kinetic and safety studies on intrathecally infused recombinant-methionyl human brain-derived...

FUNDAMENTAL AND APPLIED TOXICOLOGY 38, 8 9 - 100 (1997)

ARTICLE NO FA972314

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

Tony L. Yaksh,* Michael L. Rathbun,* Jean C. Dragani,* Shelle Malkmus,* Amy R. Bourdeau,tPhilip Richter,$ Harry Powell,§ Robert R. Myers,§ and Carl P. LeBelf

'Department of Anesthesiology, University of California, San Diego, La Jolla, California 92903; ^Department of Toxicology, Amgen, Inc., 1840DeHavilland Drive, Thousand Oaks, California 91320-1789; ^Office of Campus Veterinary Services, University of California, San Diego,

La Jolla, California 92037; and ^Department of Pathology, University of California, San Diego, La Jolla, California 92037

Received January 3, 1997; accepted April 9, 1997

Kinetic and Safety Studies on Intrathecally Infused Recombi-nant-Methionyl Human Brain-Derived Neurotrophic Factor inDogs. 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. (1977). Fundam. AppL ToxicoL 38, 89-100.

To define the kinetics and safety of spinally infused recombi-nant-methionyl human brain-derived neurotrophic factor (r-metHuBDNF), beagle dogs were prepared with lumbar intrathecalcatheters passed through the cisternal membrane to the L1-L4lumbar level. For kinetic studies, r-metHuBDNF was delivered bybolus or infusion through one catheter and lumbar CSF was sam-pled periodically through a second. As a lumbar bolus, r-metH-uBDNF displayed a biphasic clearance with t\& = 0.7 hr and ffb= 7.9 hr. Lumbar to cisternal concentrations after bolus deliverywere approximately 60:1. For safety studies, dogs received continu-ous 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) /xg/day. Control dogs showed no changes. Intrathecally infused r-metHuBDNF produced a dose-dependent increase in muscle toneand decreased coordination. Low-dose r-metHuBDNF was associ-ated with moderate increases in muscle tone after 22-28 days ofinfusion. No clinically important changes were noted in rectaltemperature, arterial pressure, respiration and heart rate, bodyweight, food consumption, stool or urine output, or change inblood chemistries measured throughout the study. Cisternal CSFprotein and glucose sampled at 28 days were not different betweendose groups and all cultures were negative. Histopathological ex-amination of the spinal cord typically revealed some degree ofchronic inflammation around the catheter, including fibrotic adhe-sions and focal accumulations of lymphoid and plasma cells, butthese effects were not dose dependent In other dogs receiving r-metHuBDNF (2000 or 4000 /ig/day), termination of infusion re-sulted in significant recovery. © 1997 Society of

r-metHuBDNF (recombinant-methionyl human brain-de-rived neurotrophic factor) is a growth factor believed to haverelevance for therapy in certain trophic diseases of the centralnervous system. Because at least one of the disorders towardwhich it is to be targeted (e.g., amyotrophic lateral sclerosis)

is strongly associated with spinal function, an appropriatedelivery paradigm for this agent is continuous deliverywithin the intrathecal space.

The preclinical systemic toxicity of r-metHuBDNF hasbeen extensively investigated in the rat (LeBel and Foss,1996) and the agent has been shown to be well tolerated.However, prior to initiation of spinal delivery of r-metH-uBDNF in humans, it is necessary to demonstrate the safetyof this material in a preclinical model. The dog was selectedbecause it is of sufficient size to permit the assessment ofpharmacokinetics, can carry a backpack pump for continuousinfusion, and will readily tolerate the extended placement oflumbar intrathecal catheters. We have employed the dogmodel in previous work to evaluate the preclinical safety ofagents for spinal delivery (Sabbe et al, 1992, 1993; Yakshet al., 1993, 1995). It is readily able to tolerate long-termplacement of catheters and pumps and is of sufficient sizeto be employed in defining the toxicokinetics of the agentunder investigation.

In the present work, we determined (i) the maximum toler-able dose of r-metHuBDNF; (ii) assessed the pharmacokinet-ics of the agent after bolus and constant intrathecal infusion;(iii) measured the effects of a range of doses of the agenton physiological parameters, peripheral and spinal pathol-ogy, over a 28-day infusion interval; and (iv) examined therecovery from changes in function after termination of infu-sion.

METHODS

These studies were carried out according to protocols approved by theInstitutional Animal Care and Use Committee of the University of Califor-nia, San Diego, in AAALAC-accredited facilities.

Animals

Male beagles were obtained from Harlan Sprague-Dawley, Inc. (RidglanFarms Inc., ML Horeb, WI). The animals were approximately 18 to 32months old and weighed 12 to 18 kg at initiation of dosing. Animals werehoused in individual runs, given ad libitum access to water and fed a drycertified canine diet (5007, PMI Feeds, Inc.). Animal room temperature and

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90 YAKSH ET AL.

photoperiod were controlled (targeted conditions: temperature range 65" to78°F, 12 hr of light and 12 hr of dark). Humidity was not controlled butrecorded regularly. Dogs were allowed to acclimate for a minimum of 7days after receipt and conditioned to vests for 3 days prior to surgery.

Intrathecal Catheterization of Animals

Each animal received penicillin G procaine (20,000 units/kg, im), atro-pine (0.04 mg/kg, im) and was sedated with xylazine (Rompun; 1-2 mg/kg, im). Each animal was then mask induced and intubated and anesthesiawas maintained under spontaneous ventilation with 1-2% halothane and60% N2O/40% O2 (approximate values). Animals were continuously moni-tored during surgery for heart rate, respiration, oxygen saturation, inspiredand end tidal values of halothane, CO2, N2O, and oxygen. After surgicalsites were shaved and prepared, using sterile technique, a skin incision wasmade and the cistema magna and dura were exposed. A sample of CSFwas taken for measurement of selected baseline chemistry values. A smallincision was made in the cisternal membrane and the intrathecal catheterinserted and passed caudally a distance of approximately 40 cm to a levelcorresponding to the L1-L4 segment The catheter was fabricated of poly-ethylene tubing (PE-50, 0.97 mm O.D., Intramedic, Becton Dickinson) andsterilized by gamma radiation after packaging. The free end of the catheterwas then tunneled subcutaneously and caudally to exit on the upper leftback at the level of the scapula. In animals prepared for kinetic studies, anadditional catheter was inserted into the cistema magna for a distance ofapproximately 2 cm and externalized in the same fashion.

