Summary
Background
Long‐term l‐dihydroxyphenylalanine (l‐DOPA) treatment of Parkinson's disease (PD) is associated with motor complications known as l‐DOPA‐induced dyskinesias (LID) and on/off fluctuations, which are linked to unsteady pulsatile dopaminergic stimulation.
Aim
The objective of this study was to improve l‐DOPA treatment by slowing and stabilizing dopamine (DA) production in the brain and increasing water solubility to provide a rescue therapy for PD.
Results
We synthesized l‐DOPA‐amide, a novel l‐DOPA precursor called DopAmide. DopAmide is water soluble and, as a prodrug, requires hydrolysis prior to decarboxylation by the aromatic l‐amino acid decarboxylase (EC 4.1.1.28; AAAD). In the 6‐OH‐dopamine (6‐OHDA)‐lesioned rats, DopAmide maintained steady rotations for up to 4 h compared with 2 h by l‐DOPA, suggesting that this rate‐limiting step generated a sustained level of DA at dopaminergic neurons. Pharmacokinetic studies showed elimination half‐life of l‐DOPA in the plasma after DopAmide treatment of t 1/2 = 4.1 h, significantly longer than t 1/2 = 2.9 h after treatment with l‐DOPA, consistent with the 6‐OHDA results.
Conclusions
The slow conversion of DopAmide to l‐DOPA provides a sustained level of DA in the dopaminergic cells, shown by the long 6‐OHDA steady rotations. The water solubility and improved bioavailability may help reduce medication frequency associated with l‐DOPA treatment of PD. Sustained levels of DA might lower the super‐sensitization of DA signaling and potentially attenuate l‐DOPA adverse effects.
Keywords: Dyskinesia, l‐Dihydroxyphenylalanine‐induced dyskinesia, Levodopa, On\Off fluctuations, Parkinson's disease
Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc). The subsequent loss of dopamine (DA) within the striatum is responsible for the deterioration and the dysfunction of the striatal circuits and motor deficits characteristic symptoms of PD.
The symptomatic therapy with the DA precursor l‐3,4‐dihydroxyphenylalanine (l‐DOPA) is very effective initially. l‐DOPA is converted to DA by the enzyme l‐amino acid decarboxylase (AAAD; EC 4.1.1.28; DOPA decarboxylase) 1, 2, relieving symptoms caused by the degradation of dopaminergic neurons in the brain. It reverses akinesia in patients with PD; however, chronic l‐DOPA treatment aggravates the symptoms, eventually causing l‐DOPA‐induced dyskinesia (LID), or involuntary aimless movements 3. The “wearing off,” motor fluctuations, and dyskinesia associated with the long‐term l‐DOPA treatment of PD 4, 5 may partly be managed by adding other medications, such as DA agonists, monamine oxidase B inhibitors, or catechol‐O‐methyltransferase inhibitors (reviewed in 6 and 7). Other developments have focused on l‐DOPA delivery formulations including duodenal infusion of l‐DOPA/carbidopa, extended‐release l‐DOPA, or oral pro‐ l‐DOPA forms 6.
All of these strategies act by prolonging the effective delivery of l‐DOPA to the brain. However, improvement in dyskinesia has been shown mainly by deep‐brain stimulation 8, and to some extent by duodenal l‐DOPA infusion, subcutaneous apomorphine, or monotherapy with DA agonists in the early phases of the disease 9.
Although the dyskinetic and antiparkinsonian actions of l‐DOPA are tied together, the initial benefits of l‐DOPA make it a favorable drug, which is widely on demand and used by all patients with PD. Therefore, a major challenge would be to utilize l‐DOPA for its beneficial effects, simultaneously lowering the “wearing effect” and dyskinesia.
In a clinical study, the administration of levodopa by intravenous infusion at a constant rate brought about a dramatic extension in the duration of mobility and reduced the frequency of fluctuations compared with oral therapy 10; see also 11.
