Michael Rathbun

Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli

We have examined the stability and sources of variation within the nociceptive model of rat hind paw withdrawal from an under-glass radiant stimulus (Hargreaves et al., 1988) using a system where stimulus intensity and floor temperature can be controlled and reproducibly changed. The current study demonstrates that: (i) increased stimulus intensity with a fixed surface temperature is associated with a monotonic decrease in mean response latency and its variance; (ii) for a fixed stimulus intensity, the mean paw withdrawal latency and variance increased as the glass floor temperature is lowered from 30 degrees C to room temperature (25 degrees C). Using subcutaneously-implanted thermocouples and a 30 degrees C glass surface, the subcutaneous paw temperature observed at an interval corresponding to the time at which the animal displayed a paw withdrawal did not differ across multiple heating rates (41-42.5 degrees C). This finding is in agreement with human studies of pain thresholds and C-fiber activity. These studies emphasize the importance of maintaining a fixed surface temperature to reduce experimental variability and the utility of this apparatus across multiple stimulus intensities to define agonist efficacy.

Efficacy of systemic morphine suggests a fundamental difference in the mechanisms that generate bone cancer vs. inflammatory pain

&NA; Pain is the cancer related event that is most disruptive to the cancer patients quality of life. Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast, prostate and lung cancers, relatively little is known about the mechanisms that generate and maintain this pain. Recently, we developed a mouse model of bone cancer pain and 16 days following tumor implantation into the intramedullary space of the femur, significant bone destruction and bone cancer pain‐related behaviors were observed. A critical question is how closely this model mirrors human bone cancer pain. In the present study we show that, as in humans, pain‐related behaviors are diminished by systemic morphine administration in a dose dependent fashion that is naloxone‐reversible. Humans suffering from bone cancer pain generally require significantly higher doses of morphine as compared to individuals with inflammatory pain and in the mouse model, the doses of morphine required to block bone cancer pain‐related behaviors were ten times that required to block peak inflammatory pain behaviors of comparable magnitude induced by hindpaw injection of complete Freunds adjuvant (CFA) (1–3 mg/kg). As these animals were treated acutely, there was not time for morphine tolerance to develop and the rightward shift in analgesic efficacy observed in bone cancer pain vs. inflammatory pain suggests a fundamental difference in the underlying mechanisms that generate bone cancer vs. inflammatory pain. These results indicate that this model may be useful in defining drug therapies that are targeted for complex bone cancer pain syndromes.

Studies on the Safety of Chronically Administered Intrathecal Neostigmine Methylsulfate in Rats and Dogs

Background The spinal delivery of the cholinesterase inhibitor neostigmine yields analgesia in rats and augments the analgesic effects of alpha2 agonists in sheep. To assess its activity in humans, preclinical toxicology studies to define its safety were required in two species. Methods Rats with chronic intrathecal catheters received daily injections of saline (vehicle) or 5 micro gram/10 micro liter or 10 micro gram/10 micro liter neostigmine HCl (n = 6/group) for 4 days and were observed for general behavior and nociception (52.5 degrees Celsius hot plate). On day 6, rats were anesthetized and submitted to whole body perfusion/fixation. For dog studies, male beagles were prepared following rigid aseptic precautions with catheters passed from the cisterna magna to the lumbar intrathecal space. Catheters were connected to an external vest‐mounted pump. Based on preliminary studies, ten implanted dogs were randomly assigned to receive infusions of neostigmine for 28 days (4 mg/4 ml/day; n = 6) or saline (4 ml/day; n = 4). At 28 days, dogs were anesthetized, cisternal cerebrospinal fluid was obtained, and dogs were submitted to perfusion‐fixation. Rat and dog spinal cords were embedded, sectioned, stained, and assessed by the pathologist without knowledge of treatment. Results In rats, neostigmine produced a dose‐dependent increase in hot plate latency, and no tolerance was observed. Mild tremor was observed but was not debilitating. Histopathology revealed a mild fibrotic reaction to the catheter with mixed signs of moderate, acute, and chronic inflammation with no differences between saline or drug groups. In dogs, neostigmine had no effect on blood pressure or on the skin twitch response but produced bradycardia and an increase in muscle tone. At sacrifice, cerebrospinal fluid protein, specific gravity, and glucose were elevated in both saline and neostigmine groups. Histopathology displayed a local reaction to the spinal catheter and a mixed acute and chronic inflammatory reaction. No group differences were observed. These results suggest that, at the neostigmine concentration of 1 mg/ml in the rat and dog and in doses up to 4 mg/day in the dog, there is no evidence of spinal tissue toxicity that can be attributed to the drug. This result, observed in two species, suggests that intrathecal neostigmine given in this manner is without distinguishable toxicity in these two models.

