Experimental Neurology 168, 171–182 (2001)doi:10.1006/exnr.2000.7592, available online at http://www.idealibrary.com on
Systemic Administration of the Immunophilin LigandGPI 1046 in MPTP-Treated Monkeys
M. E. Emborg,* P. Shin,* B. Roitberg,*,† J. G. Sramek,* Y. Chu,* G. T. Stebbins,* J. S. Hamilton,‡P. D. Suzdak,‡ J. P. Steiner,‡ and J. H. Kordower*,1
*Research Center for Brain Repair and Department of Neurological Sciences, Rush Presbyterian–St. Luke’s Medical Center,Chicago, Illinois 60612; †Department of Neurosurgery, University of Illinois, Chicago, Illinois 60612;
and ‡Guilford Pharmaceuticals, Baltimore, Maryland 21224
Received July 19, 2000; accepted October 13, 2000
Systemic administration of immunophilin ligandsprovides trophic influences to dopaminergic neuronsin rodent models of Parkinson’s disease (PD) resultingin the initiation of clinical trials in patients with Par-kinson’s disease. We believe that prior to clinical tri-als, novel therapeutic strategies should show safetyand efficacy in nonhuman models of PD. The presentstudy assessed whether oral administration of the im-munophilin 3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrollidinecarboxylate (GPI 1046)could prevent the structural and functional conse-quences of n-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine (MPTP) administration in nonhuman primates.Twenty-five rhesus monkeys received daily oral ad-ministration of vehicle (n 5 5) or one of four doses ofGPI 1046 (0.3 mg/kg, n 5 5; 1.0 mg/kg, n 5 5; 3.0 mg/kg,n 5 5; 10.0 mg/kg, n 5 5). Two weeks after starting the
rug treatment, all monkeys received a unilateral in-racarotid injection of MPTP-HCl (3 mg). Daily drugdministration continue for 6 weeks postlesion afterhich time the monkeys were sacrificed. Monkeysere assessed for performance on a hand reach task,eneral activity, and clinical dysfunction based on alinical rating scale. All groups of monkeys displayedimilar deficits on each behavioral measure as well asimilar losses of tyrosine hydroxylase (TH)-immuno-eactive (ir) nigral neurons, TH-mRNA, and TH-ir stri-tal optical density indicating that in general treat-ent failed to have neuroprotective effects.
1 To whom correspondence should be addressed at Department ofNeurological Sciences, Rush Presbyterian–St. Luke’s Medical Cen-ter, 2242 West Harrison Street, Chicago, IL 60612. E-mail:[email protected].
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INTRODUCTION
Immunophilins are protein receptors for major im-munosuppressant drugs such as cyclosporin andFK506 (41). The prototypic members of the immu-nophilin family, cyclophilin A and FKBP12, were dis-covered on the basis of their ability to form a complexthat binds to calcineurin, inhibit phosphatase activity,and increase the phosphorylation of transcription fac-tors required for interleukin-2 formation. This is themeans by which those compounds mediate immuno-suppression. Although there is essentially no sequencehomology between FKBP12 and the cyclophilins, theyshare peptideprolyl isomerase or rotamase activity,which helps the immunophilins to scaffold or chaper-one proteins with rotamase actions and facilitate pro-tein–protein binding.
Brain concentrations of immunophilins greatly ex-ceed the levels that are seen in immune tissues (42)suggesting a role for these compounds in the centralnervous system (CNS). The high levels of FKBP12 inperipheral nerves and growth cones of neonatal neu-rons (42), as well as the dramatic upregulationof FKBP12 and 43-kDa growth-associated protein(GAP-43) following damage to sciatic or facial nerves(34), support the concept that these proteins may beneurotrophic for select neuronal populations. This hy-pothesis was confirmed when FK506, rapamycin andcyclosporin showed trophic effects on PC12 cells andsensory ganglia at picomolar potencies (33). In vivo,FK506 and its analogue L685818 induce regenerationof previously crushed sciatic nerve resulting in func-tional recovery (43, 44).