Although the pathophysiology of “wearing off” or dyskinesia is not fully understood, a motor complication appeared to arise from a pulsatile stimulation of DA receptors of the striatal. It suggested that sustained‐release formulation of l‐DOPA would lead to improved control of the fluctuations observed with conventional levodopa preparations 12, 13. This treatment approach of controlling motor fluctuations is called continuous drug delivery (CDD) 14, 15. It indicates that a more‐constant blood levels through modifying the oral pharmacokinetics of l‐DOPA could enhance efficacy and reduce or prevent motor response oscillations and, possibly, also LID 12, 13.
A major contributor to LID associated with l‐DOPA treatment is the enhanced dopaminergic signaling such as the D1 DA receptors at the striatum 16, 17, 18.
A conceivable possibility for moderating striatal‐DA signaling would be by managing DA production and lowering local DA overload. A gelified version of l‐DOPA was developed for intrajejunal administration achieving constant plasma l‐DOPA concentrations over several days of infusion 19. Improving treatment and minimizing adverse effects of l‐DOPA led to the synthesis of l‐DOPA precursors, or prodrugs such as DOPA methyl ester 20, DOPA ethyl ester 21, carboxy esters, phenol esters, amides, peptides 22, 2‐tetrahydropyranylmethyl, phenoxyethyl, ethyl, 2‐hydroxypropyl 23, l‐(+)‐3‐(3‐hydroxy‐4‐pivaloyloxybenzyl)‐2,5‐diketomorpholine 24, and dimeric derivatives of l‐DOPA diacetyl esters 25, 26, see review 27.
In this study, we modified l‐DOPA structure, neutralizing the negatively charged carboxyl group to an amide. This modification made it more stable and water soluble and increased its bioavailability. In addition, it enabled a moderate and a gradual conversion to DA, leading to a more steady level of rotations in the 6‐OH‐dopamine (6‐OHDA) rotating animal model of PD.
Methods and Materials
Synthesis
l‐DopAmide, [(S)‐2‐Amino‐3‐(3,4‐Dihydroxyphenyl) Propanamide]
l‐DOPA ethyl ester [(S)‐ethyl 2‐amino‐3‐(3,4‐dihydroxyphenyl)propanoate] (1) (3 g, 13.3 mmole) was dissolved in 15 mL cold (−10°C) aqueous ammonia (25%) (16.5 mol equivalent). After 24 h, at room temperature in the dark, ethanol was added to the reaction mixture and excess ammonia and most of the solvents were removed by vacuum evaporation. The concentrated oily residue was added dropwise to a large volume of diethyl ether (~1 L), resulting in immediate precipitation of the product as an off‐white powder. The upper solvent layer was triturated, and the solid washed with three fresh portions of ether and air‐dried. The yield of product was 1.97 g (75%).
NMR analysis showed remaining starting material (ca. 10–15%). Mass spectrum identifies the presence of a hydrate in some extent, which was also supported by the elemental analysis. Lyophilization removed most of the excess water, which led to better analysis.
1H NMR (D2O, ppm): d ABX(1) 6.82 (d, 1 H, J = 8 Hz), 6.73 (d, 1 H, J = 1.8 Hz), 6.63 (dd, 1 H, J 1 = 8 Hz, J 2 = 1.8 Hz), ABX(2) 3.63 (t, 2 H, J = 7 Hz), 2.84 (dd, 1 H, J AB = 13.7 Hz, J AX = 7 Hz), 2.77 (dd, 1 H, J AB = 13.7 Hz, J BX = 7 Hz). IR (cm−1): 1681. MS (e/z): 215.15 [hydrate M+H2O+H]+, 197.56 [M+H]+, 393.43 [M‐CONH2+H]+. Elemental analysis: Calcd. C, 55.09; H, 6.16; N, 14.28. Found C, 54.07; H, 6.17; N, 13.71.