In dogs, for kinetic studies, in addition to the intrathecal catheter, a 20-gauge thin-wall needle (35 mm in total length) with a blunt tip and a huboccluded by a silicone rubber septum was implanted adjacent to the backof the skull on midline with the tip resting adjacent to the dorsal edge ofthe foramen magnum and just resting on the dura. The cannula was affixedto the back of the skull with stainless steel screws and cranioplastic cementThe hub was subcutaneous. This served as a guide cannula for cisternalpunctures.

The incision was closed in layers with 3-O Vicryl, anesthetic turned off,and recovery monitored. Butorphanol tartrate (Torbugesic; 0.04 mg/kg, im)was administered to relieve postoperative pain. Upon recovery, vests werereplaced and an infusion pump was installed and connected to the external-ized end of the catheter. Control vehicle infusion, at approximately 2.4 ml/day, was initiated.

Test Article

The test article r-metHuBDNF, Lot No. 11303G4, a clear colorless liquid^ 9 5 % pure, was provided as a solution of 15 mg/ml. Purity and proteinintegrity was assessed in randomly selected test article dilutions by reverse-phase HPLC and SDS-PAGE Silver Gel. The control vehicle was sterileDULB Dulbecco's phosphate-buffered saline, pH 7.0 (GIBCOBRL, Paisley,Scotland). The test article was refrigerated at 2° to 8°C and allowed toadjust to room temperature prior to sterile preparation. Solutions were notfiltered prior to use. Manufacturer's data indicated that the stability of drugsolution at 37°C exceeded 3 months.

Intrathecal Infusion

Animals received continuous infusion at randomly assigned dose levels.Control vehicle infusion was terminated after 3 days and test article continu-ous infusion started (Day 0). The intrathecal infusion rate remained atapproximately 2.4 ml/day. The infusate was delivered from a sterile car-tridge (Disetronic 10 ml cartridge, Cat No. 300.157) using a PANOMATC-10 infusion pump (Disetronic Medical Systems, 13005 16th AvenueNorth, Suite 500, Plymouth, MN 55441-4578) secured in the nylon vestPump flow rates were measured at a predetermined value (2.4 ml/day ±5%) before initiation and again upon completion of infusion. The infusion

TABLE 1Summary of Dose Groups for r-met-HuBNDF Safety Study

Infusion rate Duration of AnimalsGroup Average dose (/jg/day) (ml/day) dose (days) (males)

1234

Control vehicle"r-metHuBDNF:r-metHuBDNF:r-metHuBDNF:

200800

2000

2.42.42.42.4

5.28s>28s.28*28

6667

" Phosphate-buffered saline.

system was checked at least once daily. The infusion pump cartridges werereplaced at 4-day intervals.

Incubation of infusion solutions at the concentrations employed in thepresent studies indicated no absorption in the pump cartridges and cathetertubing. Solutions were changed at 4-day intervals when the pump cartridgeswere replaced.

Study Croups

Drug kinetics study. Lumbar (L1-L4) and cistemal guide cannulae weresurgically implanted in male beagle dogs. Samples in the unanesthetizeddog were taken by withdrawal from the lumbar intrathecal cannula and bythe insertion of a 24-gauge spinal needle (1.5 in) percutaneously throughthe hub of the cistemal guide cannula. On the day after surgery, each dogwas administered a bolus injection of 200 /xg r-metHuBDNF through thelumbar catheter in a total volume of 0.5 ml. The bolus dose of test articlewas followed by a vehicle control flush of 1.5 ml delivered over approxi-mately 30 sec. Samples of cerebrospinal fluid (CSF, 0.2 ml) from both thelumbar and cistemal catheters, as well as blood from the cephalic vein,were collected at 30 min prior to bolus injection, immediately prior to bolusinjection (Time 0), at 0.03, 0.1, 0.5, 1, 2, 4, and 8 hr after bolus injection.Concurrent [l4C]inulin administration in the bolus clearance study servedas a tracer of bulk CSF flow. Approximately 24 hr after the final samplewas collected in the bolus study, each dog was fitted with a nylon vestequipped with an infusion pump that was connected to the lumbar catheterfabricated with a Y-joint allowing for administration and sample collection.The same four dogs were administered a 48-hr continuous lumbar infusionof 800 /ig/day r-metHuBDNF. CSF and blood were collected periodicallyup to 8 hr after termination of the infusion. CSF and plasma were collectedfor determination of r-metHuBDNF concentration using radioimmunoassay(see below). Lumbar and cistemal pharmacokinetic profiles for both bolusand continuous intrathecal infusion of r-metHuBDNF were prepared.

Dose-ranging and safety studies. A preliminary dose-ranging studywas completed in four dogs in which r-metHuBDNF was infused at concen-trations which were incremented at 4-day intervals. Doses as high as 6000jig/day were achieved in all animals. Based on motor function side effectsobserved in this study, a daily dose of 2000 /xg r-metHuBDNF was deter-mined to be the highest usable dose for continuous infusion. Accordingly,for the safety study, three drug dose groups and a fourth control vehiclegroup were employed. These groups and their doses are summarized inTable 1.

Prior to surgery, dogs were randomly assigned to dose groups. Afteranesthesia recovery (Day —3), control vehicle infusion was initiated atapproximately 2.4 ml/day. On Day 0, initiation of infusion began at thepredetermined drug concentration. This dose was delivered continuouslyfor a minimum 28 days. Necropsy followed infusion termination.

Recovery study. To observe recovery following 28 days of infusion of2000 /Jg/day r-metHuBDNF, two sets of observations were performed.Animals of the safety study from group 4 (2000 /Jg/day r-metHuBDNF; n



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INTRATHECAL RECOMBINANT BDNF 91

= 2) were switched to control vehicle after 28 days of r-metHuBDNFcontinuous infusion for an additional 2 days before termination of all infu-sion. Group 1 (control vehicle; n = 2) continued infusion for the 2 additionaldays as well. All animals were maintained and observed for a total of 14-15 days, after the 28-day dosing phase, before necropsies were performed.In a separate recovery study, dogs were randomly assigned to receive controlvehicle (PBS; 2.4 ml/day, n = 2), r-metHuBDNF 2000 fig/day (n = 2), orr-metHuBDNF 4000 //g/day (n = 2). Each dog was infused until a motordysfunction was clearly observable in two sequential neurological examina-tions. At this time, the dog received 2 days of control vehicle before theinfusion of that dog was discontinued, and the pump and vest were removed.The dog was observed for an additional 13-14 days. All vehicle dogsreceived a minimum of 28 days of infusion.