Chronically Infused Intrathecal Morphine in Dogs

Background Despite the extensive use of intrathecal morphine infusion for pain, no systematic safety studies exist on its effects in high concentrations. The authors assessed the effects of morphine and clonidine given 28 days intrathecally in dogs. Methods Beagles with lumbar intrathecal catheters received solutions delivered by a vest-mounted infusion pump. Six groups (n = 3 each) received infusions (40 &mgr;l/h) of saline or 1.5, 3, 6, 9, or 12 mg/day of morphine for 28 days. Additional groups received morphine at 40 &mgr;l/h (1.5 mg/day) plus clonidine (0.25–1.0 mg/day) or clonidine alone at 100 &mgr;g/h (4.8 mg/day). Results In animals receiving 9 or 12 mg/day morphine, allodynia was observed shortly after initiation of infusion. A concentration-dependent increase in hind limb dysfunction evolved over the infusion interval. Necropsy revealed minimal reactions in saline animals. At the higher morphine concentrations (all dogs receiving 12 mg/day), there was a local inflammatory mass at the catheter tip that produced significant local tissue compression. All animals with motor dysfunction displayed masses, although all animals with masses did not show motor dysfunction. The mass, arising from the dura-arachnoid layer, consisted of multifocal accumulations of neutrophils, monocytes, macrophages, and plasma cells. Inflammatory cells and endothelial cells displayed significant IL1&bgr;, TNF&agr;, iNOS, and eNOS immunoreactivity. No evidence of bacterial or fungal involvement was detected. There were no other changes in spinal morphologic characteristics. In four other groups of dogs, clonidine alone had no effect and in combination with morphine reduced the morphine reaction. Conclusions The authors found that high intrathecal morphine concentrations lead to aseptic intrathecal inflammatory masses. The lack of effect of clonidine and the possible suppressive effects of clonidine on the local reaction suggest the utility of such coadministration.

Pharmacokinetics and Efficacy of Epidurally Delivered Sustained-release Encapsulated Morphine in Dogs

BACKGROUND We evaluated the epidural effects of a multivesicular liposome-based sustained-release preparation of morphine (C0401) on behavior and lumbar cerebrospinal fluid and serum kinetics of morphine. METHODS Beagle dogs were prepared with lumbar epidural catheters with subcutaneous injection ports and lumbar intrathecal catheters. Each dog (n = 6) received the following by the epidural route: 5 mg/3 ml morphine sulfate in saline (MS-5), 10 mg/3 ml C0401 (C0401-10), and 30 mg/3 ml C0401 (C0401-30). Behavioral and physiologic parameters and nociceptive responses (skin twitch latency) were evaluated, and morphine concentrations were determined in lumbar cerebrospinal fluid and serum. RESULTS All morphine treatments blocked the skin twitch response. Time to onset was 1.3 +/- 0.3 h for C0401-30; 2.6 +/- 0.6 h for C0401-10; and 0.4 +/- 0.2 h for MS-5. Duration of action was 62 +/- 0.3 h for C0401-30; 27 +/- 2 h for MS-5. All treatments produced a modest reduction in arousal, muscle tone, and coordination, with the duration of the C0401-30 preparation being longer lasting. Respiratory rate was mildly depressed by all treatments, and moderate hypotension was noted. Time to peak cerebrospinal fluid morphine concentration was 11 h for C0401-30; 3 h for C0401-10; and 5 min for MS-5. Peak lumbar cerebrospinal fluid level was 34,992 +/- 5,578 ng/ml for MS-5; 14,483 +/- 3,438 ng/ml for C0401-30; and 10,730 +/- 2,888 ng/ml for C0401-10. Morphine mean residence time in lumbar cerebrospinal fluid was 0.8 +/- 0.1 h for MS-5; 8.9 +/- 1.0 h for C0401-30. DISCUSSION Kinetics studies showed that multivesicular liposome sequestration results in a restrained and persistent release of morphine from the epidural space. This extended release corresponded with an extended duration of analgesia without an attendant increase in the incidence of side effects.