Animals
Ethics
A commercial company “Cerebricon Ltd and/or Charles River Discovery and Imaging Services,” USA, uses its ethical permission to do animal studies as a service (#C148210). All animals were treated according to the National Institute of Health (NIH) guidelines for the care and use of laboratory animals. Animal ethics committee accredits the company, and licensed veterinarians conducted the experiments. In the case that the general health status of an animal has significantly worsened, the rat is sacrificed by an overdose of CO2 and decapitated.
Experimental Procedure
Altogether 110 male Wistar rats, purchased from Charles River, Sulzfeld, Germany, and weighing 220–300 g, were used for the experiment. Animals were housed at a standard temperature (22 ± 1°C) and in a light‐controlled environment (lights on from 7 am to 8 pm) with ad libitum access to food and water. Animals were fasted overnight before day 35 in the rotation test. The bodyweight of each was measured twice a week during the study. Animals were grouped as follows:
Group 1: Ten rats treated with vehicle for carbidopa: 0.5% chemistry manufacturing and control in saline (10 mg/kg, 1 mL/kg, p.o.) 20 min before vehicle for DopAmide: water (10 mL/kg, p.o.);
Group 2: Ten rats treated with carbidopa (10 mg/kg, 1 mL/kg, p.o.) 20 min before vehicle for DopAmide: water (10 mL/kg, p.o.);
Group 3: Fifteen rats treated with carbidopa (10 mg/kg, p.o.) 20 min before l‐DOPA in water (10 mg/kg, p.o.);
Group 4: Fifteen rats treated with carbidopa (10 mg/kg, p.o.) 20 min before l‐DOPA in water (25 mg/kg, p.o.);
Group 5: Fifteen rats treated with carbidopa (10 mg/kg, p.o.) 20 min before l‐DOPA in citrate buffer (50 mg/kg, p.o.);
Group 6: fifteen rats treated with carbidopa (10 mg/kg, p.o.) 20 min before DopAmide (10 mg/kg, p.o.);
Group 7: Fifteen rats treated with carbidopa (10 mg/kg, p.o.) 20 min before DopAmide (25 mg/kg, p.o.);
Group 8: Fifteen rats treated with carbidopa (10 mg/kg, p.o.) 20 min before DopAmide (50 mg/kg, p.o.).
Unilateral 6‐OHDA Lesioning
Degeneration of dopaminergic neurons and DA depletion in the striatum was achieved by injection of 6‐OHDA to the right medial forebrain bundle (MFB). Male Wistar rats were anesthetized with 5% isoflurane (in 70% N2O and 30% O2; flow 300 mL/min) and placed in a stereotactic frame. During the operation, the concentration of anesthetic was reduced to 1–1.5%. The right brain hemisphere was exposed through a small craniectomy to the skull. The dura mater was carefully punctured with a fine needle, and 6‐OHDA (2.5 μg/μL) was injected stereotaxically into the MFB. Per rat, a total of 4 μL (10 μg) of 6‐OHDA was infused at a speed of 0.4 μL/min at the following coordinates: AP −4.4, ML −1.2, DV +8.3 mm (from skull surface). The injection needle was left in place for another 5 min before being carefully withdrawn. The rats were allowed to recover from anesthesia and were carefully monitored for possible postsurgical complications. The animals were returned to the home cages with ad libitum access to food and water.
Amphetamine Induced Rotation Asymmetry
The rats were tested for amphetamine‐induced turning behavior, at day 13 after the 6‐OHDA injections. Motor asymmetry was monitored in automated rotometer bowls (TSE Systems, Berlin, Germany) for 120 min after injection of amphetamine (5 mg/kg i.p.). The rotation asymmetry score for each test was expressed as ipsiversive rotations during the monitoring period.
6‐OHDA lesioning was tested by the amphetamine‐induced asymmetry rotation test. No significant differences between the groups were found in body weight, amphetamine‐induced rotation, or 90‐min l‐DOPA‐induced rotation, suggesting homogeneity between the test groups before the actual 300‐min rotational activity test (Data not shown).
l‐DOPA Priming
6‐OHDA‐lesioned rats were given l‐DOPA (10 mg/kg, i.p.) twice a week for 3 weeks starting at 2 weeks postlesioning. All rats also received carbidopa, 10 mg/kg, p.o, and 20 min before l‐DOPA.
