* Department of Anesthesiology, University of California, San Diego, San Diego, California 92103;
College of Science, Department of Chemistry, University of Arizona, P.O. Box 210041, Tucson, Arizona 85721-0041;
Comparative Biosciences, Inc., 2672 Bayshore Parkway, Suite 515, Mountain View, California 94043; and
University of Arizona Cancer Center, University of Arizona, P.O. Box 245024, Tucson, Arizona 85724
Received August 12, 2002; accepted November 8, 2002
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ABSTRACT |
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Key Words: DPDPE; opioid agonist; dog; intrathecal; granuloma.
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INTRODUCTION |
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Prior to undertaking delivery of novel drugs spinally in humans, it is necessary to evaluate the spinal kinetics of the agents and the toxicological profile in preclinical experiment (Yaksh et al., 1999b). Accordingly, in the present studies, we examined (1) the maximum usable dose of DPDPE delivered by chronic intrathecal delivery for 28 days in the dog prepared with chronic lumbar intrathecal catheters, and (2) the intrathecal kinetics of DPDPE in that dog model. In these studies, we unexpectedly observed a concentration dependent local toxicity in DPDPE infused dogs.
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MATERIALS AND METHODS |
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Animals.
Male and female beagles were obtained from Marshall Farms (North Rose, NY). The animals were approximately 8 to 24 months old and weighed 11 to 13 kg at the initiation of treatment. Dogs were housed in individual runs in an AAALAC accredited vivarium. Water and food (Certified Teklad 25% Lab Dog Diet, Harlan Teklad, Madison, WI) were provided except during food fasting periods prior to surgery.
Intrathecal Catheter Placement
To permit continuous infusion in the intrathecal space, test articles were delivered by a chronically placed intrathecal catheter. After receipt in the facility, animals underwent a five to seven day acclimation period and prophylactic treatment with sulfamethoxazole trimethoprim tablets (1523 mg/kg, po twice daily, 480 mg/tablet from day -5 to day -1). On day -3, after an overnight fast, animals were anesthetized with atropine (0.4 mg/kg, im) followed by xylazine (1.5 mg/kg, im). After intubation, animals were maintained by spontaneous ventilation under 12% isoflurane and 60% N2O/40% O2. Oxygen saturation, inspired and end tidal values of gas, CO2, N2O, and O2, and heart and respiratory rates were continuously monitored. Surgical areas were shaved, surgically prepared and the animal was placed in a stereotaxic headholder and draped.
Safety animals.
To catheterize the intrathecal space, a midline incision was made on the dorsal head extending 1 cm rostral from the occiput to C1 and the muscles retracted to expose the cisternal membrane. A cisternal CSF sample was then taken by puncture of the cisternal membrane with a 23G needle for clinical chemistries. A small opening was then made in the cisternal membrane to access the intrathecal space. The PE10 intrathecal dosing catheter, previously e-beam irradiated, was then inserted through the opening and passed approximately 43 cm to the lumbar enlargement. Placement in the intrathecal space was determined by sampling lumbar CSF from the catheter and confirmation at necropsy.
After catheter placement, dexamethasone sodium phosphate (0.25 mg/kg, im) was given. The externalized end of the catheter was plugged and passed subcutaneously with a trocar to exit the left scapular region. The muscle and skin were sutured. Anesthetic gases were then turned off and the animal was monitored during recovery. Butorphanol tartrate (Torbugesic® 0.04 mg/kg, im) was administered to relieve postoperative pain. The surgery required approximately 1 h to complete.
Following recovery, a nylon vest (Alice King Chatham Medical Arts, Hawthorne, CA) was replaced on the animals (animals having been previously acclimated to the vest prior to surgery) and an infusion pump placed in the vest pocket where it was connected to the externalized end of the intrathecal catheter. The intrathecal catheter was continuously infused until initiation of test article treatment with 0.9% (w/v) sodium chloride for injection, USP (saline), at approximately 100 µl/h.
Pharmacokinetic animals.
