Eric J. Pappert, Colleen Buhrfiend, Jack W. Lipton, Paul M. Carvey, Glenn T. Stebbins, and. Christopher G. Goetz. Department of Neurological Sciences, ...
Movement Disorders Vol. 11, No. 1, 1996,.pp. 24-26 0 1996 Movement Disorder Society
Levodopa Stability in Solution: Time Course, Environmental Effects, and Practical Recommendations for Clinical Use Eric J. Pappert, Colleen Buhrfiend, Jack W. Lipton, Paul M. Carvey, Glenn T. Stebbins, and Christopher G. Goetz Department of Neurological Sciences, Movement Disorders Section, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois, U.S.A.
Summary: We defined the temporal stability characteristics of levodopdcarbidopa (LD/CD) solution, and determined the effects of temperature, ascorbate, and light on LD stability over 7 days. At room temperature and without ascorbate, LD levels significantly declined by 48 h. Ascorbate prolonged stability to 72 h. Refrigeration and freezing prevented a significant decline in LD levels for
the full 7 days. Light or darkness had no effect on stability. LDlCD solution, if made daily, requires no special handling and longer stability is maintained with ascorbate, refrigeration, or freezing. Key Words: LevodopaLiquid-Parkinson’s disease-Temperature-LightAscorbate.
Levodopa (LD) remains the most effective therapy for Parkinson’s disease (PD); however, its use has been complicated by the development of motor fluctuations and dose-related dyskinesias (1). The severity of these motor effects has been related in part to variations in LD plasma levels (2,3). Continuous intravenous LD infusion (4) and controlledrelease levodopdcarbidopa (LD/CD) ( 5 ) decreased LD plasma level variations and diminished motor fluctuations. Studies evaluating LD/CD solution, administered by continuous enteral infusions (6) or very frequent oral dosings (7-9) also produced improvement in most series. Preparation and storage requirements of LD/CD tablets dissolved in water have not been rigorously studied. Investigators in prior clinical reports prepared solutions daily, maintained them under cool and dark conditions, and added ascorbate to avoid oxidation (6,7). Because long-term and widespread use of LD/CD solutions
in PD patients will necessarily require patients to prepare solutions themselves, the need for specialized preparation and storage conditions and the frequency of solution preparation must be clearly established. In this study, we defined the stability characteristics of LD/CD solution, and determined whether three environmental conditions (temperature, ascorbate, and light) were associated with maintained LD stability. METHODS Levodopa ( 1 .O mg/ml)/carbidopa (0.25 mg/ml) solutions were dissolved in water and mixed by vortex in containers shielded from light. LD/CD solutions were tested under 16 different combinations of environmental conditions, including one of three temperatures, -4, 12, or 25°C (termed freezing, refrigeration, and room temperature, respectively), exposed or shielded from ambient light (continual indoor fluorescent lighting without direct sunlight exposure), and the absence or addition of ascorbate (1,000 mg/L). Triplicate samples of each solution type were removed from each environmental condition at baseline, 8, 24, 48, 72, and 168 h. Five hundred microliters of each sample was added to
Accepted July 31, 199.5. Address correspondence and reprint requests to Dr. E. J. Pappert at Department of Neurological Sciences, RushPresbyterian-St. Luke’s Medical Center, 172.5 West Harrison, Suite 1106, Chicago, IL 60612, U.S.A.
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LEVODOPA STABILITY IN SOLUTION 2.5 ml of a standard antioxidant (HC10, 0.26 M , EDTA 1.344 @ Na,S,O, I, 0.26 pkf) and immediately stored at - 80°C. Sample LD content was determined in duplicate by high-performance liquid chromatography with electrochemical detection (10). Data were analyzed using a repeated-measures analysis of variance model. Post-hoc comparisons of individual time points were conducted using paired t test statistics with a Bonferroni corrected significance level of 0.05 (two-tailed).
RESULTS There was a significant main effect of time, and when all environments were considered together, sample LD levels significantly diminished during the 7-day period (F [5,16] = 7.80, p < 0.001) (Fig. 1). A significant main effect for temperature occurred (F [3,16] = 4.22, p < 0.05), with LD levels remaining stable in samples stored at cool environments ( - 4 or 12°C). Freezing offered no additional benefit over refrigeration ( F [5,10] = 0.71, p > 0.05). A significant interaction between temperature and time (F [15,16] = 4.035, p < 0.001) occurred in that LD levels in the refrigerated and frozen samples remained stable during the entire 7-day period, whereas LD levels in the room temperature samples were significantly diminished after 48 h (t (11) = 3.75, p < 0.01) (Fig. 2). A third main effect on LD level stability was ascorbate, and its addition diminished LD level decline (F [1,16] = 9.73, p < 0.01). This pattern occurred during the 7-day period, signifying a significant ascorbate by time interaction ( F [5,16] = 2.46, 1
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FIG. 2. Refrigerating and freezing solutions prevented (LD) levodopa degradation in comparison to those maintained at room temperature. Filled triangles, - 4°C; filled circles, 12°C; open squares, 25°C.
p < 0.05). Additionally, a significant ascorbate by temperature by time interaction occurred (F [ 15,161 = 1.81, p < 0.05). Although ascorbate had no additive stability effect in refrigerated or frozen samples, its addition maintained LD level stability in solutions stored at room temperature (F [1,5] = 526, p < 0.01) (Fig. 3). Specifically, LD levels in samples stored at room temperature with ascorbate remained stable through 72 h (t (5) = 5.73, p C 0.01); however, without ascorbate, LD levels were
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FIG. 1. The effect of time on levodopa (LD) levels in solutions in all environments.