At week 4, rats were subjected to single l‐DOPA (10 mg/kg, i.p.) dosing 20 min after carbidopa (10 mg/kg, p.o.) and were then monitored for rotation asymmetry for 90 min. After monitoring, the best responders were assigned to the various treatment groups and a 300‐min rotational test was performed on week 5.
l‐DOPA Induced Rotational Activity
On the fifth week after lesioning (Day 35), rats were evenly assigned to corresponding treatment groups based on amphetamine and 90‐min l‐DOPA rotation. Before treatments and rotation tests, the rats were fasted overnight. All rats received carbidopa, 10 mg/kg, p.o, followed 20 min later by l‐DOPA (10, 25, or 50 mg/kg, p.o, doses), DopAmide (10, 25, or 50 mg/kg, p.o.), or corresponding vehicles. Each rat was then placed in a rotometer bowl, and rotation asymmetry was monitored for 300 min. Motor asymmetry was monitored in an automatic 8‐channel rotometer (TSE systems). The rotation asymmetry score for each test was calculated by subtracting the ipsilateral turns to the lesion from the contralateral turns.
Statistical Analysis
All values were presented as mean ± standard deviation (SD) or standard error of mean (SEM), and differences were considered to be statistically significant at the P < 0.05 level. Statistical analysis was performed using StatsDirect statistical software (Cheshire, UK). Differences among means were analyzed using 1‐way ANOVA followed by Dunnet's test (comparison to the control (=l‐DOPA‐treated rats) group). Within group, comparison to the baseline was performed by 2‐way ANOVA. Nonparametric data were analyzed with Kruskal–Wallis ANOVA or Friedman ANOVA, respectively.
Aromatic l‐Amino Acid Decarboxylase Activity Assay
Native AAAD was isolated from the striatum, a region rich in AAAD 1. For each sample, one freshly dissected striatum was homogenized in 250 μL ice‐cold lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X‐100, 1 mM EGTA, 2 mM EDTA, 0.1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride, and 1 μg/mL aprotinin), incubated on ice for 30 min, and centrifuged at 15,000 g for 10 min at 4°C.
Aromatic l‐amino acid decarboxylase activity was assayed as described previously 1. In brief, tissue was homogenized in ice‐cold 0.25 M sucrose. The reaction was initiated by incubating 10 μg of the homogenate with 50 mM sodium phosphate buffer, pH 7.2, 0.1 mM EDTA, 0.17 mM ascorbic acid, 1 mM β‐mercaptoethanol, 0.1 mM pargyline, 10 μM pyridoxal‐5′‐phosphate, and l‐DOPA or DopAmide at the indicated concentrations for 20 min at 37°C. The reaction was stopped by ice‐cold solution of 0.525 M HClO4 containing 3,4‐dihydroxybenzylamine as an internal standard. DA was extracted using alumina, resolved by high‐performance liquid chromatography (HPLC), and monitored with an electrochemical detector.
PK studies were performed by XenoBiotic Laboratories, Plainsboro, NJ, USA.
Results
We chose a chemical modification approach to enable a prolonged and sustained l‐DOPA delivery. A slow rate of DA production would stabilize DA level, lower local DA concentration, and avoid overwhelming of the dopaminergic system. We chemically modified the carboxyl (COOH) group of l‐DOPA to amide (DopAmide) (Figure 1A,B). We predicted that as a prodrug, DopAmide would not be recognized directly as an AAAD substrate, but would need to be first hydrolyzed to l‐DOPA, and this hydrolysis would be rate‐limiting (Figure 1B). The structural modification was therefore expected to convey a slow and sustained rate of DA production, with a consequent improvement in motor complications. We also expected that DopAmide would be water soluble, which would improve bioavailability and allow faster and more consistent absorption than l‐DOPA, with a more rapid onset of action.
