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Systemic and brain bioavailabilities of d -Phenylglycine-l -Dopa, a sustained dopamine-releasing prodrug
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Systemic and brain bioavailabilities of D-Phenylglycine-LDopa, a sustained dopamine-releasing prodrug Chun-Li Wang a, Yang-Bin Fan b, Shi-En Lee b, Jang-Feng Lian a, Jing-Ping Liou a, Hui-Po Wang a,* a b
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
article info
abstract
Article history:
D-Phenylglycine-L-Dopa
Received 18 October 2012
dopamine, has been proven to have 31-fold higher oral bioavailability than L-Dopa in rats.
Received in revised form
To further investigate if this dipeptide enters the brain, a single-dose pharmacokinetic
(D-PhG-L-Dopa), designed as a dipeptide mimetic prodrug of
26 February 2013
study by i.v. administration, in comparison with L-Dopa, was conducted to monitor brain
Accepted 5 March 2013
availability. The dopamine level in the brain was also determined. The results indicated
Available online 1 June 2013
that both D-PhG-L-Dopa and L-Dopa entered the brain rapidly (Tmax 1 minute) with similar
Keywords:
systemic exposure than L-Dopa (AUCplasma 23.79 mmol*min/mL vs. 12.09 mmol*min/mL), it
Anti-Parkinsonism activity
thus had higher brain availability (AUCbrain 2.0 mmol*min/mL vs. 0.92 mmol*min/mL).
Brain homogenate
Although the AUCbrain
CNS bioavailability
Dopa treatment, it however exhibited higher anti-Parkinsonism activity than
D-Phenylglycine-L-Dopa
(reduction in rotation number 47.9 5.5% vs. 27.3 4.8%). Higher brain dopamine residual
degrees of penetration (AUCbrain/AUCplasma 8.83% vs. 7.61%). As D-PhG-L-Dopa had higher
dopamine
after D-PhG-L-Dopa treatment was only 24% of that from LL-Dopa
properties of D-PhG-L-Dopa treatment compared to L-Dopa treatment, namely 3.2 times
PepT1
longer MRTbrain dopamine (172.15 vs. 53.78 minutes), 10 times longer Tmax brain dopamine (30 vs. 3 minutes) and 8.36 times longer half-life (T1=2
Tmax
to
end
ðminÞ 112.51 vs. 13.46 minutes),
may explain the result. Moreover, the long terminal half-life of brain dopamine upon D-PhG-L-Dopa L-Dopa
treatment indicated its slow dopamine-releasing property compared with
treatment (112.51 vs. 44.85 minutes). It is also important to note that brain dopa-
mine level was better controlled in the D-PhG-L-Dopa group than in the L-Dopa group (Cmax/ Cmin 1.62 vs. 9.85). In conclusion, this study demonstrated that D-PhG-L-Dopa is an intrinsic dopamine-sustained-releasing prodrug. The limited concentration fluctuation of released dopamine in the brain is beneficial for the clinical management of Parkinson’s disease. Copyright ª 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.
1. L-Dopa
Introduction
(Fig. 1), used for treating Parkinson’s disease, has a wide range of inter- and intra-individual variations in the
rate and extent of absorption [1e7]. The variation is primarily from foodedrug interactions in intestinal absorption via amino acid transporters [8e10]. Many studies have been undertaken to overcome the unsatisfactory clinical outcome
* Corresponding author. School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 11031, Taiwan, ROC. E-mail address: [email protected] (H.-P. Wang). 1021-9498/$ e see front matter Copyright ª 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jfda.2013.05.001
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Fig. 1 e Structures of dopamine, L-Dopa and D-Phenylglycine-L-Dopa.
[6,11e15]. A rational approach in improving the oral bioavailability of L-Dopa is thus to prevent the drug from competing with amino acids for absorption via intestinal amino acid transporters [15]. We reported the discovery of D-Phenylglycine-L-Dopa (D-PhG-L-Dopa; Fig. 1) using D-Phenylglycine as a delivery tool for guiding L-Dopa to transport via intestinal oligopeptide transporter (PepT1). The dipeptide showed an oral absorption that was 32-fold higher than that of L-Dopa. The higher antiParkinsonism activity of this dipeptide compared to that of L-Dopa might come from its improved systemic bioavailability after oral administration [16]. To investigate if D-PhG-L-Dopa enters the brain, pharmacokinetic studies for determining the systemic and CNS bioavailability of this dipeptide, compared with L-Dopa, were conducted in rats. Brain availability of dopamine upon i.v. administration of D-PhG-L-Dopa and L-Dopa were determined. Pharmacokinetic properties of both prodrugs and in vivo antiParkinsonism activity were also analyzed.
2.2.
Pharmacokinetic studies
2.2.1.
