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Doi:10.1016/j.expneurol.2004.11.013Experimental Neurology 192 (2005) 73 – 78 Continuous dopaminergic stimulation reduces risk of motor complications Francesco Bibbiania, Lauren C. Costantinib, Raj Patelb, Thomas N. Chasea,* aExperimental Therapeutic Branch, Building 10, Room 5C103, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD 20892-1406, USA bTitan Pharmaceuticals, Inc., South San Francisco, CA, USA Received 12 August 2004; revised 5 November 2004; accepted 10 November 2004 Levodopa or short-acting dopamine (DA) agonist treatment of advanced parkinsonian patients exposes striatal DA receptors to non- physiologic intermittent stimulation that contributes to the development of dyskinesias and other motor complications. To determine whethercontinuous dopaminergic stimulation can delay or prevent onset of motor complications, four MPTP-lesioned, levodopa-naive cynomolgusmonkeys were implanted subcutaneously with apomorphine containing ethylene vinyl acetate rods. Three other MPTP-lesioned monkeysreceived daily injections of apomorphine. Animals receiving apomorphine rods showed improved motor function (dONT state) within 1 day ofimplantation, and remained continually dONT for the duration of treatment (up to 6 months) without developing dyskinesias. Injected animalsalso showed similar improvement in motor function after each apomorphine injection. However, these primates remained dONT for only 90min and within 7–10 days all developed severe dyskinesias. Implanted monkeys evidenced local irritation, which was alleviated by steroidco-therapy.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Dopamine; Parkinson’s disease; Dyskinesia; Striatum; Nigra; Implant; Polymeric; Subcutaneous; Apomorphine; Ethylene-vinyl Acetate parkinsonian monkeys, dyskinesias begin within 1–2 weeksof daily dopaminomimetic therapy (but Alterations in the motor response to standard dopami- do not appear when treated via continuous infusion for 27 nergic therapy constitute a major source of disability in patients with later stage Parkinson’s disease (PD) ( in parkinsonian primates given levodopa (90 min half-life) and Muenter, 2001; Martignoni et al., 2003; Miyawaki et than in those treated with DA agonists (with half-lives al., 1997). Increasing evidence suggests that the intermittent exceeding 4 h) that provide relatively more constant stimulation of striatal dopamine (DA) receptors contributes dopaminergic stimulation (Similarly, motor to the pathogenesis of these progressive complications complications tend to subside in PD patients when intermittent dopaminergic therapy is replaced by more 2000a,b; Olanow et al., 2000). In parkinsonian rats, 3 weeks of twice-daily levodopa alters motor responses in Hadj Tahar et al., 2000; Mouradian et al., 1990). These ways that mimic human motor fluctuations; these responses observations suggest that the risk of motor response do not occur if levodopa is administered by round-the-clock complications may be reduced by therapeutic regimens that provide steady and thus more physiologic DA replacementfor this normally tonically operating system (1989; Skirboll et al., 1990).
The only currently available means to constantly * Corresponding author. Fax: +1 301 496 6609.
E-mail address: TChase@ninds.nih.gov (T.N. Chase).
administer dopaminergic agents, by continuous infusion 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2004.11.013 F. Bibbiani et al. / Experimental Neurology 192 (2005) 73–78 has limited practicality. Recently, an ethylene vinyl acetate period of 3 h and were performed separately from those in the (EVA) copolymer system has been developed that enables EVA implanted animals. The continuously infused monkeys the continuous release of drugs at therapeutic levels over were scored once daily. Parkinsonian severity was scored on were assessed on an Abnormal Involuntary Movement Scale effective DA agonist for the relief of parkinsonian symp- choreiform and dystonic dyskinesias independently: occa- Wenning, 2000; Wenning et al., 1999), these implants have sional or mild = 1; intermittent or moderate = 2; continuous or performed safely and reliably upon subcutaneous placement severe = 3. Safety monitoring (for skin irritation) was in pigs (IDB, Titan Pharmaceuticals). Here, we evaluated performed by a different rater who was not blind to treatment.
the hypothesis that the long-term continuous administrationof apomorphine will maintain antiparkinsonian efficacy but not induce dyskinesias in previously untreated MPTPlesioned primates. In addition, the safety and tolerability Apomorphine hydrochloride (Sigma, St Louis, MO) was of these apomorphine-containing EVA rods administered by used for both injection and implantation therapy. Dexame- themselves and with steroid co-therapy was assessed in this thasone (Sigma) (0.5 mg/kg i.m.) and Triamcinolone acetonide (Kenacort A, Bristol-Myers Squibb, Anagni,Italy) (0.5 mg s.c. per injection site) were used for theprophylaxis of cutaneous inflammation.