Figure 1.
The conversion of DopAmide to dopamine (DA) requires an amino peptidase cleavage step. (A) The scheme shows the conversion of l‐dihydroxyphenylalanine (L‐DOPA) and (B) l‐Dopamide to DA. DopAmide is not a substrate for DOPA decarboxylase and requires a hydrolysis step prior to converting to L‐DOPA. This is expected to be a rate‐limiting step, slowing down DA production. (C) The aromatic l‐amino acid decarboxylase (AAAD) assay. The reaction was initiated by incubating 10 μg of AAAD homogenate with L‐DOPA or DopAmide. DA was extracted after 20 min using alumina, resolved by high‐performance liquid chromatography (HPLC) and assayed with an electrochemical detector. The graph shows the amount of DA produced over 20 min, as a function of the concentration of L‐DOPA or DopAmide.
DopAmide is Not a Substrate of AAAD
Aromatic l‐amino acid decarboxylase was prepared from freshly dissected striatum 28 (Experimental Procedure) efficiently decarboxylated l‐DOPA but only marginally decarboxylated DopAmide (Figure 1C). This established that DopAmide is not an effective substrate for AAAD, indicating that DopAmide needs to be hydrolyzed to l‐DOPA prior to becoming active, a step that would slow the rate of DA production.
DopAmide is Converted to l‐DOPA, PK Studies
Next, we confirmed that DopAmide is indeed converted to l‐DOPA, in vivo, acting as a prodrug. In pharmacokinetic studies, we measured the levels of DopAmide, l‐DOPA, and the related catecholamines, 3,4 Dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA), in the plasma of male Sprague Dawley rats after a single dose of 25 mg/kg DopAmide or l‐DOPA, given orally after carbidopa. l‐DOPA levels in the plasma reached a maximal concentration (C max) of 7130 ng/mL at 0.67 h (T max) after treatment with DopAmide (Table 1), as compared to a C max of 12900 at T max of 0.25 h after treatment with l‐DOPA (Table 2).
Table 1.
PK after single oral dose of Dopamide (25 mg/kg) to male Sprague Dawley rats
| Analyte | Subject | t 1/2 (hr) | T max (μg/mL) | C max (hr*μg/mL) | AUC0–t (hr*μg/mL) | AUC0–inf (hr*μg/mL) | F% |
|---|---|---|---|---|---|---|---|
| DopAmide | G307‐M | NC | 0.25 | 0.028 | 0.099 | NC | 0.60a |
| G308‐M | NC | 0.25 | 0.024 | 0.071 | NC | ||
| G309‐M | NC | 0.5 | 0.014 | 0.047 | NC | ||
| Mean | NA | 0.33 | 0.022 | 0.0720 | NA | ||
| SD | NA | 0.14 | 0.007 | 0.022 | NA | ||
| l‐DOPA | G307‐M | 3.67 | 0.5 | 8.49 | 13.7 | 13.7 | |
| G308‐M | 5.45 | 0.5 | 8.89 | 13.6 | 13.6 | ||
| G309‐M | 3.17 | 1 | 4.01 | 9.7 | 9.7 | ||
| Mean | 4.10 | 0.67 | 7.13 | 12.3 | 12.3 | ||
| SD | 1.20 | 0.29 | 2.71 | 2.28 | 2.28 | ||
| DOPAC | G307‐M | NC | 2 | 0.071 | 0.016 | NC | |
| G308‐M | NC | 2 | 0.099 | 0.021 | NC | ||
| G309‐M | 2.92 | 2 | 0.034 | 0.014 | 0.21 | ||
| Mean | 2.92 | 2 | 0.068 | 0.017 | 0.07 | ||
| SD | NA | 0 | 0.033 | 0.004 | 0.12 | ||
| HVA | G307‐M | 1.25 | 2 | 0.500 | 1.44 | 1.49 | |
| G308‐M | 1.82 | 2 | 0.560 | 210 | 2.29 | ||
| G309‐M | 3.06 | 2 | 0.378 | 1.98 | 2.49 | ||
| Mean | 2.04 | 2 | 0.480 | 1.85 | 2.09 | ||
| SD | 0.93 | 0 | 0.093 | 0.36 | 0.53 |
NC, not calculated; NA, not applicable; l‐DOPA, l‐dihydroxyphenylalanine; DOPAC, dihydroxyphenyl acetic acid; HVA, homovanillic acid.Bioavailability = (PO mean AUC0–t/nominal dose) ÷ (IV mean AUC0–t/nominal dose).aOral bioavailability for DopAmide is 0.60%.