For placement of two intrathecal catheters (dosing and sampling), a midline incision was made on the dorsal head extending 1 cm rostral from the occiput to C1 and the muscles retracted to expose the cisternal membrane. A small opening was then made in the cisternal membrane to access the intrathecal space. The PE50 intrathecal sampling catheter was then inserted through the opening and passed approximately 40 cm. The PE10 intrathecal dosing catheter was then inserted through an adjacent opening and passed approximately 43 cm to the lumbar enlargement. Lumbar CSF was then sampled from both catheters to confirm placement in the intrathecal space. The externalized ends of the catheters were plugged and passed subcutaneously with a trocar to exit the left and right scapular regions respectively. The muscle and skin were sutured with 3-0 VicrylTM. Anesthetic gases were turned off and the animal was monitored during recovery. Butorphanol tartrate (Torbugesic® 0.04 mg/kg, im) was administered to relieve postoperative pain. The surgery required approximately 1.5 h to complete.
Following recovery, a nylon vest was replaced on the animals (animals having been previously acclimated to the vest prior to surgery) and an infusion pump placed in each of the vest pockets where they were connected to the externalized ends of the intrathecal catheters. The intrathecal catheters were continuously infused until initiation of test article treatment with 0.9% (w/v) sodium chloride for injection, USP (saline), at approximately 100 µl/h. For PE50 sampling catheters, saline infusion was continued during nondosing intervals to maintain catheter sampling patency; the saline infusion was interrupted during intrathecal lumbar sampling intervals.
Drug.
The c[D-Penicillamine2, D-Penicillamine5]enkephalin (DPDPE; Akiyama et al., 1985; Mosberg et al., 1983
) test compound was obtained from the University of Arizona (Tucson, AZ). The test compound was provided as a 3 mg/ml or 6 mg/ml pyrogen-free, aqueous solution suitable for intrathecal administration. The pure compound, >99% DPDPE, was prepared under cGMP conditions by American Peptide, Inc. (Sunnyvale, CA). Purity and structural properties, were evaluated by quantitative amino acid analysis, mass spectra, optical rotation, and HPLC (two separate elution/column systems).
Drug preparation and delivery.
Dosing solutions were delivered as an intrathecal bolus or continuous infusion through a chronic PE10 catheter. Intrathecal boluses were delivered by syringe and needle in a volume of 1.0 ml followed by 0.5 ml saline flush. Continuous infusions were delivered via an infusion pump (Panomat C-10, T-10 or equivalent, Disetronic Medical Systems, Inc., St. Paul, MN) in a volume of 2.4 ml/day (100 µl/h).
Safety Study
Study paradigm.
The safety phase was undertaken to determine the safety as defined by the effects upon neurological function and spinal histology of two concentrations of DPDPE infused continuously into the intrathecal space for at least 28 days in beagle dogs (n = 12). Control animals were infused with saline for a minimum of 28 days.
Observations.
Food consumption, rectal temperature, presence and nature of urine and stool, and behavioral function were recorded daily throughout the in-life phase
For behavioral function, motor function and arousal assessments were performed daily throughout the in-life phase of the study as described in detail elsewhere (Yaksh et al., 1997). Physiological (heart rate, blood pressure, respiration) and nociception responses were measured at regular intervals. The thermally evoked skin twitch response was measured using a probe (approximately 1 cm surface area) maintained at approximately 62.5 ± 0.5°C. The probe was applied to shaven thoracic-lumbar areas of the back. Typically, this stimulus results in a brisk contraction of the local, underlying musculature within 13 s after probe placement. Upon appearance of this response, the probe was removed and the latency recorded. Failure to respond within 6 s was cause to remove the probe and assign that value (6 s) as the latency (Sabbe et al., 1994
).
Clinical laboratory measurements.
Blood and cisternal CSF samples were obtained prior to surgery and on the day of necropsy. Plasma samples were taken at specified time points during the infusion interval for drug analysis. Cisternal CSF samples for drug assays were obtained at sacrifice prior to sacrifice. Blood was obtained by cephalic venipuncture. Cisternal CSF was obtained while under anesthesia by sterile puncture of the cisterna magna with a 23-gauge needle at surgery and a 22-gauge 1.5-inch spinal needle at necropsy. At the end of the infusion interval, animals were euthanized and necropsied with histopathology performed on the spinal cord.