Antemortem Observations

Neurologic. Three components of canine nervous system function wereassessed and graded at each observation point: muscle tone ( - 3 to +3,flaccidity through normal to rigidity), motor coordination (0 to 3; normalto total loss of coordinated function), and arousal ( - 3 to +3; comatosethrough normal to hyperactivity).

Cardiovascular response. Heart rate and blood pressure were assessedusing the Dinamap 8100 automatic pressure monitor (Critikon) affixed witha pediatric cuff placed at the shaven base of the tail.

CSF pressure. To determine the effects of intrathecal infusion on CSFpressure in the recovery dog groups, each dog was placed in a standingharness. The infusion pump was turned off and the catheter infusion lineimmediately clamped. The line was then disconnected from the pump,elevated, undamped, and the fluid height was allowed to stabilize. Theheight of the fluid column, in centimeters relative to the auditory meatus,was then measured. The jugular veins were then lightly compressed withthe palm to induce a transient increase in CSF pressure. The jugular com-pression was then relieved and the fluid level again allowed to stabilize. Amean of the two readings was used as an indicator of CSF pressure. Theheight of the fluid column was assessed in this manner at 5 and 30 minafter the pump was turned off. These measurements were made concurrentlywith heart rate and blood pressure measurements. Pressure measurementswere taken after 3 days of control vehicle infusion from day of surgery,before the initiation of test article, on the day at which test article infusionwas discontinued, and on the day that infusion was terminated.

Clinical Pathology

r-metHuBDNF and r-melHuBDNF antibodies. Venous blood sampleswere taken in the safety study on Days 0, 7, 14, and 28. Serum and plasma(citrate) samples were prepared and stored at -70° ± 10°C until assayed.Cisternal CSF samples were taken on the day of surgery and again atnecropsy. CSF samples were stored in polypropylene tubes at —70° ± 10°Cuntil assayed.

The r-metHuBDNF was measured in blood and CSF using an enzyme-linked immunosorbent assay (ELISA). A monoclonal anti-BDNF anti-body (Regeneron RP4301) was used as the capture agent. Samples andstandards were added to the plates and incubated for 2 hr at 37°C andfollowed by washes. Rabbit anti-BDNF conjugated to HRP was addedto the plate and incubated for an additional 2 hr at 37°C. After quenching,the optical density was read at 450-650 nm. Sensitivity of the assaywas from 0.008 to 2 ng/ml.

Anti-r-metHuBDNF antibodies in sera were measured by solid-phaseRIA which were optimized for dog with different blocking solutions. Inbrief, rabbit antiserum to r-metBDNF was diluted (1:100,000). Diluted testsera and positive and negative dog control sera were added to r-met-BDNF-coated wells. After incubation, iodinated goat anti-rat IgG was added tothe sample wells containing test sera, rat negative control sera, and purifiedrat IgG. After incubation and washing, wells were disrupted and radioactiv-

ity was counted. For each BDNF-treated dog, a binding ratio was deter-mined, defined as the CPM from predose sera divided by the mean CPMfrom posttreatment serum samples. Animals with ratios greater than 2.0were considered to be seroconverted. Three or four animals were selectedfrom each treatment group to be examined over the 28-day period.

Hematology, serum, and cisternal cerebrospinal fluid chemistries. Ve-nous blood and serum samples were obtained during the safety study afteran overnight fast on Days - 7 , - 3 (day of surgery), 7, 14, and 28, just priorto necropsy but with control/test article infusion still maintained. A sterileurine sample was collected for urinalysis on Day —3 (day of surgery) andon Day 28 just prior to necropsy. Cisternal CSF samples were taken onDay —3 (day of surgery) and on Day 28, just prior to necropsy. CSF sampleswere submitted for culture and for drug analysis and chemistry.

Pathology

On the day of necropsy, after an overnight fast, animals were anesthetizedand final samples of venous blood, serum, urine, and cisternal CSF werecollected. Animals were then exsanguinated with approximately 4 liters ofsaline (0.9%) followed by perfusion of approximately 4 liters of histologicalfixative through the heart at between 160 and 200 mm Hg of pressure. Anecropsy was then performed including exposure of the spinal canal andbrain stem. Methylene blue dye was injected through the intrathecal catheterto visualize position of the intrathecal catheter and to determine the spreadof the dye around the catheter tip. The cord and brain were then removedand stored in fixative for further evaluation.

Blocks of tissue, 10 mm long, from the cervical and thoracic spinal cordand from lumbar spinal cord, immediately above and below the tip of thecatheter, were collected for further microscopic processing. A 2-mm-longsection of each block was isolated and further divided into selected 2-mm3

blocks for gluteraldehyde fixation and embedding in resin. The remainingtissue was embedded in paraffin. Complete transverse sections of the spinalcord and adjacent meninges were cut from paraffin blocks and stained withhematoxylin and eosin for identification of inflammatory cells and generaltissue morphology, or with luxol blue for neuronal analysis. One-microme-ter-thick sections of resin-embedded tissue were stained with paraphenylen-ediamine to study fine morphologic detail, especially nerve root pathologyand the integrity of myelin and axoplasm in the vicinity of the catheter tip.

Semiquantitative scoring of paraffin sections was performed indepen-dently by two investigators blinded to the dose groups. Resin-embeddedmaterial was reviewed qualitatively. Thus, tissue sections from eachblock were described qualitatively with note being taken of local reac-tions, local inflammatory cells, and pathological change such as chroma-tolysis, neuronal loss, edema, or necrosis. For purposes of comparisons,material from each animal was rated on a semiquantitative scale of 0 to4 where 0 represented a lack of pathological findings and 4 representeda severe inflammatory reaction that involved adjacent meninges, nerveroots, or spinal cord parenchyma. These values were reported and sub-mitted for a rank order comparison.

Statistical Analyses

Measured variables include body weights, clinical pathology (hematol-ogy, clinical chemistry) values, and organ weights. Summary statistics werecomputed and include group means and standard deviations (SD). For statis-tical analyses of behavioral assessments (arousal, muscle tone, and motorcoordination), an index for each was calculated (percentage of the maximumpossible effect). In this, the daily observation score for each animal wasaccumulated over a 7-day interval and this was divided by the maximumpossible score for that interval (i.e., 3 x 7 = 21) and multiplied times 100to get a percentage. In the recovery studies, shorter intervals (2 days) wereexamined (i.e., maximum score would be 2 x 3 = 6). Analysis of variance(repeated-measures ANOVA) was performed where appropriate. Wheremultiple comparisons were undertaken after the ANOVA, t tests (pairedor unpaired as required) with Bonferroni corrections were employed. The



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92 YAKSH ET AL.