New derivatives that bind to FKBP12 and displaypotent neurotrophic effects, but lack the ability to bindto calcineurin (and therefore lack immunosupressantseffects), have been recently synthesized. One of them,3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxo-pentyl)-2-pyrrollidinecarboxylate (GPI 1046) facili-tates the regeneration and remyelination of damaged
sciatic nerves (16, 17, 44). This compound also medi-ates regenerative effects upon 5-HT neurons followinglesions with p-chloroamphetamine (43) and centralcholinergic neurons following fimbria-fornix lesions(19). These neurotrophic effects resemble those of clas-sic neurotrophic proteins such as nerve growth factor,brain-derived neurotrophic factor, neurotrophin(NT)-3, and NT-4 and exert their effects at picomolardoses. Unlike other neurotrophic polypeptides (50), im-munophilin ligands have not been shown to induceaberrant sprouting of neuronal processes when admin-istered to normal animals. Further, the ability of GPI1046 to readily cross the blood–brain barrier suggeststhat this agent can be useful as a therapeutic applica-tion in neurodegenerative diseases without directCNS delivery. In that regard, systemic administrationof GPI 1046 to 6-hydroxydopamine-treated rats orn-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice resulted in sparing of dopaminergic neu-rons and fibers (43). In these studies, a two- to three-fold increase over controls levels in the density of ty-rosine hydroxylase (TH)- and dopamine transporter(DAT)-positive striatal fibers was observed. When GPI1046 was administered prior to MPTP in mice, a four-to fivefold increase over control levels of TH- and DAT-positive fibers was found (43). Based principally onthese data, a clinical trial testing the efficiency of im-munophilin compounds in patients with Parkinson’sdisease has been initiated.
The discovery of MPTP in accidentally exposed hu-mans (9, 29) allowed for a better understanding ofParkinson’s disease (PD) and the development of betternonhuman primate models of parkinsonism. MPTP-treated monkeys display features of PD such as hypo-and bradykinesia, tremor, and balance disturbance,that closely resemble parkinsonian signs and in manyways serve as the best available model of this disease.It is the prototype model for testing clinically relevanttherapeutic strategies (5, 30). Of the different ways ofadministering MPTP, a unilateral intracarotid infu-sion induces in a single dose a predictable lesion (2)that is stable overtime (13). These qualities make thisparticular model appropriate to test drugs aimed toprotect against neurodegeneration. Taking advantageof this model, we assessed whether oral administrationof GPI 1046 could prevent the structural and func-tional consequences of a unilateral intracarotid MPTPadministration in nonhuman primates.
MATERIAL AND METHODS
Subjects
Twenty-five rhesus monkeys (male, 5 years old, 3.5–5.3 kg) were used in this study. All animals were singlyhoused with a 12-h light/dark cycle. Purina monkeychow and water was available ad libitum. Diets were
supplemented with fruit during the testing sessions.The study was performed in accordance with federalguidelines of proper animal care and with the approvalof the IACUC.
Drug Administration
GPI 1046 was administered daily (7 days per week, 1PM) p.o. after the behavioral testing. Prior to the ini-tiation of this study we performed a pilot open labeldose–response experiment to establish the doses to beadministered in the present study. In the presentstudy drug administration was initiated 2 weeks priorto the MPTP lesion and continued daily for 6 weekspostlesion (Fig. 1). Based on baseline data from thehand reach task, monkeys were matched into fivegroups of five monkeys each: Group 1, vehicle; Group 2,0.3 mg/kg GPI 1046; Group 3, 1.0 mg/kg GPI 1046;Group 4, 3.0 mg/kg GPI 1046; and Group 5, 10 mg/kgGPI 1046. The animals were dosed with GPI 1046 in a10% Cremaphore solution at 1 ml/kg. The Cremaphoresolution served as the solvent for the GPI 1046 and asthe vehicle solution. Prior to dosing, GPI 1046 wassolubilized in 100% ethanol at a concentration of 100mg/ml and combined with a solution of 10% Cremaphor(w/v) in 0.9% sterile saline.