Non-erodible EVA rods capable of prolonged zero order Studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals in 7 Pharmaceuticals, Inc., South San Francisco, CA) were 26 adult male cynomolgus (Macaca fascicularis) primates mm long and 2.4 mm in diameter; each contained weighing 6.5–7.3 kg. All were housed individually, under approximately 98 mg of apomorphine. Animals were stable room conditions, with a 12-h light/dark cycle. Each sedated with 0.3 ml ketamine and a small incision (0.5 received a standard biscuit diet twice daily supplemented cm) was made between the shoulder blades. Implants were with fruit and had free access to water. All primates were positioned between the scapulae by means of a trocar, and a rendered parkinsonian by the subcutaneous administration stylet was used to slide the rod from the trocar into the of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) subcutaneous tissue. This technique allows rod placement HCl (Research Biochemicals Intl., Natick, MA) once per through a minimal incision. The first two implanted animals week at 0.5–1 mg/kg until definite parkinsonian features, received rods that were pre-washed for 8 h with saline prior including tremor, appeared (scores of 4–5 points on the to sterilization, while the third and fourth implanted animals Laval Disability Scale, where the normal state extends from received rods that had been pre-washed for 30 min with 0 to 2 points and maximum disability is 10 points) ( ethanol prior to sterilization to minimize any initial burst et al., 2001). The average cumulative MPTP dose was 4.3 drug release. The implantation procedure took approxi- mg/kg (range 2.5–7.5). Following observation for two mately 15 min and produced no complications.
additional months to ensure stable disability, animals wereselected for equivalent parkinsonian severity (disability score of 4.5–5 points). These animals, not previouslyexposed to dopaminergic agents, were randomly divided Post-implant plasma apomorphine concentrations were into two groups: the first (n = 3) received daily subcuta- measured at hours 6, 12, 24, 36, 48, then daily until day 7, neous injections of apomorphine (2 mg/kg, the minimally weekly until week 4, and monthly for the remainder of study effective dose to turn animals dONT, that is, reverse all (6 months). Plasma apomorphine levels were determined by parkinsonian signs) at rotating sites for 14 days ( extraction into an organic solvent (liquid–liquid extraction) al., 2003); each primate in the second group (n = 4) was followed by HPLC separation and MS/MS identification and treated by implantation of three apomorphine-containing quantification. Plasma samples were diluted with acetonitrile containing a known amount of an internal standard (nor-apomorphine), extracted with hexane, and the solvent evaporated. The resulting residue was reconstituted in 100AL water/acetonitrile (90:10) and injected onto an HPLC All behavioral ratings were performed by the same blinded column. The individual components were separated and investigator. Evaluations in animals receiving daily injections analyzed by MS/MS both for identity and amount by (apomorphine or placebo) occurred every 30 min over a comparison to the internal standard. Upon explantation, the F. Bibbiani et al. / Experimental Neurology 192 (2005) 73–78 residual apomorphine content in each rod was determined by injection was clinically similar (Laval scores: 0–1). How- ever, within 7–10 days (mean 8.3 days), all animals treatedby daily apomorphine injection developed dyskinesias (range 6–10 on the Involuntary Movement Scale). Incontrast, animals receiving apomorphine implants remained Sections of skin surrounding the implant site were fixed dONT for up to 6 months and never developed dyskinesias in 10% formalin, embedded in paraffin, sectioned at 5-Am (Once the EVA rods were removed, all animals were intervals, and stained with hematoxylin and eosin.
dOFFT and oral levodopa therapy was initiated.
Plasma apomorphine concentrations were measured periodically in all primates (Apomorphine levelsimmediately after implantation in animals receiving saline- The primary outcome measure of this study was the washed rods were initially high (Cmax of 10 and 27 ng/ml), appearance of dyskinesias. This essentially ball-or-nothingQ but declined to their ultimate steady state levels within a phenomenon, taken together with the limited sample size week. One of the monkeys receiving saline-washed implants precluded formal statistical analysis.
showed signs of excessive dopaminergic stimulation (vom-iting, prostration) for 2 days following rod insertion, whichresolved as drug levels reached steady state. Use of ethanol- washed rods in the remaining two monkeys, eliminated thisinitial burst release. Except as noted, apomorphine levels All MPTP-lesioned monkeys given apomorphine by remained stable in all rod-implanted animals throughout the subcutaneously implanted rods showed essentially total entire period with very little variability (0.66 F 0.08 ng/ml remission of parkinsonian signs (fully dONT) within 6–12 at steady state). During this time, the release rate of h after implantation, and continued to evidence stable motor apomorphine from the implanted rods, calculated from the benefit until explantation after 11 (first animal), 22 (second drug remaining in the rod upon explantation, was approx- animal) and 24 weeks (third and fourth animal) with no imately 0.25 mg/day/rod. In animals receiving apomorphine fluctuations in response (constant Disability Scale scores of injections, plasma drug levels rose after 20 min to 0–1). Animals treated once daily by apomorphine injection approximately 27 ng/ml, and declined to about 5 ng/ml by (2.0 mg/kg) also turned fully dONT beginning immediately 80–90 min post-injection (at which point the animals had after injection and lasting up to about 90 min. The dONT returned to the dOFFT state). Plasma levels required to state induced by apomorphine administered by rod or by maintain the dONT state in implanted animals (0.19–1.1 ng/ Fig. 1. Dyskinesia scores in animals receiving daily apomorphine injections (2 mg/kg, s.c., open circles) versus apomorphine implants (filled squares). After amean of 8.3 days of daily apomorphine injections, all animals developed dyskinesias (range 6–10 on the Involuntary Movement Scale), whereas none of theimplanted animals developed dyskinesias over 6 months of study.