70

60

50

4°30

20

10

0

70

60

50

MOTOR COORDINATION

p<0.05: 2000 800 200 Veh

QBDNF:

E^BDNF:

• BDNF:

• Vehicle

2000 ug/day

800 ng'day

200 ng/day

MUSCLE TONEp<0.05: 2000 800 200 Veh

Day -7 to 0 Day 1 to 7 Day 8 to 14 Day 15 to 21 Day 22 to 28

INFUSION INTERVAL

FIG. 1. Time course of change in muscle tone score (bottom) and loss of motor coordination (top) observed over the 28-day infusion interval forsaline, 200, 800, and 2000 /jg/day r-metHuBDNF. Each bar presents the mean and SEM of the percentage of the maximum possible effect for six orseven dogs. The %MPE of a dog for an interval is the cumulative score for that dog observed during each of the 7 days of that interval divided by themaximum possible score (e.g., 3 x 7 days) which could be obtained for that interval x 100. The larger the number, the greater the degree of dysfunction.Two-way repeated-measures ANOVA across test intervals and between treatment groups indicated significant effect related to time for both coordinationand tone (p < 0.0001) and time by treatment interactions (p < 0.0001). Treatment comparisons using Bonferroni correction indicated significantdifferences (p < 0.05) as indicated in the inset for each graph where treatment pairs not differing are joined by a common underline.

Kruskal-Wallis test was employed for nonparametric analysis (Siegel,1988).

RESULTS

Antemortem Observations

No deaths occurred during the in-life phase of this study.All animals reached their scheduled date of necropsy.

General Behavior and Neurological Picture

Arousal scores were unaffected over time in any of thedose groups. While infusion of control vehicle had no effect,the infusion of r-metHuBDNF exerted a concentration- andtime-dependent effect on motor coordination and muscletone, as indicated in Fig. 1.

An infusion of 200 /zg/day of r-metHuBDNF resulted in

a modest increase in muscle tone after a 22- to 28-day inter-val of infusion, with little effect upon coordination. Infusionof 800 and 2000 /xg/day of r-metHuBDNF caused a notablechange in hind limb muscle tone and coordination beginningat the 8- to 14-day interval and continuing throughout thestudy. During the last week of infusion, the effect uponmuscle tone and coordination, ranked according to increas-ing severity, was control vehicle, 200, 800, and 2000 /xg/day r-metHuBDNF.

The neurological picture developing over the 28-day infu-sion interval, as reflected by the general behavioral measuresdescribed above, was characterized by a hypertonicity ofmuscles of the hind limbs resulting in an upright, stiff pos-ture; a stiffened tail which was carried arched over the back;and a hunched posture with spinal curvature (concave ventraltrunk and convex dorsal trunk relative to the ground) after



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INFUSIONCOORDINATION

Vehicle(N =4)

-6 to -4 -3 to -1 1 to3 4 to 6 7 to 9

TIME (Days)10 to12 13 to15

FIG. 2. Histogram presents the %MPE (maximum possible effect) onchanges in motor coordination score presented at the two 2-day intervalsbefore termination and five 2-day intervals after the termination of theintrathecal infusion of vehicle (n = 4), r-met BDNF: 2000 /ig/day (n = 4)or r-met BDNF: 4000 /ig/day (n = 2). The %MPE of a dog for an intervalis the cumulative coordination score for that dog observed during each ofthe 2 days of that interval divided by the maximum possible score (e.g., 3X 2 days) which could be obtained for that interval X 100. The larger thenumber, the greater the degree of dysfunction. Two-way analysis of variancerepeated across time from interval —3/-1 through interval 13/15 for the2000 /ig/day and vehicle group revealed a significant time main effect (p< 0.001) and a significant interaction in treatment by time (p < 0.001).Bonferroni comparison of recovery days versus Day -3/—1 revealed asignificant recovery (*p < 0.05).

2000 fig/day r-metHuBDNF infusion. Ambulation/gait ab-normalities consisting of ataxia, hypermedia, and stiffnesswere observed.

Intrathecal r-metHuBDNF infusion was associated withthe development of a cutaneous hypersensitivity to lighttouch that included the hind limbs and tail. Vehicle infusionanimals did not show these patterns of change.

In the recovery studies, dogs being infused with r-metH-uBDNF had drug infusion terminated but continued infusionwith control vehicle for an additional 2 days before intrathe-cal infusion was terminated. Following termination of infu-sion, postural, gait, and muscle tone abnormalities decreasedmarkedly during the first week and were almost completelyresolved after the second week (Figs. 2 and 3). With termina-tion of drug delivery, hypersensitivity gradually subsidedand all dogs showed normal cutaneous sensation by the endof the study. Figures 2 and 3 summarize the muscle tone

and motor coordination scores for control vehicle (n = 4),2000 (n = 4), and 4000 n&day r-metHuBDNF (n = 2)infused dogs (pooled from the safety and recovery studies).By 14 days after drug infusion termination, all dogs hadessentially returned to the state observed prior to the initia-tion of drug infusion (p < 0.05; 2000 ^g/day group vsvehicle).

Physiological Indices

Group means for body weights (14.7 to 15.3 kg), rectaltemperatures (100.5 to 101.1 °F), heart rate (113-127 beats/min), respiratory rate (27-36 breaths/min), or mean arterialpressure (96-106 mm Hg) were not different across groupsprior to surgery. No group showed any clinically remarkablechanges in these parameters or food consumption comparedto control or across groups over the 28-day infusion period

INFUSION801

MUSCLE TONEVehicle(N = 4)

-6 to -4 -3 to -1 1 to 3 4 to 6

T IMF

7 to 9 10 to12 13 to15

FIG. 3. Histogram presents the %MPE (maximum possible effect) onchanges in muscle tone score presented at the two 2-day intervals beforetermination and five 2-day intervals after the termination of the intrathecalinfusion of vehicle (n = 4), r-met BDNF: 2000 /ig/day (n = 4), or r-metBDNF: 4000 /ig/day (n = 2). The %MPE of a dog for an interval is thecumulative muscle tone score for that dog observed during each of the 2days of that interval divided by the maximum possible score (e.g., 3 x 2days) which could be obtained for that interval X 100. The larger thenumber, the greater the degree of dysfunction. Two-way analysis of variancerepeated across time from interval - 3 / - 1 through interval 13/15 for the2000 pg/day and vehicle group revealed a significant time main effect (p< 0.001) and a significant interaction in treatment by time (p < 0.001).Bonferroni comparison of recovery days versus Day — 3/—1 revealed asignificant recovery (*p < 0.05).