The monkeys were handled for the drug administra-tion using a pole and collar handling system (1; Pri-mate Products, Redwood City, CA) by trained person-nel at the animal facility, different from the behavioraltrainers. Two to 3 weeks before starting the drug ad-ministration, the animals were trained to be removedfrom their cages, placed on a restrainer, and, afterseveral sessions, administered 1 ml/kg saline orally.Using a lubricated endogastric tube (16 frame) theanimals were intubated by mouth to avoid trauma to
FIG. 1. Schematic drawing of the experimental design used inthis experiment. All the animals were assessed throughout the studywith a fine motor test (PUT, 5 days a week), general activity monitors(ACT, 1 week period before and after MPTP), and a clinical ratingscale (CR, weekly). Two weeks before the MPTP intracarotid (ica)infusion and 6 weeks afterward all the monkeys received daily oraladministration of vehicle (n 5 5) or one of four doses of GPI 1046 (0.3mg/kg, n 5 5; 1.0 mg/kg, n 5 5; 3.0 mg/kg, n 5 5; 10.0 mg/kg,n 5 5).
173GPI 1046 IN MPTP-TREATED MONKEYS
the nasal mucosa. After a flush with 5 cc of water, thetube was gently retrieved, and the monkeys were re-turned to their home cages where their favorite treatsand monkey chow were available.
MPTP Infusion
Following baseline testing, all monkeys received uni-lateral lesions of the substantia nigra as previouslydescribed (3, 12, 13, 24). The monkeys were first tran-quilized with ketamine (10 mg/kg, im) and then anes-thesia was induced and maintained with isofluorane(1–2%). The animal was positioned in the supine posi-tion with neck hyperextended and slightly turned left.Under sterile conditions, a number 15 blade was usedto cut through the skin along the medial edge of thesternocleidomastoide muscle. The carotid sheath wasopened using fine iris scissors and the common carotidartery, internal jugular vein, and vagus nerves wereidentified. The common carotid was exposed below thecarotid bifurcation. Silk (2-O) thread was loopedaround the common carotid artery while the externalcarotid artery was identified with the superior thyroidartery seen branching distal to the bifurcation andtemporarily clamped. A 27-gauge butterfly needle wasinserted into the common carotid artery in a directionretrograde to the blood flow, and 20 ml of saline con-taining 3 mg of MPTP-HCl was infused at a rate of 1.33ml/min. After the infusion was completed, a 3-ml post-flush of saline was delivered. The clamps were removedfrom the superior thyroid and external carotid arteries.The needle was withdrawn from the carotid artery, anda small piece of Gelfoam was used to apply focal pres-sure to the penetrated vessel. The musculature, sub-cutaneous tissues, and skin were then closed in a rou-tine fashion. Each animal was given cefazolin (25mg/kg im) and buprenex (0.01 mg/kg im) upon wakingup response and 24 h postsurgery.
Clinical Rating
Once a week a clinical rating scale (CR scale) wasused to assess the clinical status of the monkeys byusing a previously validated measure (13, 14, 21, 27,28). These evaluations occurred prior to MPTP, follow-ing MPTP, and for the entire GPI treatment period. Allthe ratings were performed by a trained observerblinded to the treatment conditions. The scale consistsof ratings of tremor (0–3 for each arm), posture (0–2),gait (0–5), bradykinesia (0–5), balance (0–2), grossmotor skills (0–4 for each arm), defense reaction (0–2),and freezing (0–2). The score was obtained as the sumof the features, with a total possible score of 32 points.Occurrence of dyskinesias, psychological disturbances,and vomiting was also recorded.
Activity Monitoring
Collection of activity data was performed beforeMPTP infusion and 4 weeks post-MPTP infusion. Eachmonkey was tranquilized with ketamine (15 mg/kg, im)and fitted with a primate vest that contained a PAM2activity monitor (IM Systems, Baltimore, MD; 14) inthe inside back pocket. These monitors measure accel-eration. Every time a monitor senses an accelerationthat exceeds a threshold of 0.1g, an electrical pulse isgenerated and recorded. Thus, each pulse represents234 ms of acceleration above the 0.1g threshold. Thenumber of pulses is expressed for a preselected timeperiod (1 min). After a 1-week period, the animals wereagain tranquilized with ketamine, the jacket was re-moved, the activity monitor was interfaced with aMacintosh computer, and the data were downloaded.The data were expressed as the mean of each 12-hlight/dark cycle.
