TREATMENT OF PARKINSON S DISEASE BY CELL TRANSPLANTATION

TREATMENT OF PARKINSON S DISEASE BY CELL TRANSPLANTATION Pierre Cesaro, Marc Peschanski, Jean-Paul N Guyen Department of Medical Neurosciences and INSERM U 421, Henri Mondor Hospital, Créteil, France Reprint requests to: Prof. P. Cesaro, Service of Neurology, Henri Mondor Hospital, 51 avenue du Maréchal de-lattre-de Tassigny, 94010 Créteil Cedex, France. e-mail: pierre.cesaro@hmn.ap-hop-paris.fr Many environmental and occupational chemicals are known to affect the central and/or peripheral nervous system, causing changes that may result in neurological and psychiatric disorders. Because of the limited accessibility of the mammalian nervous tissue, new strategies are being developed to identify biochemical parameters of neuronal cell function, which can be measured in easily obtained tissues, such as blood cells, as potential markers of the chemically-induced alterations occurring in the nervous system. This review includes a comparative analysis of the effects of mercurials on calcium signalling in the neuroadrenergic PC12 cells and rat splenic T lymphocytes in an attempt to characterize this second messenger system as a potential indicator of subclinical toxicity. The suitability of neurotransmitter receptors in blood cells, such as the sigma binding sites, as biological markers of psychiatric disorders is also discussed. KEY WORDS: Cell therapy, dopamine, graft, Parkinson s disease, pilot trial, stereotaxy. FUNCT NEUROL 2001;16: 21-27 INTRODUCTION Brain transplantation of neurons may act by two different mechanisms: 1) by a drug delivery system: e.g. dopaminergic (DA) neurons in Parkinson s disease (PD) or indirect gene therapy in Huntington s disease (HD), and 2) by o rganotypic reconstruction: e.g. striatal neurons in HD. Parkinson s disease is not a lethal disease, and surgery has to be safe, and eff e c t i v e for many years. To d a y, surgery is considered only in severe PD patients at stages IV and V of the Hoehn and Yahr scale, after optimization of drug therapy. Several preclinical phases are mandatory: 1) a biotechnological one, e.g. transfection of cells, vector design for gene therapy; 2) preclinical trials: in vitro, in vivo r o- dents, then large animal (primate) studies; finall y, 3) clinical trials, classifiable into three categories: phase 1 or pilot, phase 2, often controlled, including dose-response assessment, and phase 3, which involve large cohorts of patients who are randomized to receive the study treatment or a control treatment. In this paper, we do not consider adrenal autografts, which have been abandoned, but concentrate on the heterotopic allografting of fetal mesencephalic DA neurons (1). FUNCTIONAL NEUROLOGY (16)1 2001 21

P. Cesaro et al. LONG-TERM REPORTED RESULTS Table I - Long-term brain fetal allograft studies with clinical and PET follow up Defer et al., 1996 (ref. 2) 5 unilateral Wenning et al., 1997 (ref. 3) 10 unilateral Hauser et al., 1999 (ref. 4) 6 bilateral Hagell et al., 1999 (ref. 5) 5 bilateral Most published reports are phase 1, pilot trials that have included few patients, and assessed uni- or bilateral transplant; the patient serves as his own control, and Positron Emission Tomography (PET) using radiolabelled Levodopa is mandatory to evaluate the results. Restricting ourselves to published trials with a long-term follow up and with PET performed in all patients, we find that only few are available (2-5), amounting to a total of fewer than 30 patients (Table I). The results can be summarized as follows: not all patients are responders, grafting is followed by a 30% decrease in daily L-dopa intake, with an improvement in duration and severity of defined off periods and a decrease in the UP- DRS score measured in off periods. The trials also demonstrated good safety. Since the first pilot trials began more than 10 years ago, one question must be asked: why, with this treatment, has there been no progression to phase 2-3 trials or clinical practice, as in the case of deep brain stimulation (DBS)? Technical and ethical concerns may be the main reasons. Only one phase 2 trial has been completed (Freed et al., as yet unpublished): 36 PD patients were randomized, and a control group receiving sham surgery (general anesthesia, incomplete craniotomy) allowed a double blind follow up by self-reporting, neurological evaluation, and PET scanning. The design was very costly, but gave statistically significant results. A moderate improvement of motor symptoms was observed along with a decrease in daily antiparkinsonian drug intake. The patients own answers constituted the major endpoint of the trial, and statistically more patients in the treatment group reported, after one year, that they believed themselves to have been grafted, whereas no diff e r- ence emerged between the groups after 3 months. Ethical concerns relating to sham s u rgery have been discussed elsewhere (6). CAN THE BRAIN ALLOGRAFT BE A LONG-TERM TREATMENT FOR PD PATIENTS? The answer to this question must be based on long-term clinical results, as well as on longterm PET follow-up of grafted areas and evolution of non-grafted striatum. We look at the results of a case with a long follow up: this male, 49 years old, stage V of the Hoehn and Ya h r scale, was unable to walk in off state and complained of severe drug induced dyskinesias in on ; two unilateral transplantations were performed at an 18-month interval (1993 and 1994). Three months after the first graft, the percentage of daily on periods increased from 40 to 70%, and the patient was able to suspend the subcutaneous injections of apomorphine. This percentage rose to 80% after the second graft, while off phases became less severe (his Hoehn and Yahr score dropped from 5 to 4 in o ff ), and daily drug intake decreased, over 5 years, from 1200 to 250 mg, with concomitant reduction in dyskinesias. Immunosuppression was reduced 6 months after, and stopped one year after, the second graft. Two severe adverse events occurred: a confusional state with excitation and hypersexuality immediately after the first graft, a transient frontal syndrome with minor bleeding along the needle tract after the second session. Both were transient. No major cognitive change was measured at the 1-, 2- and 3- year follow ups. No improvement was noticed in vegetative symptoms, and the patient had to be administered drugs for orthostatic hypoten- 22 FUNCTIONAL NEUROLOGY (16)1 2001