Pharmacokinetics Study
Study paradigm.
Pharmacokinetic animals (n = 3) with dual lumbar catheters were prepared to determine (1) the clearance of DPDPE from lumbar CSF after a bolus intrathecal injection, (2) steady-state lumbar CSF levels produced by continuous infusion of two concentrations of DPDPE, and (3) the time course of clearance in lumbar CSF of DPDPE after termination of a continuous intrathecal infusion of DPDPE. Animals received an intrathecal bolus (1 ml) of the 3 mg/ml DPDPE followed by a 0.5 ml saline flush. Samples were taken periodically over the ensuing 24 h. After the 24-h sampling interval, an intrathecal infusion of 3 mg/ml (2.4 ml/day) DPDPE for 24 h was initiated. Following this infusion/sampling interval, the infusion dose was increased to 6 mg/ml (2.4 ml/day) for the following 24 h. After the 24 h infusion of DPDPE (6 mg/ml), drug infusion was terminated and samples were collected at intervals over 24 h after termination of the drug during the clearance period.
Necropsy.
On the scheduled date of necropsy of a safety study animal, each animal was anesthetized with sodium pentobarbital (35 mg/kg or to effect, iv). A percutaneous puncture of the cisterna magna was performed to collect cisternal CSF (approximately 2.5 ml). The chest was then opened, aorta catheterized, and the blood cleared by perfusion at 80160 mmHg of pressure with approximately 4 l of saline (0.9% sodium chloride) followed by approximately 4 l of 10% formalin. The spinal cord dura was exposed by laminectomy of the spinal canal. Methylene blue dye was injected through the catheter to confirm catheter placement and integrity. For the pharmacokinetic studies, a similar protocol was followed with the principal exceptions being that subjects received an overdose of sodium pentobarbital (50 mg/ml, iv), cardiac perfusions were not performed, and both intrathecal catheters were assessed for patency and location by dye injection.
Drug assay.
DPDPE was quantified by an atmospheric pressure ionization liquid chromatographic-mass spectrometric assay (see below; Rossi and Yaksh, 2002). Samples (5 µl) containing DADLE as internal standard (IS) were de-salted by reverse phase C18 solid phase extraction using ZipTip micro-cartridges. One µl aliquots of extracted eluate were injected onto an Agilent Zorbax SBC-18 column (30 x 2.2 mm; 3.5 Fm) at a flow rate of 0.4 ml/min. The isocratic mobile phase of methanol and 10 mM ammonium formate (pH 3; 75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadruple mass spectrometer, and DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r2 >0.991) over the concentration range 11000 ng/ml. The efficiency of extraction recovery was 97% and the intra-assay and inter-assay precision was within 9% relative SD. DPDPE and IS were stable in the injection solvent at 4°C for at least 48 h.
Histopathology.
For safety animals, four blocks of spinal cord (cervical, thoracic, lumbar [catheter tip region], and distal lumbar) were embedded in paraffin, sectioned, stained with haematoxylin-eosin, and examined by light microscopy by a board-certified veterinary pathologist unaware of the experimental treatment. Semiquantitative evaluations were made as to the degree of inflammation and the pathological changes in the meninges, vessels, nerve roots, and spinal cord parenchyma found in these sections (Yaksh et al., 1997). Observed tissue was assigned an individual pathology score based on the evaluation. The pathology score was on a whole number scale from 0 to 4, where 0 represented no injury or inflammation and 4 represented very severe injury and/or inflammation. Additionally, a single overall pathology ranking was assigned to each animal, using a semiquantitative scale of 1 to 12. This score reflected the ranking of that individual animal as compared with all other animals in the study.