Table 2.
PK after single oral dose of l‐DOPA (25 mg/kg) to male Sprague Dawley Rats
| Analyte | Subject | t 1/2 (hr) | T max (μg/mL) | C max (hr*μg/mL) | AUC0–t (hr*μg/mL) | AUC0–inf (hr*μg/mL) | F% |
|---|---|---|---|---|---|---|---|
| DopAmide | G4‐10‐M | NC | NC | NC | NC | NC | |
| G4‐11M | NC | NC | NC | NC | NC | ||
| G4‐12‐M | NC | NC | NC | NC | NC | ||
| Mean | NA | NA | NA | NA | NA | ||
| SD | NA | NA | NA | NA | NA | ||
| l‐DOPA | G4‐10‐M | 1.9 | 0.25 | 11.2 | 15.5 | 15.5 | 149a |
| G4‐11M | 5.0 | 0.25 | 15.5 | 18.8 | 18.8 | ||
| G4‐12‐M | 1.8 | 0.25 | 11.9 | 22.9 | 22.9 | ||
| Mean | 2.9 | 0.25 | 12.9 | 19.1 | 19.1 | ||
| SD | 1.83 | 0.00 | 2.31 | 3.7 | 3.7 | ||
| DOPAC | G4‐10‐M | NC | 0.25 | 0.053 | 0.153 | NC | |
| G4‐11M | 14 | 0.25 | 0.040 | 0.099 | 0.42 | ||
| G4‐12‐M | NC | 0.25 | 0.065 | 0.199 | NC | ||
| Mean | 14 | 0.25 | 0.053 | 0.150 | 0.14 | ||
| SD | NA | 0.00 | 0.012 | 0.05 | 0.24 | ||
| HVA | G4‐10‐M | NC | 4 | 0.500 | 1.57 | NC | |
| G4‐11M | 1.71 | 2 | 0.560 | 1.57 | 1.69 | ||
| G4‐12‐M | 5.87 | 4 | 0.378 | 3.14 | 3.30 | ||
| Mean | 3.79 | 3.33 | 0.480 | 2.09 | 1.66 | ||
| SD | 2.94 | 1.15 | 0.093 | 0.91 | 1.65 |
NC, not calculated; NA, not applicable; l‐DOPA, l‐dihydroxyphenylalanine; DOPAC, dihydroxyphenyl acetic acid; HVA, homovanillic acid.Bioavailability = (PO mean AUC0–t/nominal dose) ÷ (IV mean AUC0–t/nominal dose).aOral bioavailability for l‐DOPA is ~100%. The >100% result may be due to assay variability.
Notably, the elimination half‐life (t 1/2) of l‐DOPA after DopAmide treatment was 4.1 h (Table 1), significantly longer than the elimination half‐life (t 1/2) of 2.9 h after direct treatment with l‐DOPA (Table 2).
DopAmide itself was detected in the plasma at very low levels, probably due to its conversion to l‐DOPA. Alternatively, DopAmide could be rapidly delivered across the blood–brain barrier (t max = 0.67 h; Table 1).