Fine Motor Test
Each monkey was tested for fine motor performancein both upper limbs by using a modification of a previ-ously described food pick up task (14, 24). Testingoccurred in a modified home cage. The animals werepresented with a 3 3 3 matrix of recessed food wellsembedded in a Plexiglas board. During each trial, sixcubic pieces of apple (0.5 cm) were placed within thesame six food wells, and the time it took for the mon-keys to retrieve all six pieces (speed of movement) wasascertained. The test board is configured in such man-ner that the monkeys could only retrieve the food re-ward by using the arm being evaluated. Monkeys re-ceived 10 trials per arm in each test session with thearm being tested alternated for each trial. Each animalwas tested by the same investigator at the same time ofthe day, 5 days a week throughout the course of thestudy. Five researchers trained the animals. Everytrainer was responsible for five animals that werespread across the different doses. All trainers wereblinded to the treatment their animals received.
Necropsy and Preparation of Tissue
Six weeks postoperatively, the monkeys were anes-thetized with pentobarbital (25 mg/kg, iv) and perfusedtranscardially with warm (100 ml) followed by ice-cold(400 ml) normal saline. The brain was rapidly removedfrom the calvaria and immersed in ice-cold saline for 10min. Then the brain was slabbed (5 mm thickness) inthe coronal plane using a calibrated Lucite brain sliceapparatus. The slabs were then immersed in a 4%Zamboni’s fixative for 48 h of fixation, cryoprotected byimmersion in a graded (10–40%) sucrose/0.1 M phos-phate-buffered saline (PBS, pH 7.2) solution. The tis-sue slabs were cut frozen (40 mm) on a sliding knife
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174 EMBORG ET AL.
microtome. All the sections were stored in a cryopro-tectant solution before processing.
Immunohistochemistry
Sections through midbrain and striatum were pro-cessed in parallel for immunohistochemical staining ofTH according to our previously published procedures(14, 22, 24, 25). Endogenous peroxidase activity wasremoved with a 20-min incubation in 0.1 M sodiumperiodate. After 3 3 10-min washes in PBS plus 0.05%
riton-X (dilution medium), background staining waslocked with a 1-h incubation in a Tris-buffered salineolution containing 3% normal horse serum, 2% bovineerum albumin, and 0.05% Triton X-100. The sectionsere then incubated with a monoclonal TH (1:20,000;hemicon Inc., Temecula, CA) primary antibody for8 h at room temperature. Sections were then incu-ated for 1 h in horse anti-mouse (TH) biotinylatedecondary antibodies (1:100; Vector Laboratories, Bur-ingame, CA). After 12 3 10-min washes in dilution
edium, the sections were placed in the avidin–biotinABC, “Elite” kit, Vector Laboratories) substrate (1:,000) for 75 min. Sections were then washed in a 0.1
imidazole/1.0 M acetate buffer, pH 7.4, and theneacted in a chromogen solution containing 0.05% 3,39-iaminobenzidine and 0.05% H2O2.Controls consisted of processing tissue in an identi-
cal manner except the primary antibody solvent or anirrelevant immunoglobulin G (IgG) was used in lieu ofthe primary antibody. Sections were mounted on gela-tin-coated slides, dehydrated, and coverslipped withPermount. Additional sections were stained for Nisslsubstance using cresyl violet acetate.