Additional immunohistochemical analysis was performed on paraffin-embedded sections using following primary antibodies: monoclonal mouse GFAP (1:1000), rabbit TNF- (1:1000, Serotec, Raleigh, NC), mouse antihuman macrophages MAC (1:200, Serotec). Briefly, sections were deparaffinized in xylene and rehydrated through graded alcohols. Nonspecific immunolabeling was blocked using 5% normal goat serum (NGS) in phosphate buffered saline containing 0.2% Triton X-100, pH7.4. Excess blocker was removed and mouse and rabbit primary antibodies were applied (24 h/4°C). Afterwards, sections were washed in PBS-TX 100 and incubated with fluorescence secondary antibodies, goat antimouse IgG Alexa Fluoro-488, or goat antirabbit Alexa Fluoro-594 (Molecular Probes, Eugene, OR). In addition, a nuclear marker for neuronal and nonneuronal cells, Dapi (4', 6-diamidino-2-phenylindole dihydrochloride, Molecular Probes, OR) was added to the solution and incubated together with secondary antibodies 1 h at room temperature. Finally, sections were rinsed and cover slipped with antifade mounting medium kit (Molecular Probes, OR) and observed under a Leica fluorescence microscope. To confirm specificity of immunolabeling, sections were processed without primary antibodies or by substituting normal mouse or rabbit serum for mouse or rabbit primary sera.
Data analysis.
Data are expressed as means and standard errors of the respective observations (e.g., mmHg, beat/min, µg/ml). For motor function, the cumulative coordination scores were obtained by adding the positive value of the individual daily motor function score for the 28 days of test article delivery. 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 < 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.
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RESULTS |
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Assessment of general behavioral indices throughout the study showed no systematic changes in arousal or muscle tone (data not shown) within any of the three treatment groups. No evidence of urinary retention or changes in daily stool were noted at any time during this study.
Neurological Assessments
No significant neurological abnormalities or motor dysfunction associated with infusion were observed in three of the four the saline animals. One saline animal displayed a mild hind limb ataxia beginning on day 4 that persisted unaltered throughout the in-life phase of the study. There was a concentration-dependent development of motor dysfunction in the DPDPE treatment groups (Fig. 1). This dysfunction evolved over the 28-day interval of infusion with the mean time of onset for motor dysfunction being 11 ± 5 and 6 ± 1 days for the 3 mg/ml and 6 mg/ml DPDPE treatment groups respectively. Neurologically, the abnormalities noted included decreased hind limb reflexes (e.g., wheelbarrow reflex, tactile placing reflex), decreased righting reflex, and pain and discomfort upon ambulation and manipulation.
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Respiratory rate, blood pressure, and heart rate.
Mean respiration rates ranged from 16 to 32 breaths/min and did not vary from baseline measures over the course of the infusions. Mean arterial pressures ranged from 96 to 127 mmHg and did not vary from baseline over the course of the infusions. Heart rates ranged from 58162 beats/min and did not vary significantly from baseline over the infusion interval (see Table 1).
Nociceptive response.
By the seventh day of infusion, skin twitch latencies in DPDPE-treated animals had increased in a dose-dependent fashion and remained elevated as compared to the vehicle control (see Fig. 2).
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Gross pathology.
At necropsy, vehicle and 3 mg/ml DPDPE-treated animals showed no remarkable changes in external spinal cord morphology upon gross examination. Two of four 6 mg/ml DPDPE treated animals spinal cords showed some discoloration and erosion of the dura in the vicinity of the catheter tip.
Histopathology
Overview.
In vehicle-treated animals, a modest pericatheter reaction was observed at all levels of the catheter with no significant masses or changes in spinal cord morphology (Fig. 3). This experimental group showed minimal reaction as defined by glial reaction (GFAP) or macrophage infiltration. In contrast, in the high dose DPDPE-infused animals, significant localized collections of inflammatory cells (granulomas) occurred with severe multifocal inflammation and mild to moderate damage to the neuropil (Figs. 3
and 4
). In the 3 mg/ml DPDPE group, lesions were similar in nature though considerably less severe. Dorsal root ganglia were not affected.
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Additional immunocytochemical staining characterized the cellular composition of granuloma masses with respect to glial elements, pro-inflammatory cytokines, and macrophages. Normal patterns of parenchymal staining, as evidenced by Dapi immunoreactivity intermixed with GFAP positive astrocytes, was observed in spinal cord segments above the catheter tip (Fig. 5C). At the level of the inflammatory mass, GFAP positive cells were observed in adjacent tissue and not in the mass. The perivascular cuffs in the parenchymal white matter (Fig. 5A
) were surrounded by normal astrocytic figures (Fig. 5B
). In contrast, occasional reactive gliosis limited to the regions of vessels in the spinal cord parenchyma (Figs. 5D
and 5E
) or to the spinal cord compression could be detected. However, small areas of attenuated GFAP staining could be detected in nearby regions of significant compression indicating significant cell loss (Fig. 5E
). No evidence of bacterial or fungal involvement could be detected, based on histological stains (data not shown).