Animal experiments
Male Wistar rats (200e250 g) were fasted for at least 18 hours prior to the study. The rats were put under a heating lamp to maintain their body temperatures at 37 C throughout the experiment. A single dose of D-PhG-L-Dopa (100 mg/kg equivalent to 0.3 mmol/kg) or L-Dopa (59.74 mg/kg equivalent to 0.3 mmol/kg) was dissolved in 2.5 mL of normal saline and administered i.v. into the tail vein. Blood samples were withdrawn from the carotid artery at time intervals of 1, 3, 5, 10, 15, 30, 45, 60 and 90 minutes before the rats were sacrificed for the determination of systemic bioavailability. The whole brain was harvested for the determination of CNS bioavailability. All procedures involving the use of animals were in compliance with the guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of Taipei Medical University.
2.2.2.
2.
Methods
2.1.
Materials
D-PhG-L-Dopa was synthesized in our laboratory [16]. Analytical grade chemicals for biological studies were from Sigma-Aldrich (St. Louis, MO, USA), E. Merck KG (Darmstadt, Germany), Fluka Chemika (Buchs, Switzerland), Acros (Morris Plains, NJ, USA) and Wako (Richmond, VA, USA). Acidwashed alumina was purchased from RiedaL-de Haen Company (Spring Valley, CA, USA). High-performance liquid chromatography (HPLC) grade acetonitrile and methanol were purchased from Alpus Pharmaceutical Industries Co. (Gifu, Japan). Branson Sonifier 450 sonicator (Danbury, CT, USA), Kubota 2010 (Tokyo, Japan), Eppendorf AG 5415C centrifuge (Hamburg, Germany), Model 905 incubator and Ystral Laboratory series 10/20 homogenizer (BallrechtenDottingen, Germany) were used in the preparation of biological samples. Male Wistar rats (200e250 g) for pharmacokinetic studies and rotational behavior studies were purchased from the Laboratory Animal Center of National Taiwan University (Taipei, Taiwan). The animals were pathogen-free and allowed to acclimate to the environmentally-controlled quarters (24 1 C and 12:12 hour lightedark cycle) for at least 5 days before the experiments. The animal studies were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Analytical sample preparation
Heparin sodium (25 IU/mL in 0.3 mL of saline) was added to the blood samples and centrifuged (5585g) at 4 C for 8 minutes. The plasma was frozen immediately and kept at e78 C until they were analyzed, whereupon 200 mL of the plasma sample in a 10-mL test tube was mixed with 500 mL of 1 M Tris-HCl buffer (adjusted to pH 8.6 by adding disodium EDTA), 10 mL of dihydroxybenzylamine (DHBA, 2 mg/mL, as internal standard) and 100 mg of alumina. The tube was shaken for 15 seconds and the supernatant was decanted. The alumina was washed four times with 5 mL of water. The compounds adsorbed on the alumina were eluted with 200 mL of acid buffer comprising 0.9 mL of glacial acetic acid in 4 mL of 1.0 M phosphate buffer; 30 mL of the solution was then analyzed with HPLC. The isolated brain was immersed in Ringer’s solution, cleaned and homogenized. After centrifugation (at 5585g and 4 C for 8 minutes), the clear supernatant from the brain homogenate was subjected to alumina extraction using the same procedure as that used for preparing the plasma sample.
2.2.3. Chromatography 2.2.3.1. Assay methods. Assays were performed using a HPLC system with an ion exchange column coupled with an electrochemical detector. The HPLC system consisted of an autosampler (AS950, Jasco, Tokyo, Japan), a Waters Model 600E solvent delivery pump (Millipore, Milford, MA, USA), a Model LC-4C electrochemical detector with a glassy-carbon electrode (Bioanalytical Systems, Inc., West Lafayette, IN, USA), and an integrator (Macintosh LC II with Macintegrator I). A Nucleosil 10 SA cationic ion-exchange column (10 mm, 300 4.0 mm; Macherey-Nagel, Du¨ren, Germany) with a mobile phase
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comprising NaCl (50 mM) and Na2-EDTA (1 mM) in 0.1 M phosphate buffer (pH 2.0) at a flow rate of 2.0 mL/min was used for the analysis of D-PhG-L-Dopa and L-Dopa. A C18 reversed phase microbore column (particle size 5 mm, 150 1 mm I.D.; Bioanalytical Systems, West Lafayette, IN, USA) was used for the analysis of dopamine in brain homogenate. The eluents were filtered through a Millipore 0.22-mm filter and degassed prior to analysis. The flow rate was set at 0.05 mL/min.
2.2.3.2. Validation of assay methods. The lower limit of detection (LOD) and limit of quantification (LOQ) of D-PhG-LDopa and L-Dopa were determined. Assay methods were validated by determining the precision and accuracy of intraday and inter-day analyses of serum standards over a period of 6 days. The coefficients of variation for inter- and intra-day assays were less than 18%. 2.2.3.3. Data treatment. Pharmacokinetic parameters (Cmax, Tmax, AUC, AUMC, elimination rate constant, Vdss, T1/2) were calculated using the logelinear trapezoidal rule. Plasma concentrations after i.v. administration of drugs were calculated using WINNONLIN software by non-compartment model. Data analyses were performed on Microsoft Excel and represented as mean SD for n experiments. Treatment differences were evaluated by paired t-test. 2.2.4.