Optical Disector Procedure
Counts of TH-immunoreactive (ir) neurons were per-formed by a single investigator, blinded to the animal’streatment group. Quantitative estimates of the totalnumber of nigral TH-ir neurons in the substantia nigrawas determined using an unbiased, stereologic cellcounting method as described in detail elsewhere (8,14, 20, 35, 36). TH-ir neurons within the ventral teg-mental area were excluded from this analysis. Briefly,the optical disector system consisted of a computer-assisted image analysis system including an OlympusBX-60 microscope hard-coupled to a Prior H128 com-puter-controlled x-y-z motorized stage, a high-sensitiv-ity Hitachi 3CCD video camera system (Hitachi, Ja-pan), and a Macintosh 8500 computer. Neuronalcounts were performed by using NeuroZoom stereo-logic software (4). Before each series of measurements,the substantia nigra was outlined under a low magni-fication (1.253), and 5% of the outlined region wasmeasured with a systematic design of disector countingframes (5040 mm2). The total number of nigral TH-irneurons was estimated by using a 603 planapo oil
mmersion objective with a 1.4 numerical aperture.he substantia nigra was demarcated by the caudaldge of the mamillary bodies rostrally, the cerebraleduncle ventrally and laterally, and the third cranialerve rootlets medially. Based on pilot experimentseveral sections of each sample containing the substan-ia nigra were used for analysis. Neurons were sam-led by optical scanning using a uniform, systematic,nd random design. Four equally spaced sections wereampled from each subject. The absolute thickness ofach section was determined empirically. To determinehe section thickness, the top of the section was firstrought into focus. Then, the NeuroZoom program wassed to step through the z-axis in 1-mm increments
until the very bottom of the section was in focus. Sec-tion thickness averaged 37.93 6 0.37 mm. The volumeof the substantia nigra was calculated according to theprocedure of Cavalieri (6). Once the top of the sectionwas in focus, the z-plane was lowered at 1–2 mm.Counts were then made while focusing down through5-mm-thick dissectors. Care was taken to ensure thatthe bottom forbidden plane was never included in theanalysis. The total number of TH-ir neurons (N) in thentire substantia nigra was calculated by using theormula N 5 N v 3 V sn, where N v is the numerical
density and V sn is the volume of the substantia nigra(20, 35, 36). The variability within groups was assessedby means of the coefficient of error (CE; 49).
TH-ir Striatal Optical Density
The density of TH-ir fibers was quantified within thecaudate nucleus and putamen using the NIH Image1.60 analysis system. Nine to 10 sections per case wereanalyzed. Optical density measurements were per-formed at a total magnification of 2503. The opticaldensity for TH-ir was then averaged per structure.Background staining was measured in the corpus cal-losum (an area low in TH-positive fibers) and sub-tracted from the averaged total to result in a net opticaldensity.
Data Analysis
Differences between groups were evaluated usingeither repeated measures or factorial analysis of vari-ance (ANOVA) models. Where appropriate, groupscomparison was performed using least significance testemploying a Bonferoni correction factor to control formultiple comparisons.
RESULTS
General Observations
All animals tolerated the drug administration andthe MPTP lesion without complications. The animalsmaintained a constant weight throughout the study
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175GPI 1046 IN MPTP-TREATED MONKEYS
and did not display evidence of nausea, vomiting, diar-rhea, signs of weakness, fever, or infection associatedwith potential immunosuppression. Throughout thestudy, they were cooperative during test sessions andresponsive to food stimuli.
Clinical Rating
Prior to the administration of MPTP all the animalsdisplayed behavior indicative of normal young adultmale rhesus monkeys (Table 1). They were fast withsteady movements and did not show any neurologicalimpairment. As assessed using the rating scale, all theanimals scored 0 in the pre-MPTP condition. Therewere no changes in clinical rating scores during the 2weeks period prior to MPTP treatment, when the mon-keys were primed with GPI 1046 or vehicle.
After the intracarotid MPTP infusion, there was sig-nificant variability in the parkinsonian status of thedifferent animals in the GPI 1046-treated groups.Some animals appeared unaffected by the MPTP,while others appeared severely hemiparkinsonian, pre-senting tremor, flexed posture, and impaired motorskills in the hand contralateral to the infusion, as wellas balance disturbance, stopped posture, bradykinesia,and slow spontaneous circling ipsilateral to the lesionside.