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Cisternal CSF.
Cisternal CSF in the control animals displayed a slight increase in protein concentration. DPDPE infused groups displayed marked increase in protein concentrations compared to baseline measures. There were no significant changes in cisternal glucose concentrations or white blood cell counts in any group (see Table 3). Comparison of the rank-ordered increase in cisternal protein concentrations with the Jonckheere statistical test for ordered significance revealed the increases to be proportional to dose (p < 0.05).
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Pharmacokinetics
Bolus treatment.
The bolus kinetics data for individual animals are presented in Figure 7. The calculated values for the three animals and the mean parameters are presented in Table 4
. As indicated, after bolus delivery of 3 mg/ml, there was biphasic clearance of the DPDPE from the lumbar intrathecal space, with T1/2D and T1/2E values of 14.0 ± 10.3 and 100.0 ± 17.2 min, respectively. Examination of the concurrent inulin data and the DPDPE/inulin ratio indicate that DPDPE was cleared more rapidly than the extracellular marker during the initial distribution phase.
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DISCUSSION |
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Over the period of infusion there was a progressive loss of the inhibition of the skin twitch response latency. Previous work has shown that such extended exposure to µ and opioid agonists leads to a pharmacological tolerance wherein the antinociceptive effects are diminished (Russell and Chang, 1989
; Sabbe et al., 1994
; Stevens and Yaksh, 1992
). While pharmacological tolerance is a likely mechanism of the observed changes in antinociception, the cisternal levels of DPDPE (and perhaps in the lumbar intrathecal space) were substantially lower than expected in those safety animals receiving 28-day high concentration infusions. As will be noted below, all of these high concentration animals displayed significant inflammatory masses and we hypothesize that this lower DPDPE concentration might reflect a rapid clearance of the drug from the lumbar CSF. If so, this increased clearance might also contribute to the loss of antinociception observed here.
In addition to the antinociceptive action, over the 28-day infusion interval, dogs receiving the higher concentrations of DPDPE showed a reliable and progressive onset of hind limb dysfunction. The increased motor tone and time course suggested a gradually developing upper motor neuron type lesion.
Histological Effects of Intrathecal DPDPE Infusion
After the 28-day intrathecal infusions, histology revealed modest pericatheter reactions in vehicle infused dogs, consistent with a typical local intrathecal meningeal reaction to a foreign body, such as the catheter (Weller, 1999). In contrast, in animals receiving the
opioid agonist DPDPE, we observed a concentration-dependent development of a compressive intrathecal mass proximal to the catheter tip. Inspection of serial sections suggested that the inflammatory mass appeared to take origin from the dura-arachnoid layer. Cellular constitution of the mass consisted of multifocal accumulations of neutrophils, monocytes, macrophages, and plasma cells, resembling a granuloma (Adams and Hamilton, 1989
; Sheffield, 1990
).
No evidence of bacterial or fungal involvement could be detected, based on histological stains or CSF cultures. This inflammatory response was associated with increased cisternal CSF protein and the progressive development of neurological signs consistent with a compressive spinal cord lesion.
These effects were most evident in the groups receiving 6 mg/ml. Given the restricted distributional kinetics of the intrathecal space, the current thinking points to the importance of local drug concentration as an important variable in the tissue toxicity produced by intrathecally infused drugs (Yaksh et al., 1999b). The steady state CSF concentration of DPDPE adjacent to the catheter tip observed in the present study at 6 mg/ml was on the order of 25 µg/ml.