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FIG. 3. The addition of ascorbate reduced (LD) levodopa degradation in solutions maintained at room temperature. Filled circles, ascorbate; open squares, no ascorbate.
Movement Disorders, Vol. 11, N o . I , 1996
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E. J . PAPPERT ET AL.
significantly diminished by 48 h (t (5) = 3.00, p < 0.05). There was no main effect of light versus dark environments (F [1,16] = 0.65, p > 0.05) on LD stability. DISCUSSION This study confirmed that LD in LD/CD solution is an unstable compound and degrades naturally over time. Our focus was not on the mechanism of such degradation, but rather on the environmental factors that promoted stability. Of the factors examined, refrigeration, freezing, and the addition of ascorbate reduced degradation. Ambient light had no significant impact on stability. Few studies have examined LD stability in LDlCD solution and none has assessed the time course of degradation and stability in different environments. Cedarbaum reported 100% recovery of LD at 24 h when LD/CD solutions were stored in the dark under refrigeration (6). With added ascorbate (2,000 mg/L), Kurth concurred that stability persists through 24 h (7). Based on these observations, LD/CD solutions have been prepared daily, refrigerated, and kept in the dark for most clinical studies. The inconvenience of these procedures for daily production and storage has precluded the use of LD solutions for many disabled PD patients. Our findings demonstrated that LD/CD solutions, if made daily, require no special handling in terms of temperature, ascorbate, or light exposure and can be stored in simple containers at room temperature. For longer stability, solutions can be prepared once weekly, if stored in a refrigerator or freezer. If refrigeration facilities are unavailable, ascorbate can be added to the solution to maintain stability for 3 days. As new formulations are increasingly assessed in PD, stability characteristics for each delivery system need to be established. Because many of these preparations will never be commercially developed,
Movemenl Disorders, VoI. 11, No. I, 1996
patients will ultimately need to prepare them independently and at home. Because of the ease of preparation and storage of LD/CD solutions, clinicians may now expand their use beyond a strict experimental setting to the more widespread population of PD patients with motor fluctuations. REFERENCES Fahn S. “On-off” phenomenon with levodopa therapy in parkinsonism. Neurology 1974;24:431441. Fabbrini G, Mouradian MM, Juncos JL, Schlegel J, Mohr E, Chase TN. Motor fluctuations in Parkinson’s disease: central pathophysiological mechanisms, part 1. Ann Neurol 1988;24:366-371. Mouradian MM, Juncos JL, Fabbrini G , Schlegel J, Bartko JJ, Chase TN. Motor fluctuations in Parkinson’s disease: central pathophysiological mechanisms, part 11. Ann Neurol 1988:24:372-378. 4. Nutt JG, Woodward WR, Hammerstad JP, Carter JH, Anderson J. “On-off ’ phenomenon in Parkinson’s disease: relationship to L-DOPA absorption and transport. N Engl J Med 1984;3 10:483488. 5 . Goetz CG, Tanner CM, Shannon KM, et al. Controlledrelease carbidopa/levodopa (CR4-Sinemet) in Parkinson’s disease patients with severe fluctuations. Neurology 1988; 38: 1143-1 146. 6. Cedarbaum JM, Silvestri M, Kutt H. Sustained enteral administration of levodopa increases and interrupted infusion decreases levodopa dose requirements. Neurology 1990;40: 995-997. 7. Kurth MC, Tetrud JW, Irwin I, Lyness WH, Langston JW. Oral levodopaicarbidopa solution versus tablets in Parkinson’s patients with severe fluctuations: A pilot study. Neurology 1993;43:103&1039. 8. Singh A, Tanner CM, Wasserstein P, Kurth M, Tetrud J. Orally administered levodopa-carbidopa (LD-CD) solution reduces “off-time” in Parkinson’s disease (PD) [Abstract]. Neurology I993;43 :A 197. 9. Metman LV, Hoff J, Mouradian MM, Chase TN. Fluctuations in plasma levodopa and motor responses with liquid and tablet levodopdcarbidopa. Mov Disord 1994;9:463465. 10. Wagner J, Vitali P, Palfreyman MG, Zraika M, Huot S. Simultaneous determination of 3,4-dihydroxyphenylalanine, 5-hydroxytryptophan, dopamine, 4-hydroxy-3-methoxyphenylalanine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, and 5-hydroxyindoleacetic acid in rat cerebrospinal fluid and brain by highperformance liquid chromatography with electrochemical detection. J Neurochem 1982;38:1241-1254.