Throughout the drug-treatment regimen the ani-mals, for the most part, displayed a relatively consis-tent PD score for each of the five PD ratings (repeatedmeasures ANOVA, F , 1). Due to the extensive vari-bility between animals, a factorial ANOVA based onose failed to reveal a significant effect (F(1, 4) 5
2.23; P 5 0.1012; Table 1). Sign by sign analyses didot reveal any improvement to specific tests.
ctivity
Prior to any treatment, spontaneous general activityevels in the home cage measured with personal activ-
TAB
Clinica
GPI 1046 (mg/kg)0
(mean 6 SE)0.3
(mean 6 SE)
remorR hand 0.2 6 0.18 0.1 6 0.09L hand 1.0 6 0.00 1.0 6 0.00
ty monitors located in primates jackets were similar tohat was observed in previous studies (e.g., 14).After the MPTP treatment a repeated measuresNOVA revealed a significant effect of time (pre vsost) (F(1, 20) 5 35.843; P , 0.001). There were no
significant effects for GPI 1046 dose (F(4, 20) 5 1.863;P 5 0.16) or dose 3 time interaction (F , 1; Fig. 2).Vehicle-treated animals displayed a 60% reduction inhome cage activity. A similar nonsignificant dose effectwas found for the percentage of reduction in activityscores following MPTP administration (F , 1).
Fine Motor Task
A pick up test was used to compare the speed ofmovement in monkeys between treatment conditions(Fig. 3). All the animals were first trained to a plateau
FIG. 2. Group means and SEM activity recorded with personalmonitors during the day period, before and after MPTP administra-tion, and vehicle (n 5 5) or one of four doses of GPI 1046 (0.3 mg/kg,n 5 5; 1.0 mg/kg, n 5 5; 3.0 mg/kg, n 5 5; 10.0 mg/kg, n 5 5).
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level of performance with both hands. All the animalslearned the task relatively quickly, taking 15 to 20sessions to achieve asymptomatic performance, andthere were no significant differences in the time nec-essary to complete the task between the left and righthands (repeated measures ANOVA, F , 1).
After the intracarotid MPTP administration, virtu-ally all the monkeys displayed a slowing of perfor-mance in the left hand (contralateral to the MPTPinfusion) while maintaining relatively normal functionwith the right. Some animals stopped using the handcontralateral to the MPTP infusion or used it sporadi-cally in slower fashion. Relatively stable performancewas seen bilaterally over the 6-week postlesion timecourse. A repeated measures analysis of variance ofhand reach performance revealed a significant maineffect of time (pre- vs 6 weeks post-MPTP; F(1,16) 519.92; P , 0.02), a significant main effect of side(ipsilateral vs contralateral; F(1,16) 5 22.59; P ,0.01). The main effect of dose was not significant (F(4,16) , 1). The only reliable interaction was time 3 side(F(1, 16) 5 29.08; P , 0.01). Analysis of the relativepercentage of increase (affected hand vs intact hand)by group revealed a smaller nonsignificant increase for1.0 and 10.0 mg/kg doses (vehicle, 209%; 0.3 mg/kgdose, 180%; 1.0 mg/kg, 81%; 3.0 mg/kg, 215%; 10.0mg/kg, 42%). The main effect for dose was not signifi-cant (F , 1); the main effect for side was significant(F(1, 20) 5 24.85; P , 0.001); and the dose by sideinteraction was not significant (F(4, 20) 5 1.46, P 50.252.
FIG. 3. Group means and SEM latency to perform the fine motorask with the hand contralateral to ica MPTP infusion after thedministration of vehicle or one of four doses of GPI 1046 (0.3 mg/kg,5 5; 1.0 mg/kg, n 5 5; 3.0 mg/kg, n 5 5; 10.0 mg/kg, n 5 5).