Intrathecal DPDPE Kinetics
The intrathecal delivery of DPDPE resulted in a biphasic decline in concentration, reflecting an initial redistribution and a second slower clearance that reflect the movement for the injection site by bulk redistribution in the CSF and into the tissue from the CSF (Shafer and Shafer, 1999). Systematic examination for the presence of DPDPE metabolites was not undertaken in the present study. However, previous work has indicated it to be remarkably stable after incubation in blood or brain tissue, a finding consistent with its cyclic structure (Weber et al., 1992
). The T1/2E calculated after bolus delivery was comparable to that observed when the clearance from steady state was determined in the continuous infusion experiments, suggesting that the kinetics of he agent after bolus or continuous infusion were similar. The half life of DPDPE resembles that previously reported in this model for molecules such as inulin, brain derived nerve growth factor, and ziconotide, an N-type calcium channel blocker (Yaksh et al., 1997
, 1999a
). With continuous infusion, steady states were achieved by 8 h, a finding consistent with the estimated lumbar CSF elimination half-life of DPDPE.
In the safety studies, cisternal CSF was sampled at the time of sacrifice. As indicated, the levels of cisternal DPDPE in the high-dose animals were considerably less than those observed in animals receiving lower doses. This unexpected finding has been similarly observed in recent work with intrathecal morphine where a similar granuloma formation in dogs showing high levels of granuloma formation supraspinal redistribution was attenuated as evidenced by reduced cisternal concentrations (Yaksh et al., in press). While lumbar CSF levels in animals displaying inflammatory masses have not yet been assessed, this reduced cisternal redistribution may reflect an increased local clearance and/or metabolism. It is currently appreciated that intrathecal drug can diffuse though the meninges (Ummenhofer et al., 2000
; Zenker et al., 1994
). To the degree that the inflammatory mass damages local meningeal integrity in the vicinity of the catheter tip, a more pronounced clearance of local drug may occur.
Nonspecific Mechanisms Initiating Intrathecal Inflammatory Reactions
Several variables contributing to the triggering of this intrathecal granuloma may be considered. (1) Absence of positive CSF or injectate cultures or histological signs of fungal or bacterial infection suggests that the granuloma is not an infectious process. (2) The DPDPE solutions delivered spinally in the dog studies are essentially isotonic with CSF (Artru, 1999) and comparable in pH and osmolarity to the 0.9% saline vehicle. (3) The results were not due to the catheter or infusion alone. Granuloma growth was not present in saline animals and was reliably evident at higher DPDPE infusion concentrations. In other studies with this canine model, infusion for 28 days of other agents including baclofen (2 mg/ml; Sabbe et al., 1993
), clonidine (2 mg/ml, present study), adenosine (5 mg/ml; Chiari et al., 1999
), neostigmine (2 mg/ml; Yaksh et al., 1995
), and brain derived nerve growth factor (2 mg/ml; Yaksh et al., 1997
) did not cause such inflammatory masses. However, as noted above, in recent studies with continuous infusions of high concentrations of intrathecal morphine, comparable masses were noted (Yaksh et al., in press
).
DPDPE Mechanisms for Inducing an Intrathecal Granuloma
The mechanism by which DPDPE produces these intrathecal masses is not known. Given the pharmacology of DPDPE, we suggest the speculative hypothesis that this agent could be acting through a opioid receptor to influence inflammatory cell migration through the meningeal and superficial parenchymal vasculature. Delta opioid receptor mRNA and binding are present in a variety of inflammatory cells including peripheral mononuclear, T-cell, B-cell, and monocyte cell lines, and peripheral dendritic cells in blood and other peripheral organs (Chuang et al., 1994
; Gaveriaux et al., 1995
; Sharp et al., 2000
; Wick et al., 1995
), as well as on vascular endothelial cells (Stefano, 1998
). In recent preliminary work we have identified significant binding of
opioid receptor targeted antibody in dog granulomas (Sommers, Cizkova, and Yaksh, unpublished observations).