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94 YAKSH ET AL.

TABLE 2Clinical Chemistry of Cistemal Cerebrospinal Fluid Harvested at Termination in Nonrecovery and Recovery Dogs

Control (Day-3) meanNonrecovery0

Vehicle control meanBDNF 200 ^g/day meanBDNF 800 /ig/day meanBDNF 2000 Mg/day mean

RecoveryVehicle control meanBDNF 2000 Mg/day meanBDNF 4000 Mg/day mean

Necropsyday

N/A

28282828

38-43*24-43*

28"

Subjectnumber

25

4665

442

Specificgravity

1.005 ± 0.001

1.004 ±0.0011.005 ±0.0011.005 ± 0.0011.005 ± 0.001

1.005 ± 0.0001.005 ± 0.0011.005 ± 0.000

Protein(mg/dl)

33 ± 20

55 ± 2565 ± 5754 ± 4850 ± 29

39 ± 1161 ± 1870 ± 39

Glucose(mg/dl)

84 + 84

69 ± 570 ± 969 ± 371 ± 4

76 ± 1067 ± 1169 ± 11

WBC(NoJmm3)

2 ± 2

88 ± 83374 ± 87943 ± 5719 ± 23

4 ± 3298 ± 530

8 ± 11

RBC(Noimm3)

248 ± 489

166 ± 120487 ± 552547 ± 672165 ± 132

1658 ± 17762048 ± 2337930 ± 99

Culture(No. yes)

0/25

0/40/60/60/5

on0/2N/D

Note. N/A, not applicable; N/D, not determined." ANOVA comparisons between treatment groups were not statistically significant (p > 0.05).* Animals were allowed to survive 15-17 dyas after termination of intrathecal drug infusion and CSF samples were determined at that time (necropsy).

(p > 0.05). Over the 28-day infusion period, urination anddefecation patterns, as well as stool, were not changed.

Clinical Pathology

For the safety study, examination of the hematology andblood clinical pathology (CBC, RBC morphology, serumchemistry, or urinalysis) results reveal that parameter valuesfell within ranges considered to be clinically unremarkablewith no systematic trends evident for any parameter whencompared across group or over time. Two exceptions to thiswere the LDH level and the albumin/globulin ratio. For thealbumin/globulin ratio, levels were higher than normal popu-lation values; for the LDH the levels tended to be higher forall groups. As these levels were different from the normalpopulation values, even in two control (Day —7 and —3)measurements, and because there was no subsequent changewith dose, these differences are not considered to be clini-cally relevant.

With regard to CSF for both the safety and recovery stud-ies, summary values are provided in Table 2. Data for Day—3 were not different for the several study groups and werepooled. As indicated, using ANOVA, protein, glucose, spe-cific gravity, white blood cell (WBC) count and red bloodcell (RBC) count show no significant (p > 0.05) differencesbetween dose groups for both Days —3 and Day 28. Withindose groups, comparisons of specific gravity, protein, andRBC for Days - 3 and 28 were not significant. For glucose,all dose groups showed significant differences when Day—3 is compared with Day 28. The control vehicle groupshowed significant differences for WBC, Day - 3 versus Day28. Elevated RBC levels are associated with contaminationthat can occur with cistemal sample collection. No dose-related differences were associated with the clinical chemis-try of the respective cerebrospinal fluid.

Anatomical Pathology

Peripheral Tissues

In the safety study, terminal body weights, absolute organweights, and organ-to-body weight percentages were not sta-tistically different between dose groups for any organ (datanot shown). Histopathological examination of the peripheraltissues indicates that intrathecal infusion of r-metHuBDNFfor 28 days was not associated with any evident systemictoxicity.

Brain

Necropsy revealed mild, usually mixed meningitis in thebrain with no difference in the frequency and severity be-tween groups in the safety study. Based on the incidence ofmeningeal inflammation, this finding is thought to be a resultof chronic placement of an externalized catheter/pump deliv-ery system. Focal hemorrhages were noted in several animalsbut were not associated with any particular dose. No differ-ences were apparent among the dose groups with regard tomyelination of the brain.

Spinal Cord

At necropsy, all catheters were found to be intact andlocated at the rostral lumbar spinal cord at the level of T9-L3. Catheters were observed to be patent and injected dyeshowed a free rostrocaudal distribution. Several small, super-ficial, well-organized hematomas were observed at the cervi-cal cord level in two animals and were believed to be second-ary to catheter placement. Occasional mild (eechymotic)bleeding was noted in one animal.

Histopathological examination of the spinal cord usinghematoxylin and eosin stain typically revealed a fibrosis



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FIG. 4. (A) Paraffin section stained with hematoxylin and eosin show-ing catheter tract ( • ) adjacent to dura (D) and nerve roots in intrathecalspace (arrow). Fibrosis defines the catheter tract and also extends along thedura for a short distance. Spinal cord (bottom) and nerve roots appearnormal. The overall pathology score for this animal was 1 + (animal received

around the catheter (Figs. 4A and 4B) and occasional con-centrated accumulations of plasma and lymphoid cells (Fig.4C). All dogs displayed a mild chronic inflammatory reac-tion judged to be secondary to the presence of the catheter.Thickening of the dura was also a consistent finding acrossdose groups. Several animals displayed a more severe reac-tion in which inflammatory cells invaded spinal nerve root,but there was no dose group relationship. The severest reac-tion was typically observed in the sections containing thecatheter tip or just caudal to the catheter tip.

Analysis of resin-embedded tissue sections showed goodpreservation of myelin except in occasional nerve roots adja-cent to the catheter tip where there was physical injury and/or inflammation judged to be due to the presence of thecatheter. An occasional degeneration axon was seen enteringthe dorsal horn and this was presumed to be secondary tothe catheter-induced injury of the associated nerve root.

The rank ordering of animals according to severity ofpathology score is shown in Table 3. This ranking revealedno statistically significant ordering based on r-metHuBDNFdose or control vehicle, thus indicating no systemic effecton spinal histopathology for any dose of intrathecal r-metH-uBDNF.