Pharmacology and Toxicology of Chronically Infused Epidural Clonidine · HCl in Dogs

To evaluate the physiological effects and toxicity of epidural clonidine.HCl, male Beagle dogs were prepared with chronic lumbar epidural catheters and administered constant infusions of either saline (N = 10), or 80 micrograms/hr (N = 6), 200 micrograms/hr (N = 6), or 320 micrograms/hr (N = 12) clonidine.HCl at a rate of 4 ml/24 hr for 28 days. Saline infusion had no effect upon any behavioral measure. Epidural clonidine produced a dose-dependent increase in thermal skin-twitch response latency (antinociception), lowering of respiration rate, heart rate, and blood pressure, and increased sedation. The effects were maximum from approximately Day 1 to Day 3 when, with the exception of respiration which remained depressed, a progressive adaptation was observed over the course of the study. There were no negative effects on body weight, body temperature, motor function, bowel or bladder function, or clinical pathology values. After 28 days of continuous infusion, the dogs were deeply anesthetized and terminated. Cisternal cerebrospinal fluid taken at termination displayed no clinically significant differences in protein or glucose concentration. All groups, including control, had dogs which had a chronic inflammatory response in the epidural space, as represented by fibrosis, foreign body giant cells, and lymphocytes, but no spinal cord pathology. Both the steady-state plasma and CSF concentrations of clonidine were proportional to the dose; the ratio of CSF to plasma concentration was approximately 0.5. The failure to see any change in CSF composition, significant spinal cord pathology, or signs of tissue or organ toxicity emphasizes the safety of epidurally administered clonidine at infusion rates up to 320 micrograms/hr and at infusate concentrations up to 2 mg/ml.

Safety Assessment of Encapsulated Morphine Delivered Epidurally in a Sustained-Release Multivesicular Liposome Preparation in Dogs

We have shown that the epidural (EPI) delivery of morphine encapsulated in multivesicular liposomes (DepoFoam™ drug delivery system) produces a sustained clearance of morphine and a prolonged analgesia. We have sought to subsequently determine the likelihood of deleterious effects on local tissue of repetitive epidural injections of this encapsulated morphine preparation (C0401). Beagle dogs were prepared according to protocol approved by the Institutional Animal Care and Use Committee under volatile general anesthesia with chronic lumbar EPI catheters and subcutaneous injection ports. Male and female dogs (three groups) received a total of 4 EPI injections at 8-day intervals of 3 mL of C0401 (10 mg/mL morphine) (N = 6), DepoFoam vehicle (N = 6), or 0.9% sodium chloride (N = 6). Following EPI-C0401, but not saline or DepoFoam vehicle, there were transient (< 72 hr) decreases in food consumption, arousal, hindlimb muscle tone, and body temperature. Heart rate was unaltered, but there were modest decreases in blood pressure and respiratory rate, which persisted for 24-72 hr after C0401. No persistent changes in sensory/motor function, body weight, or stool/urine production were observed. Cerebrospinal fluid, blood chemistry, and urinalysis performed at surgery and on the day of sacrifice (24 hr after the last dose) were within normal ranges. Gross pathology at necropsy was unremarkable. Spinal histopathology findings were judged to be minimal (e.g., modest pericatheter inflammation and fibrosis) and present in all dogs. However, a statistical trend in the rank order of pathology scores was noted (Saline < DepoFoam vehicle < C0401). Repeated EPI injection of C0401 at the maximum dose that could be administered (30 mg) resulted in moderate, transient behavioral and physiological effects after each injection, consistent with morphine administration, and a modest effect on cord histopathology. This level of pathology is reflected in the lack of change observed in cerebrospinal fluid and lack of neurological findings. These results suggest that C0401 is without significant pathological effects at this dose after repeated epidural delivery in dogs.

Kinetic and Safety Studies on Intrathecally Infused Recombinant-Methionyl Human Brain-Derived Neurotrophic Factor in Dogs☆

To define the kinetics and safety of spinally infused recombinant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF), beagle dogs were prepared with lumbar intrathecal catheters passed through the cisternal membrane to the L1-L4 lumbar level. For kinetic studies, r-metHuBDNF was delivered by bolus or infusion through one catheter and lumbar CSF was sampled periodically through a second. As a lumbar bolus, r-metHuBDNF displayed a biphasic clearance with t(1/2)a = 0.7 hr and t(1/2)b = 7. 9 hr. Lumbar to cisternal concentrations after bolus delivery were approximately 60:1. For safety studies, dogs received continuous intrathecal infusion (2.4 ml/day) for 28 days of saline (n = 6), r-metHuBDNF at 200 (n = 6), 800 (n = 6), or 2000 (n = 7) microg/day. Control dogs showed no changes. Intrathecally infused r-metHuBDNF produced a dose-dependent increase in muscle tone and decreased coordination. Low-dose r-metHuBDNF was associated with moderate increases in muscle tone after 22-28 days of infusion. No clinically important changes were noted in rectal temperature, arterial pressure, respiration and heart rate, body weight, food consumption, stool or urine output, or change in blood chemistries measured throughout the study. Cisternal CSF protein and glucose sampled at 28 days were not different between dose groups and all cultures were negative. Histopathological examination of the spinal cord typically revealed some degree of chronic inflammation around the catheter, including fibrotic adhesions and focal accumulations of lymphoid and plasma cells, but these effects were not dose dependent. In other dogs receiving r-metHuBDNF (2000 or 4000 microg/day), termination of infusion resulted in significant recovery.