While the role played by a opioid receptor in the development of a granuloma with intrathecal DPDPE is not certain, it is clear that
opioid agonists have potent effects upon thymocyte activation and T-cell proliferation (Bidlack, 2000
). Thus, T-lymphocyte chemotaxis is activated by DPDPE in a concentration-dependent fashion (Heagy et al., 1990
). Endothelial cells also display
opioid binding, and local application of
opiate agonists resulted in enhanced granulocyte adherence (Stefano, 1998
). Consistent with this activating influence on inflammatory cells,
agonists can evoke the formation of pro-inflammatory cytokines (TNF-
and IL1ß) from macrophages and free radical formation by neutrophils (Gomez-Flores et al., 2001
; Haberstock and Marotti, 1995
; Nelson and Lysle, 2001
). As noted in the present studies, significant expression of TNF-
was observed in the granuloma and in macrophages in the perivascular cuffs. Accordingly, given the observed origin of the mass, we suggest that the inflammatory cells derive from this dural microvasculature, which lies in proximity to the catheter tip (Kerber and Newton, 1973
). Given the above body of observations, we are inclined to speculate that with persistent exposure to high concentrations of a
(or µ) opioid agonist, a cascade is initiated that is mediated in part by an action upon local endothelial cells that serve to increase cytokine release. This then leads to a local increase in T cell and granulocyte migration through the dural capillary network. We recognize that in the chronic implant model there is a modest ongoing stimulus that is provided by the catheter. This low level stimulation may, on occasion, also provide moderate levels of cytokines that become sufficient to initiate a local aseptic reaction that leads to enhanced inflammatory cell migration. These inflammatory cells lead to an additional local release of cytokines (TNF-
) that can initiate additional inflammatory responses, including the activation of local dendritic cells (McMenamin, 1999
). Mu and delta opioid agonists have been shown to facilitate the stimulatory effect of dendritic cells on T-cell proliferation (Makarenkova et al., 2001
). We again emphasize that additional studies are required to reliably implicate the role of
opioid receptors in this proposed cascade.
Clinical Relevance of Intrathecal Granuloma Formation
We believe that the present findings indicating a concentration-dependent granulomatous reaction to continuous infusions of intrathecal DPDPE and recent parallel findings with chronic intrathecal morphine have particular relevance to the clinical utilization of these agents. While DPDPE has not been administered in humans, morphine has been widely used as a continuously infused agent since 1981 (Onofrio et al., 1981). Until the 1990s, no untoward events as noted here were reported (Wallace and Yaksh, 2000
). At that time, the concentrations of morphine placed in pumps were increased to obtain longer pump refill intervals. Since that time, six clinical case reports describe 13 catheter patients in whom a local compressive lesion was initially manifested as a progressive motor or sensory dysfunction (Anderson et al., 2001
; Bejjani et al., 1997
; Blount et al., 1996
; Cabbell et al., 1998
; Coombs et al., 1985
; Langsam, 1999
; North et al., 1991
). Recently, a comprehensive retrospective review has been published that reflects experiences with an additional 45 instances of human chronic morphine infusion patients with comparable symptomatology (Coffey and Burchiel, 2002
). In the published case reports, the mass was identified by imaging and/or by subsequent resection. There is no evidence of infection (Anderson et al., 2001
; Bejjani et al., 1997
; Cabbell et al., 1998
). Histology has emphasized the presence of macrophages, neutrophils, and monocytes, with a necrotic center, a description closely paralleling that which we have noted with intrathecal DPDPE and morphine in dogs (present study; Allen et al., 2002
). Importantly, as with the previous work with dog infusions, this problem has not been reported in patients receiving baclofen for spasticity, suggesting a specific drug effect (Coffey and Burchiel, 2002
; Penn, 1992
).
The importance of the present studies is that they reveal the potential of granuloma formation as an important component of potential toxicity in the use of spinal delivery. The intrathecal space has become increasingly appreciated as an important route of delivery for drugs to manage a variety of syndromes including pain, spasticity, and obesity for the treatment of meningeocarcinomatosis, spinal infections, and neuropathologies, such as degenerative disorders with growth factors (see Yaksh, 1999). In many cases, the proposed therapy requires a persistent drug exposure. Aside from the potential direct tissue toxicity that could arise from such a local drug action, the current evidence suggests that an important factor may well be the evolution of space occupying granulomas. Our understanding of how this collection of inflammatory cells develops will be important in further implementing this route of drug delivery. The observations here suggest that the canine model will provide an important vehicle for assessing those mechanisms.
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ACKNOWLEDGMENTS |
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NOTES |
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