Cell transplantation treatment in PD sion. On the basis of favourable results of this kind, which cannot be reproduced in all cases, transplantation may be viewed as a palliative treatment for motor symptoms in PD, and has been shown to improve quality of life (7). BRAIN IMAGING MRI shows the tracts and a hypersignal in the region of the graft during the first 12 months. After several years, this turns into a T2 hyposignal, with a minimal growth of the size of the image (12). PET imaging with labelled fluorodopa typically shows an increase in Ki, meaning an increase of uptake in the striatum as compared to the cortex. We reported a statistical correlation between PET data and finger dexterity measurements, or daily on, self-reported by the patients (8). This supported the physiological function of the transplanted neurons, recently demonstrated by Piccini et al. (9). In one patient, who had received a transplant in the right putamen 10 years earlier, grafts had restored both basal and drug-induced dopamine release to normal levels. This was associated with sustained, marked clinical benefit and normalized levels of dopamine storage in the grafted putamen. This elegant clinical experiment confirms that the brain allograft can become a long-term therapy for severe PD patients. MECHANISMS Several clinical observations give insights into mechanisms; the increase of endogenous release of dopamine may be explained by the reduction in daily off, by the improvement in motor off UPDRS, and also by the improvement in movement speed in off. Alteration of exogenous L-dopa metabolism is illustrated by the increase in the duration of the on period induced by a single dose of L-dopa, and also by an increase in contralateral peak-dose dyskinesias. This confirms the PET results, and points to some functional integration of the graft. Dyskinesias seen in some patients suggest either a lesion effect in the striatum, or indicate that the heterotopy of the grafted neurons may be partially harmful to the motor pathways (2). MINIMAL AMOUNT OF GRAFTED TISSUE Few post mortem studies have been performed that include a full histochemical analysis of the grafted striatum. Kordower et al. reported well-documented data indicating that 80,000 and 130,000 tryosoine hydroxylase (TH) -positive neurons remained alive in the brains of grafted patients who died more than one year after surgery and after discontinuation of immunosuppressive treatment, respectively (10). On the c o n t r a r y, xenografts with porcine neurons led to poor survival (less than 1,000 per grafted hemisphere) as compared to grafts with human neurons (11 ). In our series (unpublished), some patients received one mesencephalic area in one hemisphere, whereas others had 2-4 per side. There was a striking difference between the two groups, with positive results in on-off self-report, off UPDRS, motor speed and drug intake. It is generally accepted that at least 80,000 neurons per side are the minimal amount (around 20% of normal values) needed to obtain positive results. In these double blind trials, the number of surviving neurons was lower than 80,000 in one patient, which is perhaps explained by a long time elapsing between abortion and transplant (more than 1 week), with a low survival rate as a consequence. Placement of tissue A systematic striatal somatotopic study has been performed by our group (12), showing that d i fferent targets can be defined according to the FUNCTIONAL NEUROLOGY (16)1 2001 23