Toxicology Profile of N -Methyl-d-aspartate Antagonists Delivered by Intrathecal Infusion in the Canine Model

Background:Intrathecal N-methyl-d-aspartate antagonists have antihyperalgesic efficacy. The authors examined toxicity in a canine model of chronic lumbar intrathecal infusion. Methods:Dogs (10–16 kg) were prepared with lumbar intrathecal catheters connected to vest-mounted pumps (100 &mgr;l/h). In phase 1, stepwise incrementations in infusion concentration were performed at 48- to 72-h intervals to determine an infusion dose with minimal but detectable behavioral effects. In phase 2, the dose/concentration defined in phase 1 was infused for 28 days. Behavioral function during infusion and histopathology at sacrifice was assessed. Drugs examined were 2-amino-5-phosphono-valorate (AP5), MK801, memantine, amitriptyline, S-methadone, and saline. Results:In the phase 1 dose ranging, the minimum effect doses for the several agents were as follows: AP5, 1 mg/day; amitriptyline, 1 mg/day; ketamine, 10 mg/day; MK801, 1 mg/day; and memantine, 4 mg/day. In phase 2, infusion of these doses typically resulted in mild hind limb weakness by 3–5 days after initiation of infusion, which progressed over the 28-day infusion interval. In a limited number of animals, a similar effect was observed with S-methadone. Histopathologically, vehicle-infused animals displayed a minor local catheter reaction. With the drug treatments, a gradient of increasing pathology from cervical to lumbar segments was noted. Pathology ranged from local demyelination to necrotizing lesions of spinal parenchyma near the catheter tip. All drugs given at their respective doses produced pathology scores significantly worse than saline controls. Conclusions:These drugs given for 28 days at acutely tolerable doses lead to spinal pathology. These data suggest a reevaluation of the use of these agents in chronic spinal delivery.

Development of a canine nociceptive thermal escape model

Acute nociceptive models which have been validated for large animal species are limited, yet nociceptive assessment in non-rodent species is important in analgesic drug development where larger animals may be necessary because of the technical requirements of the study. Here we report development and validation of a canine hind paw thermal escape model and the effect of analgesics on withdrawal latencies. Individual focused projection bulbs were used as left and right voltage-adjusted thermal stimuli placed below a glass plate in a specifically designed canine holding apparatus. After acclimation, dogs were lightly restrained in a fabric sling while standing on the glass plate. The anterior center of the metatarsal pad of the left and right hind paw was positioned on the glass over each light, and duration of stimulation tolerance timed. For every trial, the escape latency from lamp actuation to paw withdrawal was recorded twice for each hind paw. The mean population baseline withdrawal latency of 9.3+/-1.7s (mean+/-S.D., n=12 dogs) was shown to be repeatable between paws, within and between individual animals, and between test days. This latency corresponded to a glass surface temperature of 49.5 degrees C. A cut-off time of 20s (corresponding to a glass surface temperature of 56.5 degrees C) was set to prevent tissue damage. Intravenous administration (mg/kg) of morphine (1.0), hydromorphone (0.2), butorphanol (0.4), fentanyl (0.01), and dexmedetomidine (0.01) significantly (p<0.05) increased withdrawal latency from baseline within 15-30 min of administration while buprenorphine (0.03) produced a delayed, modest but significant latency increase. Rank order of opioid analgesic duration was morphine=hydromorphone>butorphanol>bupenorphine>fentanyl=saline. A dose-effect curve for hydromorphone was generated and corresponded to previously described dose-effect relationships in other species. The non-analgesic tranquilizer acepromazine (0.1mg/kg) produced mild sedation, but no significant increase in latency from that of saline. The model yielded a clear distinction between analgesia and sedation for all agents tested. These studies provide validation of a canine thermal escape model and have demonstrated the efficacy of clinically relevant doses of analgesics in elevating escape latencies. This model will facilitate quantification of the effects of parenterally and neuraxially administered analgesics in dogs.