Toxicokinetics of Intrathecal r-metHuBDNF

Bolus Delivery

BDNF. The clearance profile of r-metHuBDNF from theCSF in the cisternal and lumbar space following bolus in-trathecal infusion in beagle dogs is presented in Figs. 5and 6. After bolus delivery, there was an initial acute peakobserved in lumbar CSF followed by a prolonged clearancewith the appearance of a somewhat delayed peak in cisternalCSF. The following clearance rates (with SD) were derivedfrom curve-fitting of the BDNF bolus curves (n = 4): fja =0.7 ± 0.3 hr, tjb = 7.9 ± 1.1 hr, Vd = 17.1 ± 5.2 ml, Cl =9.2 ± 3 . 1 ml/hr. At no time during these study phases wasr-metHuBDNF detected in plasma following intrathecal ad-ministration.

Evaluation of the clearance of r-metHuBDNF from CSFat the cisternal level after lumbar bolus (Fig. 5) showedmaximal levels within the first 10 min, with mean cisternal

intrathecal BDNF 200 /ig/day). (B) Paraffin section showing catheter tract( • ) with fibrosis depressing subjacent white matter of spinal cord whichotherwise appears normal. Pathology score 2+ (animal received intrathecalvehicle). (C) One-micrometer-thick section from resin-embedded tissueshowing invasion of inflammatory cells into nerve root in intrathecal space(indicated by arrow). Paraphenylenediamine stain shows normal-appearingaxoplasm and dark staining myelin. Note one thinly myelinated axon atperiphery of nerve root (arrow). Inflammatory cells and fibrosis dominatethe intrathecal pathology of this animal. Pathology score, 4 (animal receivedBDNF 800



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96 YAKSH ET AL.

TABLE 3Rank Ordering of Histopathology in Dogs Receiving 28 Days of Infusion of Excipient or One of Several Doses of r-metHuBDNP

Least

1A

2C

3C

4A

5B

6B

7B

8B

9C

10C

11A

12D

13B

14D

15A

16B

17D

18C

19D

20D

21D

22D

23A

24C

Most

25A

" Ordered from least to most severe signs: A, vehicle; B, 200 /ig/day; C, 800 /ig/day; D, 2000 /ig/day. Kniskal—Wallace nonparametric analysis, meanof rank sum: vehicle = 13.2, 200 /zg/day = 9.2, 800 /ig/day = !•• ' . 2000 Mg/day = 17.8, H crit value = 4.997, p = 0.17.

concentrations approximately 60 times lower than meanlumbar concentrations drawn at equivalent times, indicatinga large concentration gradient up the neuraxis in dogs, andthis ratio tended to decline over the 8-hr interval (see Fig.6, top; one-way ANOVA: p < 0.08). Plasma levels afterbolus intrathecal delivery were uniformly below the limitsof detection (<0.16 ng/ml).

Inulin. As a marker for extracellular fluid, [14C]inulinwas administered simultaneously with r-metHuBDNF as apart of the lumbar bolus in four dogs. As with r-metH-

10000i

1000:

E

a.

o

Q.

a

TBDNF

Lumbar

BDNF/INULIN RATIO

*

om

2 4 6 8TIME (hours)

FIG. 5. The concentrations from the lumbar and cisterna space of r-metHuBDNF (200 /ig/top) with [14C]inulin (middle) delivered intrathecallyas a bolus. The calculated BDNF/inulin ratio (//g/CPM) is presented in thebottom panel. Each point presents the mean and SD of four dogs.

uBDNF, lumbar [l4C]inulin samples displayed a biphasicrate of clearance with the following calculated values (SD):/5a = 0.49 ± 0.25 hr, Vc = 9.1 ± 6.2 ml, Cl = 10.4 ± 4.3

CISTERNAULUMBAR RATIO

.001

600-

_, 400-

ZatUJI-

G 200-|

2 4 6TIME (hours)

INULIN (DPM/40uL)y • 89 2S4 + 7 8892e-2x RA2 - 0.757

BDNF (ng/mL)y 13 284 + 1.8433a-2x R"2 . 0.888

0 2000 4000 6000 8000 10000 12000 14000

LUMBAR

FIG. 6. (Top) The log ratio in cistemal/lumbar samples of inulin andBDNF sampled concurrently plotted versus time of sampling after boluslumbar intrathecal delivery. Each point presents the means and SD of datafrom four dogs. (Bottom) Data from top graph are replotted, presenting thelevels of inulin (in DPM/40 /JI) or BDNF (ng/ml) as measured in the cistemaversus levels sampled concurrently in the lumbar space. Circled valuesindicate samples taken at T = 3 min and the last sample was taken at 8 hr.SD not shown for clarity. Slopes of best-fit regression line are statisticallydifferent (p < 0.05).



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ml/hr. [l4C]Inulin levels in the cisternal CSF revealed a 10-fold concentration gradient between lumbar and cisternalconcentration and this ratio tended to increase over time (seeFig. 6, top; one-way repeated measures ANOVA, p < 0.05).

BDNF/inulin ratios. The r-metHuBDNF/[14C]inulin ra-tio in the lumbar CSF after bolus delivery over the 8-hrinterval after delivery was stable (Fig. 5, bottom; one-wayrepeated-measures ANOVA p > 0.20). In contrast, the r-metHuBDNF/[l4C]inulin ratio in the cisterna was approxi-mately 6- to 10-fold less (consistent with the lumbar to cis-terna ratios for either agent) and there was a trend for thisratio to decline over time (Fig. 5, bottom; one-way repeated-measures ANOVA, p < 0.07). To further display this trend,the concurrent levels over the 8-hr sampling interval in cis-terna versus lumbar of [l4C]inulin and r-metHuBDNF wereplotted. As indicated in Fig. 6 (bottom), both showed anarithmetically linear relationship with the slope of inulin >r-metHuBDNF. This emphasizes over the entire time coursethat cisternal r-metHuBDNF levels were less than those an-ticipated for inulin, based on a given lumbar level.

Continuous Infusion

Delivery of r-metHuBDNF at a rate of 800 fig/day for 2days by infusion resulted in a lumbar intrathecal level of 10± 3 /ig/ml. In the continuous infusion kinetic study, plasmalevels were not detectable (<0.16 ng/ml).

