by an initial distribution followed by clearance. Inulin, a

small molecule shows a similar biphasic clearance with similar

T1/2 values (Yaksh et al., 1997). Importantly, continuous infu-

sion of 0.5 and 5 mg/ml gave rise to lumbar CSF concentration of

approximately 4 and 53 mg/ml respectively, suggesting that the

CSF kinetics of intrathecally infused ketorolac are linear.

Intrathecal Ketorolac Efficacy

In the dog, the available pain models involve thermal escape

(e.g., skin twitch). COX inhibitors have little effect upon such

acute nociceptive processing (Svensson and Yaksh, 2002).

Accordingly, based on rodent data (Svensson and Yaksh,

2002), we anticipated that IT ketorolac would have no effect

upon this end point. However, measurement of prostaglandins

levels in the lumbar cerebrospinal fluid revealed depression of

the basal levels after bolus and infusion. Suppression was great-

est with continuous infusion. Delivery of 1.2 mg over 24 h (e.g.,

0.5 mg/ml) resulted in a greater reduction in CSF PGE2

levels than when 5 mg was delivered as a bolus. As suppression

of prostanoid synthesis reflects the essential mechanism of

ketorolac, we conclude that achieving CSF concentrations of

4 mg/ml of ketorolac in the CSF leads to a prominent biological

action.

Intrathecal Ketorolac Therapeutic Ratio

In the rat, it has been shown that intrathecal; ketorolac (10 mg/

10 ml) bolus can produce a potent analgesia (Malmberg and

Yaksh, 1992a.b., 1993). In the present study, we undertook

332 YAKSH ET AL.

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repeated bolus delivery of 50 mg/10 ml) of ketorolac. This is the

maximum dose that can be delivered given the standard injection

volume of 10 ml and available ketorolac concentration. At this

maximum concentration/dose, no untoward spinal toxicology

was noted. Accordingly, we suggest that the therapeutic index

(toxic dose/effective dose) for analgesia in the rodent exceeds

five. In so far as concerns the dog, the suppression of PGE2

concentration is a surrogate marker. We assert that a maximal

suppression of CSF-PGE2 concentration as compared to control

was observed with continuous infusion of 0.5 mg/ml yielding a

CSF concentration of 4 mg/ml. As spinal tissue toxicity was not

observed at a mean steady CSF concentration of ketorolac of

approximately 50 mg/ml (produced by 5 mg/ml), we suggest that

the therapeutic ratio as measured by ketorolac concentration in

the CSF or infused dose readily exceeds a factor of 10.

Relationship of Preclinical Safety Data to Humans

The ‘robustness’ of the assertion of intrathecal safety is

defined by the degree to which the preclinical exposure exceeds

that to which the human will be exposed. Accordingly, to the

degree that local concentration defines toxicity, we conclude that

28 days of exposure to a local CSF concentration of approxi-

mately 50 mg/ml (as produced by the continuous infusion of

5 mg/ml) has minimal drug related toxicity. As for bolus

delivery, we note that after the bolus delivery of 5 mg in 1 ml,

the initial CSF concentration was approximately 3000mg/ml or a

factor of 60 (i.e., 3000/60) greater. In previous work, we noted

that the local acute dilution volume of the lumbar space of the

dog is conservatively 1/3 of the human, i.e., for any given dose

the human will dilute the bolus injection by a factor three times

greater than the dog (Sabbe et al., 1993). Therefore, assuming

linear kinetics we would argue that the initial bolus dose of

ketorolac in humans would be 5 mg/ml 3 1/60 3 3 5 250

mg/ml. These are admittedly crude calculations, but provide a

conservative first approximation of the dose and concentration

for bolus delivery that should give a local concentration in the

human that was tolerated for 28 days in the preclinical model.

Based on these studies, a Phase 1 bolus dose ranging trial

was undertaken with intrathecal ketorolac in humans with no

significant events (Eisenach et al., 2002).

With regard to continuous infusion, it is clear that in the dog,

the limiting side effects at the 5 mg/ml concentration was total

dose, At doses greater than 6 mg/day (corresponding to approxi-

mately 0.6 mg/kg over 24 h in a 10 kg dog or chronic serum

concentrations of 1.1 mg/ml) systemic gastrointestinal effects

and reductions in hematocrit were evident. In humans, a single

bolus analgesic dose of ketorolac (approximately 0.4 mg/kg

yields peak plasma levels of 2–3 mg/ml; Brocks and Jamali,

1992) can indeed yield significant and long lasting (24 h)

changes in coagulation parameter (Niemi et al., 2000). Doubt-

less chronic exposure with these serum concentrations would be

poorly tolerated and such considerations must temper any

chronic ketorolac application in humans.