Neuroanatomical Analysis
TH-ir cell counts. Sections through the midbrainshowed varying degrees of degeneration of TH-immu-noreactive neurons within the substantia nigra parscompacta ipsilateral to the intracarotid MPTP infusion(Table 2). Some animals displayed a comprehensiveloss of TH-ir neurons within the central and ventrolat-eral portions of the A9 region while A10 ventral teg-mental area was minimally affected (Fig. 4). Somecases displayed minimal neuronal degeneration withinthe substantia nigra pars compacta (Fig. 6B). Whenanalyzed as a group, a factorial ANOVA failed to dis-cern a main effect of dose (F(4, 16) 5 1.01; P 5 0.43)or a dose by side interaction (F , 1.0). In contrast, asignificant lesion-side interaction emerged (F(1, 4) 5164.64; P , 0.001). However, it is notable that someanimals in the GPI-treated groups displayed increasedTH-ir cell counts compared to the vehicle group. Vehi-cle-treated animals displayed a range of cell loss ofbetween 45 and 79.5%. In contrast, 7 of the 20 GPI1046-treated animals, but none of the vehicle-treatedanimals, displayed small (23.8 to 30.3%) losses inTH-ir cell number.
TH-ir optical density within the striatum. Quanti-tative measurements of TH-ir staining within the cau-date nucleus and putamen failed to reveal significantdifferences between groups (Table 3, Fig. 5). For thecaudate, a factorial ANOVA with nested factors re-vealed a significant effect of side (F(1, 4) 5 130.40;P , 0.001) illustrating the effectiveness of the lesion.However, there was no significant effect of dose (F ,1.0), nor a dose by side interaction (F(4, 16) , 1.0).For the putamen, an ANOVA again revealed a signif-icant effect of side (F(1, 4) 5 285.44; P , 0.001), butnot a significant effect of dose (F , 1.0) nor a signifi-cant dose by side interaction (F(4, 16) 5 1.06; P 50.41).
Qualitative analyses of TH-ir staining in the stria-tum revealed a pattern of TH-ir fibers and terminalsthat corresponded to the quantitation of TH-ir neuronsin the substantia nigra. The seven monkeys displayingmodest to negligible cell loss displayed robust TH-irwithin the caudate nucleus and putamen at an inten-
sity and pattern which was often indistinguishablefrom the intact side (Fig. 6A). Many animals in allgroups displayed significant losses of TH-ir nigral neu-
FIG. 4. Microphotographs through the midbrain of tyrosine hydp.o. GPI 1046- (B) treated rhesus monkeys. CP, cerebral peduncle. B
The present study demonstrates that, when ana-lyzed as groups, systemic administration of GPI 1046does not provide neuroprotective effects upon the hostnigrostriatal system in unilateral MPTP-treated mon-keys. Behaviorally, the monkeys did not display a sta-tistically significant attenuation of MPTP-induced im-pairment measured with a fine motor coordinationtask, activity monitors, and clinical rating. Further,neuroanatomical studies failed to reveal a significant
FIG. 5. Microphotographs through the striatum of tyrosine hydr.o. GPI 1046- (B) treated rhesus monkeys. AC, anterior commisure
protection of TH-ir nigral perikarya or striatal fibers byGPI 1046. This suggests that clinical trials employingimmunophilins might be premature.
Several reports had described differences in the de-gree and duration of MPTP-induced parkinsonism innon human primates (10, 11, 37, 40, 47, 48). However,the finding that control MPTP-treated monkeysshowed a significant TH-ir cell loss (mean 64.17%,range 45–79.5%) that corresponded with a high PDscore (9.3 6 .604), low general activity (a decrease of60% of pre-MPTP level), and increased time to com-
lase (TH)-immunostained sections from vehicle- (A) and 1.0 mg/kgar, 210 mm.
oxy. B
i
179GPI 1046 IN MPTP-TREATED MONKEYS
plete the fine motor task (209% increase of pre-MPTPtimes), confirm the effectivity of the ica MPTP lesion-ing, as previously reported (2, 12, 13, 24).