In the 28-day infusion safety study, r-metHuBDNF con-centrations were measured in plasma at Days —7, - 3 , 7, 14,and 28. Cisternal CSF levels were measured from samplesobtained on Day 28 prior to infusion termination at timeof necropsy via cisternal tap. There was no r-metHuBDNFdetected in plasma or CSF obtained from any dog prior toinitiation of infusion or in control vehicle infused animalsat any time. Cisternal CSF concentrations, while variable,were typically highest in dogs receiving the highest infusionconcentration. The range of r-metHuBDNF concentrationsin cisternal CSF levels by dose group at the time of termina-tion were vehicle group, 0 -0 ng/ml; low dose, 0-91 fig/day; medium dose, 0 -7 /ig/ml; high dose, 2-16, 227 ng/ml. Concentrations of r-metHuBDNF in CSF obtained fromcontrol vehicle or high-dose animals after the 2-week recov-ery period were below the limit of detection.

Plasma samples were collected on Days 7, 14, 28, and onDays 42/43 for recovery dogs only. Plasma levels evaluatedfrom control vehicle and low-dose animals revealed r-metH-uBDNF concentrations below limits of assay detection at allsampling intervals. Examination of data derived from ani-mals administered the middle dose showed that three of sixanimals, on Day 28, had low plasma concentrations thatwere consistent throughout the dosing phase of the study(range of means, 0.8-3.5 ng/ml). Animals receiving the highdose, 5 of 7, on Day 28, had plasma concentrations of r-metHuBDNF that were twofold greater than those measured

TABLE 4Number of Dogs Displaying Appearance of Anti-r-metHuBDNF

Antibodies in Serum after Receiving Continuous Intrathecal Infu-sion of Vehicle or 220, 800, or 2000 /ig/day of r-metHuBDNF

Intrathecal dosing

Study day

07

1428

0 /tg/day

0/31/3°

on0/3

200 /ig/day

0/30/31/30/3

800 /ig/day

0/30/30/31/3

2000 /ig/day

0/31/43/43/4

" Number of dogs positive versus number of dogs examined at the threetime points.

for the middle dose group (range means, 3.7-7.8 ng/ml).In one of two high-dose animals that underwent a 2-weekrecovery from dosing period, concentrations were low butdetectable (1.2 ng/ml) in plasma.

CSF Pressure

In the recovery study, baseline CSF pressures on Day 0,taken after 4 days of control infusion and prior to initiationof r-metHuBDNF infusion, were (mean ± SD: n = 6) 10 ±5 and 10 ± 3 cm of H2O when measured 5 and 30 min afterinfusion interruption. In control animals (n = 2) after 28days of infusion, CSF pressures at 5 and 30 min after thetermination of infusion were 16 (18 and 15) and 14 (16and 13) cm of H2O, respectively. For r-metHuBDNF-infusedanimals (n = 4) the respective values were 11 ± 7 and 12 ±8 cm of H2O. Thus, extended intrathecal infusion of controlvehicle or drug had no effect upon lumbar CSF pressures.

Serum Antibodies to r-metHuBDNF

Dogs (three or four) from each treatment group were se-lected for systematic examination for seroconversion. Mea-surement of anti-r-metHuBDNF in sera obtained on Days 0,7, 14, and 28 in animals receiving continuous infusion ofvehicle or the several doses of r-metHuBDNF showed atendency for a dose- and time-dependent incidence of sero-conversion (e.g., serum activity ratios 3= 2) (Table 4).

DISCUSSION

In the present study, dogs received 28 days of continuousinfusion of vehicle or r-metHuBDNF ranging from doses of200 to 2000 ugld&y. Behaviorally there was a time-depen-dent increase in hind limb motor tone, the latency to onsetand magnitude of which were related to infusion concentra-tions and were not observed with the excipient. Thesechanges were reversible even when the drug was terminatedafter a length of exposure extending well past the onset of



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98 YAKSH ET AL.

the deficit and in several animals receiving twice the highestdose examined in the safety study (4000 /zg/day). Thoughthe intrathecal catheters displayed local reactions, the histo-pathology was not different across excipient or dose groups.Further, there were no significant intergroup differences inCSF clinical chemistry consistent with any unusual inflam-matory reaction to the drug infusion. Measurement of lumbarCSF pressure revealed no significant increase with the infu-sion of either excipient or r-met-BDNF. These points thusdo not support the hypothesis that a nonspecific componentof the infusion or a local toxic reaction of the spinal cordwas responsible for the observed behavioral effects of r-met-BDNF. Our kinetics studies suggest that these delays in onsetor offset cannot be accounted for by the spinal half-life ofr-metHuBDNF. Accordingly, it seems possible that the agentinduced some alteration in spinal function. It is importantto emphasize that r-metHuBDNF interacts as a growth factorwith several specific families of growth factor receptorsknown to exist in primary afferent terminals in the spinaldorsal horn and on dorsal root ganglion cells (Ernfors et al,1993; Altar et al., 1993). The nature of the coupling of thereceptors in the dog, in the absence of injury, is not known.However, in other models, the delivery of r-metHuBDNFwithin the CNS by infusion led to increases in spinal dorsalhorn substance P (Siuciak et al, 1995). Substance P, con-tained in and released from small afferents, is believed toserve as a nociceptive transmitter (see Yaksh and Malmberg,1994 for review). A time-dependent, reversible upregulationin substance P content or release produced by chronicallyinfused growth factor might yield an enhanced reflex compo-nent or some of the signs of hypersensitivity observed at thehighest dose. In any case, over the range of concentrationsof agent examined, 28-day exposure failed to detect anyevident spinal pathology, either detectable in the local histol-ogy or in the CSF chemistry, induced by the extended contin-uous exposure to the agent.