ACKNOWLEDGEMENT

This work was sponsored by NIH GM48085.

REFERENCES

Brocks, D. R., and Jamali, F. (1992). Clinical pharmacokinetics of ketorolac

tromethamine. Clin. Pharmacokinet. 23(6), 415–427.

Chiari, A., Yaksh, T. L., Myers, R. R., Provencher, J., Moore, L., Lee, C. S., and

Eisenach, J. C. (1999). Preclinical toxicity screening of intrathecal adenosine

in rats and dogs. Anesthesiology 91, 824–832.

Devoghel, J. C. (1983). Small intrathecal doses of lysine-acetylsalicylate relieve

intractable pain in man. J. Int. Med. Res. 11, 90–91.

Devoghel, J. C. (1993). Intrathecal injection of lysine-acetylsalicylate in man

with intractable cancer pain. In Progress in Pharmacology and Clinical

Pharmacology (I. Jurna and T. L. Yaksh, Eds.), Vol. 10, pp. 111–118. Gustav

Fischers Verlag.

Eisenberg, E., Berkey, C. S., Carr, D. B., Mosteller, F., and Chalmers, T. C.

(1994). Efficacy and safety of nonsteroidal antiinflammatory drugs for cancer

pain: A meta-analysis. J. Clin. Oncol. 12, 2756–2765.

Eisenach, J. C., Curry, R., Hood, D. D., and Yaksh, T. L. (2002). Phase I safety

assessment of intrathecal ketorolac. Pain 99(3), 599–604.

Gallivan, S. T., Johnston, S. A., Broadstone, R. V., Jortner, B. S., and Reimer, M.

(2000). The clinical, cerebrospinal fluid, and histopathologic effects of

epidural ketorolac in dogs. Vet. Surg. 29, 436–441.

Gillies, G. W., Kenny, G. N., Bullingham, R. E., and McArdle, C. S. (1987). The

morphine sparing effect of ketorolac tromethamine. A study of a new,

parenteral non-steroidal anti-inflammatory agent after abdominal surgery.

Anaesthesia 42, 727–731.

Horais, K., Hruby, V., Rossi, S., Cizkova, D., Meschter, C., Dorr, R., and

Yaksh, T. L. (2003). Effects of chronic intrathecal infusion of a partial

differential opioid agonist in dogs. Toxicol. Sci. 71, 263–275.

Malmberg, A. B., and Yaksh, T. L. (1993). Pharmacology of the spinal action of

ketorolac, morphine, ST-91, U50488H, and L-PIA on the formalin test and

an isobolographic analysis of the NSAID interaction. Anesthesiology 79,

270–281.

Malmberg, A. B., and Yaksh, T. L. (1992a). Antinociceptive actions of

spinal nonsteroidal anti-inflammatory agents on the formalin test in the rat.

J. Pharmacol. Exp. Ther. 263, 136–146.

Malmberg, A. B., and Yaksh, T. L. (1992b). Hyperalgesia mediated by spinal

glutamate or substance P receptor blocked by spinal cyclooxygenase inhibi-

tion. Science 257, 1276–1279.

Niemi, T. T., Backman, J. T., Syrjala, M. T., Viinikka, L. U., and Rosenberg, P. H.

(2000). Platelet dysfunction after intravenous ketorolac or propacetamol.

Acta Anaesthesiol. Scand. 44(1), 69–74.

Pellerin, M., Hardy, F., Abergel, A., Boule, D., Palacci, J. H.,

Babinet, P., Wingtin, L. N., Glowinski, J., Amiot, J. F., Mechali, D., et al.

(1987). [Chronic refractory pain in cancer patients. Value of the spinal

injection of lysine acetylsalicylate. 60 cases]. Presse Med. 16,

1465–1468.

Rice, A. S., Lloyd, J., Bullingham, R. E., and O’Sullivan, G. (1993). Ketorolac

penetration into the cerebrospinal fluid of humans. J. Clin. Anesth. 5(6),

459–462.

Sabbe, M. B., Grafe, M. R., Mjanger, E., Tiseo, P. J., Hill, H. F., and

Yaksh, T. L. (1994). Spinal delivery of sufentanil, alfentanil, and morphine

in dogs. Physiologic and toxicologic investigations. Anesthesiology 81,

899–920.