Prolonged and Steady Rotations of 6‐OH‐DA‐Lesioned Rats
We next examined the effects of DopAmide in an in vivo model of PD. In agreement with the pharmacokinetic data, we anticipated that the effects of DopAmide in the brain would be prolonged, as compared to the effects of l‐DOPA. For these studies, we used the 6‐hydroxy‐dopamine (6‐OHDA)‐lesion rat model 29. In this model, unilateral injections of 6‐OHDA cause widespread depletion of the dopaminergic system in the MFB, by deafferentation of the substantia nigra pars compacta (SNc) and ventral tegmental‐area (VTA) terminal fields 29. The rats show rotational behavior in response to the administration of DA agonists.
In this assay we compared the rates of rotation induced by DopAmide and l‐DOPA. On the fifth week after lesioning, the rats were randomly assigned to eight treatment groups and orally (p.o.) treated with carbidopa (10 mg/kg, p.o. 1 mL/kg), followed 20 min later by DopAmide (10, 25 or 50 mg/kg, 10 mL/kg) or l‐DOPA (10, 25 or 50 mg/kg, 10 mL/kg). Control groups were treated with vehicle alone or with carbidopa followed by vehicle. The rotational behavior of the rats was followed for the duration of 300 min (Experimental Procedure). All groups showed rotations in response to the oral administration of DopAmide or l‐DOPA (Figure 2A). The rotational activity induced by DopAmide was dosage dependent, and a dose of 50 mg/kg induced significantly more rotations than a dose of 25 mg/kg. Unlike DopAmide, l‐DOPA induced maximal rotational activity at a dose of 25 mg/kg, with no significant increase at 50 mg/kg (Figure 2A–F). The net rotations of all the rats during the 300 min test are shown in Figure 2B. DopAmide at 50 mg/kg dose showed ~35% higher net rotational behavior compared with similar dose of l‐DOPA (P = 0.078).
Figure 2.
Rotation induced asymmetry test in rats 35 days after 6‐OH‐dopamine (6‐OHDA) lesioning (A) The data of all groups are presented as net rotational activity/5 min over the course of 300 min. (A) Veh + Veh (B) carbidopa + Veh (C) l‐dihydroxyphenylalanine (L‐DOPA) (10 mg/kg) (D) L‐DOPA 25 (mg/kg) (E) L‐DOPA 50 (mg/kg) (F) DopAmide 10 (mg/kg) (G) DopAmide 25 (mg/kg) (H) DopAmide 50 (mg/kg) (B) Net rotational (300 min CCW‐CW) of DopAmide and L‐DOPA vehicle, vehicle, L‐DOPA, or DopAmide induced rotation asymmetry test 35 days after 6‐OH‐dopamine lesion. Total rotations CCW‐CW. Data are presented as mean ± SEM; groups as in (A) (C) vehicle, or DopAmide induced rotation asymmetry test 35 days after 6‐OHDA lesioning. The 300‐min time curves (rotations/5 min) for (A) Veh + Veh (B) carbidopa + Veh (F) DopAmide 10 (mg/kg) (G) DopAmide 25 (mg/kg) and (H) DopAmide 50 (mg/kg) (D) vehicle, or L‐DOPA induced rotation asymmetry test 35 days after 6‐OHDA lesioning. The 300‐min time curves (rotations/5 min) for (A) Veh + Veh (B) carbidopa + Veh (C) L‐DOPA (10 mg/kg) (D) L‐DOPA 25 (mg/kg) and (E) L‐DOPA 50 (mg/kg) (E) Net rotational activity/5 min during 300 min of DopAmide (50 mg/kg) induced rotation asymmetry test 35 days after 6‐OHDA lesioning. The rate of rotation is indicated by the straight line. The horizontal slope indicates a steady rate of rotations, depicting a steady level of dopaminergic activity in the brain (F) Net rotational activity/5 min during 300 min of L‐DOPA (50 mg/kg) induced rotation asymmetry test 35 days after 6‐OHDA lesioning. The steep slope (straight line) indicates a decrease in the rate of rotation, depicting a decline in dopaminergic activity in the brain. Vehicle + vehicle, carbidopa + vehicle induced rotation are shown (See [A]).