Certain findings in this study suggest that GPI 1046might be providing some measure of behavioral andneuroanatomical neuroprotection. However, thiswould require further investigation. Individual ani-mals in each group of the GPI 1046-treated subjects,but none in the vehicle group, displayed normal behav-ior as measured by the parkinsonian rating scale, aswell as activity and hand reach task times in a rangeappropriate for their age. These same animals dis-played either modest or no reductions in TH-ir nigral
FIG. 6. Microphotographs through the striatum (A) and the Mmmunostained sections from 1.0 mg/kg p.o. GPI 1046-treated rhesu
mm, (B) 350 mm.
neurons and modest to no losses in striatal opticaldensity (see Fig. 6). One caveat to this hypothesis is therelatively small sample size in the present study. Sig-nificant effects in the 1.0 mg/kg dosing may emergewith larger samples.
Neurotrophic therapies aimed to protect and re-store dopaminergic nigrostriatal function may be amore parsimonius way to treat PD than DA replace-ment (by L-dopa treatment or cell transplantation) ordisruption of basal ganglia circuit (by pallidotomy ordeep brain stimulation). Different proteins with do-paminergic trophic actions had been discovered inthe last decade but methods for the delivery still
P-treated side of the midbrain (B) of tyrosine hydroxylase (TH)-onkey. AC, anterior commisure. CP, cerebral peduncle. Bar (A), 210
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180 EMBORG ET AL.
need to be better characterized and optimized. Crit-ically, trophic factors such as glial derived neurotro-phic factor (GDNF) cannot be delivered systemically.In order to avoid the blood– brain barrier, invasivesurgery is required to place syringes or cannulae fordirect infusion of GDNF (15) or to implant cells (45)or viral vectors (3, 7) genetically modified to synthe-sized the factor. Intracerebrovascular delivery of tro-phic factors has been accepted as a suitable methodfor clinical trials. However, this route can often bedose limiting (31, 32) and produce unacceptable sideeffects (26). Drugs with trophic actions that can crossthe blood– brain barrier to reach their nigral targetare an ideal therapy for the millions of patientsacross the world that suffer Parkinson’s disease. At-tempts to link a molecular carrier to trophic factorshave been performed, in particular using nervegrowth factor (NGF). NGF–antitransferrin receptorcomplex was successful at sustaining the growth ofintraocular septal transplants (18) and to protectcholinergic striatal cells against quinolinic acid le-sioning in a model of Huntington’s disease (23). Al-though this alternative to intracerebral interven-tions is promising, the production of the carriers aswell as the size and molecular structure of the fac-tors limit the use of these links. Gangliosides, suchas GM1 (46), have shown dopaminotrophic actionsand can cross the blood– brain barrier; however,their effects in nonhuman primates are still contro-versial. Intramuscular administration of GM1 tononhuman primates was reported to protect againstMPTP lesioning when administered before the neu-rotoxin (39). But when GM1 was administered tomonkeys that received an intracarotid infusion ofMPTP 1 year before, it failed to induce recovery (12).
Immunophilins have shown potential to induce pro-tection or recovery of dopaminergic nigral cells afterneurotoxin challenge. Similar to what we presentlyobserved in some of our monkeys, pretreatment withGPI 1046 elicited four- to fivefold increase density ofstriatal TH-positive fibers and dopamine transporterin MPTP-treated mice (43). Unlike our animals thatreceived oral treatments, mice were injected subcuta-neously with GPI 1046. These results suggest that ifGPI 1046 provides some measure of behavioral andneuroanatomical neuroprotection, factors affecting bio-availability such as route of administration, brain size,and other differences between species could affect theoutcome.
In summary, the present study showed that whenanalyzed in groups, GPI 1046 did not provide neuro-protective effects from MPTP neurotoxicity inhemiparkinsonian rhesus monkeys. However, selectanimals displayed modest reductions in TH-ir nigralneurons and modest to no losses in striatal dopaminethat corresponded to lack of motor parkinsonian fea-
tures suggesting that this approach may warrant fur-ther investigation.
ACKNOWLEDGMENTS
We thank Theodora Kladis for histological technical assistanceand James Stansell for assistance with behavioral testing. Thisresearch was funded by Guilford Pharmaceuticals.
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