Sabbe, M. B., Grafe, M. R., Pfeifer, B. L., Mirzai, T. H., and Yaksh, T. L. (1993).

Toxicology of baclofen continuously infused into the spinal intrathecal space

of the dog. Neurotoxicology 14, 397–410.


Dow



Siesjo, B. K., and Nilsson, B. (1982). Prostaglandins and the cerebral circulation.

Adv. Prostaglandin. Thromboxane. Leukot. Res. 10, 367–380.

Svensson, C. I., and Yaksh, T. L. (2002). The spinal phospholipase-cyclooxy-

genase-prostanoid cascade in nociceptive processing. In Annual Reviews in

Pharmacology and Toxicology (A. K. Cho, T. Blaschke, P. A. Insel, and

H. H. Loh, Eds.), Vol. 42, pp. 553–583. Annual Reviews, Palo Alto, CA.

Weller, R. O. (1999). Reaction of intrathecal and epidural spaces to infection

and inflammation. In Spinal Drug Delivery (T. L. Yaksh, Ed.), pp. 297–315.

Elsevier Science B.V., Amsterdam.

Yaksh, T. L. (1982). Central and peripheral mechanisms for the antianalgesic

action of acetylsalicylic acid. In Acetylsalicylic Acid: New Uses for an Old

Drug (J. M. Barnet, J. Hirsh, and J. F. Mustard, Eds.), pp. 137–152. Raven

Press, New York.

Yaksh, T. L., Dirig, D. M., Conway, C. M., Svensson, C., Luo, D., and

Isakson, P. C. (2001). The acute antihyperalgesic action of NSAIDs and

release of spinal PGE2 is mediated by the inhibition of constitutive spinal

COX-2 but not COX-1. J. Neurosci. 21, 5847–5853.

Yaksh, T. L., Grafe, M. R., Malkmus, S., Rathbun, M. L., and Eisenach, J. C.

(1995). Studies on the safety of chronically administered intrathecal neostig-

mine methylsulfate in rats and dogs. Anesthesiology 82, 412–427.

Yaksh, T. L., Horais, K. A., Tozier, N. A., Allen, J. W., Rathbun, M., Rossi, S. S.,

Sommer, C., Meschter, C., Richter,P. J., and Hildebrand, K. R. (2003). Chroni-

cally infused intrathecal morphine in dogs. Anesthesiology 99, 174–187.

Yaksh, T. L., Hua, X. Y., Kalcheva, I., Nozaki-Taguchi, N., and Marsala, M.

(1999a). The spinal biology in humans and animals of pain states generated by

persistent small afferent input. Proc. Natl. Acad. Sci. USA. 96, 7680–7686.

Yaksh, T. L., and Malkmus, S. A. (1999). Animal models of intrathecal

and epidural drug delivery. In Spinal Drug Delivery (T. L. Yaksh, Ed.),

pp. 317–344. Elsevier Science B.V., Amsterdam.

Yaksh, T. L., Rathbun, M., Jage, J., Mirzai, T., Grafe, M., and Hiles, R. A. (1994).

Pharmacology and toxicology of chronically infused epidural clonidine HCl in

dogs. Fundam. Appl. Toxicol. 23, 319–335.

Yaksh, T. L., Rathbun, M. L., Dragani, J. C., Malkmus, S., Bourdeau, A. R.,

Richter, P., Powell, H., Myers, R. R., and Lebel, C. P. (1997). Kinetic

and safety studies on intrathecally infused recombinant-methionyl

human brain-derived neurotrophic factor in dogs. Fundam. Appl. Toxicol.

38, 89–100.

Yaksh, T. L., Rathbun, M. L., and Provencher, J. C. (1999b). Preclinical safety

evaluation for spinal drugs. In Spinal Drug Delivery (T. L. Yaksh, Ed.),

pp. 417–437. Elsevier Science B.V., Amsterdam.

Yaksh, T. L., and Rudy, T. A. (1976). Chronic catheterization of the spinal

ub-arachnoid space. Physiol. Behav. 17, 1031–1036.

Yaksh, T. L., and Svensson, C. (2001). Role of spinal cyclo-oxygenases in

nociceptive processing. In Therapeutic Roles of Selective COX-2 Inhibitors

(J. R. Vane and R. M. Botting, Eds.), pp. 168–190. William Harvey Press,

London.

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