P. Cesaro et al. clinical goal of the graft. Further studies are needed to document the usefulness of a strategy aiming at graft placement tailored to the major symptoms of the patient in accordance with the known data on striatal somatotopy. It has been claimed that a complete filling of the putamen, or caudate and putamen, should be reached. In this regard, a single injection seems to fill a striatal area included in a sphere of 5 mm diameter. Injection tracks should thus be separated by 1 cm (4,13). The heterotopic placement of a graft may well prevent a physiological reorganization of striatal DA innervation. Other targets have been proposed: the ventral tegmental area and substantia nigra itself. C u r r e n t l y, we are not able to promote the elongation of DA axons from the nigra to the striatum; it has been shown that this is age dependent (14). Further studies are needed to ascertain whether co-grafting in both the striatum and the nigra is clinically useful. Dissection and selection of tissue and number and age of transplanted neurons Several methods have been used for fetal DA transplantation: solid pieces of tissue, cell suspensions, or both. We use a mix of cell suspension and micropieces obtained following mechanical dissociation of tissue. We do not know what is the best method at this time. Conservation of fetal mesencephalic areas should not exceed 48h because of reduced viability of neurons and increase of contamination by germs. Cryopreservation is also a major factor in reduced viability. Post mitotic cells are used today; stem cells or progenitor cells need an ex vi - v o d i fferentiation prior to transplantation. At this time, preclinical data are not convincing enough to launch trials with these cells (13). Present data on xeno transplantation are not convincing, with a lack of congruence between improvement and graft development assessed by PET. It is generally accepted that neuroprotection may improve graft survival and thus, therapeutic e ff i c a c y. Preclinical trials or pilot trials have pointed to tirilazad or flunarizine (15). The use of neurons after in vitro preservation with glial cell line-derived neurotrophic factor (GDNF) has also been published (16). However, we still do not have standardized protocols that might allow graft survival and the magnitude of the expected clinical improvement to be predicted with reasonable certainty. Selection of patients To d a y, severe PD patients (Hoehn and Ya h r stages IV and V) are included in graft trials. Preference should be given to patients under the age of 60. There is now evidence that multiple system atrophy (MSA) and other degenerative diseases do not benefit from DA neuron grafting. Longterm follow up of cohorts of grafted patients must indicate whether patients will be protected against deterioration of motor signs. A long-term comparison with results of subthalamic deep brain chronic stimulation (STN DBS) is also warranted. Available data indicate that over a period of 5 years, STN DBS allows better control of motor symptoms in most patients (17). If long-term results indicate no major deterioration after graft, and no major side effect, grafts could be attempted in younger patients at stage III, before the occurrence of severe on-off fluctuations, dystonia and dyskinesias. The same considerations apply to STN DBS. Table II compares the effects of grafting and DBS, findings obtained following comparison of cohorts included in different trials. No comparative trial has been performed at the present time. It must also be borne in mind that whereas no major cognitive or psychiatric side effects follow striatal DA grafts, vegetative, axial, cognitive as well as psychiatric disturbances are not improved by grafting, with the exception of the partial benefit obtained by reduction of DA drug i n t a k e. It can be concluded, in short, that grafting remains a palliative treatment for motor impairment in PD. 24 FUNCTIONAL NEUROLOGY (16)1 2001

Cell transplantation treatment in PD Table II - Comparison of improvement following DA graft and STN DBS (literature findings) Clinical signs GRAFT STN DBS Akinesia +++ +++ Rigidity ++ ++ Tremor +/- +++ Speech +/- +/- Writing + + Autonomic impairment - - Gait +/- + Axial impairment - - Cognitive impairment - - Psychiatric impairment - - L-dopa intake ++ ++ Abbreviations: DA = dopaminergic; STN DBS = subthalamic deep brain stimulation. SIDE EFFECTS In two patients in our series, we observed a drastic motor improvement in the 8 to 15 days following the graft. Because of severe dyskinesia, the patients had to suspend, transiently, their DA treatments. In patient 5 this occurred after the first graft, with confusional status, and in patient 11 after the 2 grafts, without any confusional symptom. The mechanism of their improvement remains unclear, but the suggestion is that there was an acute DA release either by the grafted neurons or by the remaining DA striatal t e r m i n a l s. Immediate side effects in our series were in line with known risks of stereotaxy: 1 symptomatic frontal hematoma among 20 surgical sessions; transient frontal symptoms (4/20); transient psychiatric events: confusion, aggressiveness, schizophrenia: (6/20); and one severe postoperative infectious status in one patient. Long-term side effects are, in most cases, explained by the disease or concomitant pathology: e.g. recurrence of previously experienced events (delusions in one patient) or sural phlebitis (one patient). In patients with a long interval between the first engraftment and bilateralization, asymmetry of motor signs was common, but it could also be observed in other patients with shorter intervals. In the event of asymmetry of motor signs, PET must be used to check for graft growth asymmetry (2-4). Dyskinesias usually improve in those patients who can reduce their DA drug intake. In two patients, spontaneous dyskinesias, observed in defined o ff state, i.e., 12 hours after cessation of drugs, occurred on the most affected side. This indicates a spontaneous release of dopamine by the graft itself, and suggests a possible deleterious eff e c t of the heterotopic grafted neurons: long-term follow up of patients is also warranted to assess such eff e c t s. CONCLUDING REMARKS It is now more than ten years since the first results in PD patients were published, and while long-term results where there is improvement of motor symptoms and, in some cases, reduction or cessation of DA drugs may be reached in severe patients, transplantation continues to be a matter of technical and ethical concern that remains within the domain of medical research (1). At the same time, chronic DBS in the STN (first trial in humans in 1993) has been performed in more FUNCTIONAL NEUROLOGY (16)1 2001 25