F. Bibbiani et al. / Experimental Neurology 192 (2005) 73–78 Fig. 2. Plasma drug concentration in animals receiving daily apomorphine injections (2 mg/kg s.c., open circles) versus apomorphine implants (filled squares).
Peaks and troughs are observed with daily injections: plasma levels in animals receiving apomorphine injections peaked after 20 min at approximately 27 ng/ml,then declined to about 5 ng/ml by 80–90 min post-injection (at which point they returned to the dOFFT state). In all implanted animals, apomorphine levelsevidenced very little variability (0.66 F 0.08 ng/ml at steady state). Plasma levels required to maintain the dONT state in implanted animals (0.19–1.1 ng/ml) weresignificantly lower than those required in injected animals (5.0 ng/ml).
ml) were substantially lower than those required in injected apomorphine concentrations required for optimal motor improvement in the injected animals were an order of Local irritation was observed clinically in the first 2 magnitude higher than those required in the rod implanted implanted primates. The first implanted monkey manifested primates. Conceivably, higher agonist levels may be signs of irritation within 2 days that continued until required with intermittent stimulation to prolong the time explantation at 11 weeks. Histological examination of the DA receptors are exposed to suprathreshold stimulation. It is implant sites revealed a dermal zone of necrosis surrounded also possible that that postsynaptic DA receptors in a system by viable and degenerated neutrophils, macrophages, multi- that normally functions tonically, respond more efficiently nucleate giant cells, and plasma cells. The second implanted to stable physiologic-range stimulation than to intermittent animal was pretreated systemically with dexamethasone (0.5 pulsatile activation, possibly owing to restore changes mg/kg i.m.) 1 day before implantation, on the day of occurring within or downstream from striatal dopaminocep- implantation, and 2, 4, and 6 days thereafter. This primate showed no irritation until 22 weeks when the implants were The present results further suggest that the continuous removed. The third and fourth implanted animals were also administration of dopaminomimetics, at doses sufficient to pretreated systemically with dexamethasone and, in addi- ameliorate parkinsonian signs can substantially reduce the tion, received local triamcinolone acetonide (0.5 mg risk of inducing dyskinesias. All apomorphine-injected injection s.c. per implant site) at 2, 4, and 6 months after primates developed dyskinesias within 1–2 weeks of treat- insertion. No clinical signs of irritation were evident in these ment initiation, consistent with the findings of earlier animals that were explanted at 6 months, as per protocol.
investigations in which DA agonists were intermittently Microscopic examination, however, revealed a mild inflam- matory response immediately surrounding each implant.
2003; Smith et al., 2003). In contrast, no apomorphine There were no systemic adverse reactions to the implants or implanted animal developed dyskinesias over treatment to the anti-inflammatory treatments.
intervals lasting up to 6 months. Previous studies ofcontinuous dopaminergic administration in rodent andprimate models of PD ( 1994) showed protective results for up to 4 weeks. Since noother therapeutic regimen has been reported capable of Results from this study indicate that subcutaneous maintaining a similar degree of dyskinesia-free antiparkin- implanted EVA rods can stably release apomorphine for sonian efficacy over such an extended period, findings from up to 6 months, allowing continuous dONT time in MPTP- this study provide additional evidence in support of the lesioned primates without the appearance of dyskinesias.
hypothesis that the intermittent and thus, non-physiologic Apomorphine injections produced equivalent, although stimulation of striatal dopaminergic receptors contributes to transient improvement in parkinsonian signs, but lead to the onset of dyskinesias and related motor response the development of dyskinesias within 7–10 days. Plasma complications (The nonphysiologic stimula- F. Bibbiani et al. / Experimental Neurology 192 (2005) 73–78 tion of dopamine receptors on medium-sized spiny neurons, Baronti, F., Mouradian, M.M., Davis, T.L., Giuffra, M., Brughitta, G., first due to the progressive denervation caused by nigro- Conant, K.E., Chase, T.N., 1992. Continuous lisuride effects on centraldopaminergic mechanisms in Parkinson’s disease. Ann. Neurol. 6, striatal system degeneration and subsequently due to the intermittent high-intensity stimulation associated with dop- Bezard, E., Brotchie, J.M., Gross, C.E., 2001. Pathophysiology of aminomimetic treatment, has been shown to activate levodopa-induced dyskinesia: potential for new therapies. Nat. Rev., signaling cascades that regulate the phosphorylation state of coexpressed ionotropic glutamatergic receptor subunits Bibbiani, F., Oh, J.D., Chase, T.N., 2001. Serotonin 5-HT1A agonist improves motor complications in rodent and primate parkinsonian models. Neurology 10, 1829 – 1834.
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