Most prominent were the differences in the rotation kinetics induced by DopAmide and l‐DOPA (Figure 2E–F). The slopes depicted by the straight lines illustrate the rates of rotations. Animals treated with 50 mg/kg DopAmide showed a maximal rotation rate of 60‐rotations/5 min, which initially decreased slightly and was then maintained at a steady rate of 35‐rotations/5 min over the course of 3 h (horizontal line, Figure 2E). The rate of rotations then gradually declined, reaching 25‐rotations/5 min about 255 min (4.2 h) after treatment. At the same dose of l‐DOPA, there was a continual gradual decay in rotational rate, from a maximum of 43‐rotations/5 min to 25‐rotations/5 min at 120 min (2.0 h) (Figure 2F). At the lower dose of 25 mg/kg, as well, DopAmide‐treated rats maintained rotational activity at a steadier rate than rats treated with l‐DOPA (Figure 2A).
These results strongly confirm targeting of DopAmide to dopaminergic neurons in the brain. The hydrolysis step of DopAmide to l‐DOPA, which is required for decarboxylation by AADC, appears to slow DA production. It would be interesting to explore whether the aromatic L‐type amino acid transporter also requires DopAmide hydrolysis to enable transport into the cell 30. Most probably DopAmide is hydrolyzed at the periphery, inferred by the PK studies. However, future studies should confirm whether a small percentage of DopAmide does cross the Blood‐Brain‐Barrier.
In summary, l‐DOPA levels in the plasma were maintained for longer periods after treatment with DopAmide than after treatment with l‐DOPA. DopAmide had a slightly larger maximal effect than l‐DOPA on rotational behavior. More importantly, rotations induced by DopAmide were sustained at a higher rate and extended over a much longer period than rotations induced by l‐DOPA.
We therefore suggest that the slow DA production and the steady level of DA as demonstrated by the dopamide‐induced rotations of the 6‐OHDA‐lesioned rats could downregulate excessive activation of dopaminergic signaling.
Conclusions
We have shown that a novel l‐DOPA derivative, DopAmide, which must be hydrolyzed prior to decarboxylation by AAAD, slows DA production in the brain, demonstrating a high and steady level compared with l‐DOPA. The addition of a rate‐limiting hydrolysis step led to a 2‐fold increase in the half‐life of l‐DOPA in the plasma following dosage with DopAmide, as compared to the administration of l‐DOPA. The higher DA activity in the brain is indicated by a 35% increase in total rotations in the DopAmide‐6‐OHDA‐lesioned rats, compared with l‐DOPA ‐treated animals. These data strongly show effective DA production over a long period of time, compared with l‐DOPA.
Similar to l‐DOPA, DopAmide demonstrates dopaminergic activity sufficient to support antiparkinsonian action. In contrast with l‐DOPA, we suggest that DopAmide could reduce fluctuations by keeping steady level of DA production. Hence, the slower, steadier kinetics of DA production in the brain could benefit patients with PD by reducing motor fluctuations and LID. Furthermore, the need for less frequent dosage with DopAmide that acts as l‐DOPA precursor could reduce neuronal degeneration, which is associated with higher doses of l‐DOPA treatment. Finally, DopAmide is soluble in water, unlike l‐DOPA, which should improve bioavailability and allow rescue therapy of PD.
Conflict of Interest
The study was supported by a “Proof of concept”‐Grant by “Yissum”, The Research & Development Company of The Hebrew University of Jerusalem Ltd. The authors declare no conflict of interest.
Acknowledgments
The author thanks Dr. Maria and Dr. Norton Neff for performing the AAAD assay, Iris Ben‐David for helping in DopAmide synthesis, Katia Lejnev for the graphic work, and Dr. Shoshana Klein for editing the manuscript. “Yissum”, The Research Development Company of The Hebrew University of Jerusalem, and Teva Pharmaceutical Industry, Ltd, for supporting the study.
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