P. Cesaro et al. than 2,000 PD patients worldwide. However, many questions remain unanswered, and these concern, namely: the best location and size of transplantation; the quantity of grafted neurons; the need for and duration of immunosuppressive treatments; the best source of neurons for transplantation and the long-term effect as compared to other medical or surgical treatments. Nervertheless, we can be sure that this method is not harmful to patients and thus deserves new clinical trials aimed at giving logical and documented answers to the many questions raised above. It may be that a better understanding of neural repair can promote neurotrophic treatments without transplantation. Furthermore, results obtained in PD may promote research into transplantation therapy for other diseases of the central nervous system (13). No trial of striatal transplantation in H u n t i n g t o n s disease could have been launched without the information provided by trials completed in PD patients. REFERENCES 11. Hallett M, Litvan I and the Task Force on S u rgery for Parkinson s Disease. Evaluation of surgery for Parkinson s disease. Neurology 1999;53:1910-1921 12. Defer GL, Geny C, Ricolfi F et al. Longterm outcome of unilaterally transplanted parkinsonian patients. I. Clinical Approach. Brain 1996;11 9 : 4 1-5 0 13. Wenning GK, Odin P, Morrish P et al. Shortand long-term survival and function of unilateral intrastriatal dopaminergic grafts in P a r k i n s o n s disease. Ann Neurol 1997;42:95-1 0 7 14. Hauser RA, Freeman TB, Snow BJ et al. Long term evaluation of bilateral fetal nigral transplantation in Parkinson s disease. Arch Neurol 1999;56:179-187 15. Hagell P, Schrag A, Piccini P et al. Sequential bilateral transplantation in Parkinson s disease. Brain 1999;122:11 2 1-11 3 2 16. Freeman TB, Vawter DE, Leavertun PE et al. Use of placebo surgery in controlled trials of a cellular-based therapy for Parkins o n s disease. N Engl J Med 1999,341: 988-999 17. Hagell P, Crabb L, Pogarell O et al. Healthrelated quality of life following bilateral intrastriatal transplantation in Parkinson s disease. Mov Disord 2000;15:224-229 18. Remy P, Samson Y, Hantraye P et al. Clinical correlates of 18FI fluorodopa uptake in five grafted parkinsonian patients. Ann Neurol 1995;38:580-588 19. Piccini P, Brooks DJ, Bjorklund A et al. Dopamine release from nigral transplants visualized in vivo in a Parkinson s patient. Nature Neurosci 1999;2:1137-1140 10. Kordower JH, Freeman TB, Snow BJ et al. Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinsons disease. N Engl J Med 1995;332:1118-1124 11. Schumacher JM, Ellias SA, Palmer EP et al. Transplantation of embryonic porcine mesencephalic tissue in patients with PD. Neurology 2000; 54:1042-1050 12. Palfi S, Nguyen JP, Brugieres P et al. MRI-stereotactical approach for neural grafting in basal ganglia disorders. Exp Neurol 1998;150:272-281 13. Björklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nature Neurosci 2000;3:537-544 14. Bentlage C, Nikkhah G, Cunningham MG, Bjorklund A. Reformation of the nigrostriatal pathway by fetal dopaminergic micrografts into the substantia nigra is critically dependent on the age of the host. Exp Neurol 1999;159:177-190 15. Kaminski Schierle GS, Hansson O, Brundin P. Flunarizine improves the survival of grafted dopaminergic neurons. Neuroscience 1999;94:17-20 1 6. Mendez I, Dagher A, Hong M et al. En- 26 FUNCTIONAL NEUROLOGY (16)1 2001

Cell transplantation treatment in PD hancement of survival of stored dopamine rgic cells and promotion of graft survival by exposure of human fetal nigral tissue to glial cell line-derived neurotrophic factor in patients with Parkins o n s disease. Report of two cases and technical considerations. J Neurosurg 2 0 0 0 ; 9 2 : 8 6 3-8 6 9 17. Limousin P, Krack P, Pollak P et al. Electrical stimulation of the subthalamic nucleus in advanced Parkinson s disease. N Engl J Med 1998;339:1105-1111 FUNCTIONAL NEUROLOGY (16)1 2001 27