Toxicokinetics

The sampling of r-metHuBDNF levels and [l4C]inulin inthe lumbar CSF after bolus delivery revealed a similar bipha-sic clearance from the vicinity of the catheter tip. The initialreduction in drug concentration likely reflects the local dilu-tion of the injectate into the local volume around the cathetertip. An acute bolus of injectate at 200 ^g/2 ml is expectedto provide an initial concentration of 20 /xg/ml. At approxi-mately 30 min postinjection, the local concentration was onthe order of 9 £tg/ml, a value similar to that observed forinulin. This suggests that the local acute volume of distribu-tion with these injection parameters is about 2 - 3 ml. Thisvalue is slightly less than that of local dilution values thathave been established for other polar agents in this model,such as morphine (Sabbe and Yaksh, unpublished). The sec-ond prolonged phase of clearance observed with both r-

metHuBDNF and inulin likely reflects the true clearance offree drug from the local intrathecal space. Support for thisidea is further derived from the observation that consider-ation of clearance, after terminating a continuous infusionthat provides a psuedo-steady-state, reveals a long, singlehalf-life that is in the range of the ^b calculated for theclearance of r-mefHuBDNF bolus delivery. The prolongedhalf-life of this agent and its similarity to that of inulinargues that the drug is cleared as CSF. Drugs delivered intothe intrathecal space, after local dilution in the adjacent CSF,are cleared by movement into tissue and then into the vascu-lature and, by movement with bulk CSF flow up the cerebro-spinal axis into the cisterna and over the cerebral convexitieswhere it enters the systemic circulation after absorption intothe subarachnoid granules (see Yaksh, 1981; Durant andYaksh, 1986). In contrast, small lipid soluble molecules arerapidly absorbed and display short residence times in theCSF and display correspondingly high plasma levels (seeHerz and Teschemacher, 1971; Durant and Yaksh, 1988;Sabbe et al, 1993).

A significant craniocaudal distribution of BDNF after bo-lus or infusion was noted in the present study. This gradienthas been similarly reported by Dittrich and colleagues(1996). Unexpectedly, in spite of the similar clearance fromthe lumbar CSF, the cisternal/lumbar ratio for inulin wasconsiderably greater than that for r-metHuBDNF. This sug-gests that r-metHuBDNF did not diffuse as readily as inulininto the cistemal space. This may reflect a local mechanismwhereby BDNF is cleared from the lumbar CSF and retainedat the spinal level in a manner not exhibited by inulin. Asseroconversion appeared to occur with some incidence atthe higher dose and even then with a brief delay, it seemsunlikely that this would account for the apparent loss of freeBDNF after acute treatment. Whether the difference betweeninulin and BDNF reflects upon local receptor binding by thelatter in the spinal cord is not known.

A presumed target of BDNF and other growth factors inspinal neurological disorders is in the motor horn cell. Recentwork by Dittrich and colleagues (1996) demonstrated thatBDNF displayed poor direct penetration into the spinal pa-renchyma (a finding consistent with its high molecularweight), but did accumulate in motor neurons. They suggestlocal absorption and retrograde transport to the cell bodies.

Robustness of Safety Model

Excluding specific assumptions regarding the mechanismsof action, we have argued that the likelihood of observinga toxic event with a spinally delivered agent is related to thelocal concentration of the agent in the spinal space and theduration of exposure (Sabbe et al, 1993; Yaksh et al, 1994).This local concentration depends upon the kinetics of the testmodel and the concentration of the delivered agent. Previouswork has suggested that the kinetics of bolus spinal agents



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is remarkably similar across species. The use of continuousinfusion permits the assumption that the local tissue wasitself continuously exposed to the highest tolerable drug con-centration for an extended period. This minimizes the likeli-hood that species differences in metabolism or binding mightinfluence the concentration to which the test model was ex-posed. With regard to the concentration, the highest system-atically studied dose was 2000 //g/2.4 ml/day for 28 days.This corresponds to a local drug concentration at the tip ofthe catheter of approximately 833 fxg/m\. We would arguethat reducing the local concentration would reduce the localbiological effects produced by the continuous infusion ofthis agent even though this was not actually studied. Previouswork in humans, with the commonly employed agent lido-caine, has in fact supported the concept that, for this agent,it is the local concentration and not the total dose per se thatcan lead to a local toxic reaction (Rigler and Drasner, 1991;Ross et al, 1992; Rigler et al, 1991).

With regard to the dog spinal safety model, it has beenextensively employed for epidural and intrathecal drugsafety evaluation for a number of agents including baclofen(Sabbe et al, 1993), morphine, sufentanil and alfentanil(Sabbe et al, 1994), clonidine (Yaksh et al, 1994), andneostigmine (Yaksh et al, 1995). In those studies, agentsgiven in the maximal tolerable dose failed to display agent-related toxicity. In contrast, in other investigations, the deliv-ery of the trifluroacetic acid salt of the opioid peptideDPDPE (Sabbe, Tiseo, and Yaksh, unpublished observa-tions) or the tricyclic antidepressant amitryptylline (Yakshand Eisenach, unpublished observations) did result inmarked spinal pathology. These data suggest that this caninemodel can indeed discriminate between classes of agentswith and without effects upon spinal histology when givenintrathecally.

Safety of Spinally Delivered BDNF

BDNF is known to be constitutively expressed and re-leased from brain and spinal systems. Considerable data haveindicated that it may play a role in maintaining normal neu-ronal function (Koliatsos et al., 1993) and promoting regen-eration after neuronal injury (Ernfors et al, 1993; Schnellet al, 1994). While there have been few systematic studiespublished as to the safety of extended exposure to growthfactors such as r-metHuBDNF, there are evolving data thathave suggested in the rat and in culture that some growthfactors, such as BDNF, might serve to augment neuronaldeath induced by excitatory amino acids (Koh et al, 1995;Lindholm et al, 1993; but see Cheng et al, 1994a,b). Inaddition, some studies have indicated that "high" doses ofgrowth factors may lead to a reduced preservation of motorneurons after nerve injury (Vejsada et al, 1994).

Such observations suggest the importance of consideringthe safety of high doses of these endogenous agents that

might be anticipated in their therapeutic implementation. Itis important to note therefore that the present studies suggesta lack of toxicity for r-metHuBDNF at 200 Mg/day deliveredcontinuously to the surface of the lumbar spinal cord in aconcentration of 83 /ig/ml for a period of 4 weeks, with theexception of a slight increase in muscle tone toward the endof the 28-day sequence. The clinical significance of thischange in tone is not known. Nevertheless, the safety of thisdose is suggested by the following observations: (i) whilethere was a mild increase in muscle tone at 22 to 28 days,there was no change in coordination at 200 /ig/day; (ii) whileinfusion at 800 and 2000 ^ig/day results in readily observableincreases in muscle tone and reduced coordination after 7days, these changes are reversed within 14 days of infusiontermination of 2000 /zg/day; (iii) despite the direct applica-tion of the agent at 10-fold higher doses to the surface ofthe lumbar spinal cord for 28 days, there were no clinicallysignificant abnormal findings in nonnervous tissue, brain, orspinal cord that were distinguishable from control vehicle-treated dogs.

ACKNOWLEDGMENT

This work was supported by funds from Amgen/Regeneron Partners.

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