Rotational behavior of rats [17e20]
Male Wistar rats (180e200 g) were anesthetized with pentobarbital sodium (30 mg/kg body weight, i.p.) and the heads were fixed in a David-Kopf steric taxic frame. A solution of 6hydroxydopamine (6-OHDA, 2.0 mg/mL 8 mL) in saline was infused using Paxinos and Watson coordinates (AP 5.3, L 2.0, H 7.8 mm [17]) into the unilateral substantia nigra compacta (SNc) of the brain with a syringe pump through a 30-gauge stainless steel needle at a flow rate of 2 mL/min. After 2 weeks’ recovery period, the 6-OHDA treated rats were placed in a spherical bowl (radius 20 cm) and secured by a thoracic harness which was connected to a 486 PC computer for automatic recording of rotation induced by (þ)-methamphetamine (MA; from SigmaAldrich, St. Louis, MO, USA). The rotational behavior of rats was recorded 10 minutes after MA treatment (MA in saline, 4 mg/kg body weight of rat, s.c.). The number of turns recorded was defined as the control value (T0) for each individual animal. Only animals showing a T0 greater than 400 were chosen for further experiments. After 2 weeks’ wash-out period, the animals were subjected to drug treatment. A single dose (0.051 mmol/kg) of each test compound was administered i.p. to rats 5 minutes prior to MA treatment (4.0 mg/kg body weight, s.c.). The rotation counted for a period of 110 minutes starting 10 minutes after MA treatment was recorded as Td for each tested rat. The percentage of reduction in rotation for each animal was calculated and presented as (Td T0)/T0 100%.
3.
Results
3.1.
Chromatography and validation of assay methods
The HPLC chromatogram for the analysis of D-PhG-L-Dopa, L-Dopa and dopamine is depicted in Fig. 2. The LODs of
Fig. 2 e Chromatogram of D-PhG-L-Dopa, L-Dopa, internal standard dihydroxybenzylamine (DHBA) and dopamine in brain sample.
D-PhG-L-Dopa in plasma samples and brain homogenates were 0.1 mg/mL and 0.25 mg/mL, respectively. The LOQs were in the range of 0.5 mg/mL and 12.5 mg/mL for plasma samples (R2 ¼ 0.9999 intra-day; R2 ¼ 0.9947 inter-day) and brain homogenates (R2 ¼ 0.9973 intra-day; R2 ¼ 0.9999 inter-day), respectively. The LOD of L-Dopa was 0.05 mg/mL in plasma samples and brain homogenates. LOQ was in the range of 0.05 m/mL for plasma samples (R2 ¼ 0.9994 intra-day; R2 ¼ 0.9999 inter-day) and 2.5 mg/mL for brain homogenates (R2 ¼ 0.9996 intra-day; R2 ¼ 0.9987 inter-day). The LOQ values of dopamine for brain homogenates were in the range of 0.05 mg/mL and 1.25 mg/mL (R2 ¼ 0.9950 intra-day; R2 ¼ 0.9990 inter-day), respectively. The coefficients of variation of all assays were less than 18%.
3.2. Systemic and brain concentrationetime profile upon i.v. administration of D-PhG-L-Dopa The plasma and brain concentrationetime profile of D-PhG-LDopa after i.v. administration (50 mg/kg) is depicted in Fig. 3.
Fig. 3 e The concentration-time curves of D-PhG-L-Dopa in rat blood and brain after i.v. administration (50 mg/kg). The values represent the group mean ± SD (n [ 4).
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Fig. 4 e Concentrationetime curve of (A) plasma and (B) brain homogenate after i.v. administration of D-PhG-L-Dopa (n [ 6) or L-Dopa (n [ 4) in rats.
3.3. Comparison of CNS bioavailability of D-PhG-L-Dopa and L-Dopa Equal molar (0.3 mmol/kg) of D-PhG-L-Dopa or L-Dopa was administered to the rats by i.v. injection. For comparison, the concentrationetime curves in plasma (Fig. 4A) and in brain homogenate (Fig. 4B) are depicted. The dopamine profile in brain homogenate upon administration of D-PhG-L-Dopa or LDopa is depicted in Fig. 5. The pharmacokinetic parameters of D-PhG-L-Dopa and LDopa are summarized in Table 1. AUCbrain/AUCplasma was calculated as an indicator of the portion of prodrugs that penetrated into the brain. As time factor is important for the biotransformation of the prodrugs to parent dopamine, AUMCbrain/AUMCplasma of the prodrugs was also calculated for comparison. The key pharmacokinetic parameters of dopamine in brain upon i.v. administration of D-PhG-L-Dopa and L-Dopa are summarized in Table 2. The CNS bioavailability of dopamine released from both prodrugs was compared. MRTbrain dopamine, T1/2 and Tmax are also listed for the comparison of dopaminereleasing properties.
3.4.
model measured in 6-OHDA-treated unilateral striatallesioned rats in which rotations were elicited with (þ)-MA. The reduction in rotational counts was used as an indicator of Parkinsonism activity. Both D-PhG-L-Dopa and L-Dopa demonstrated inhibition of MA-induced rotation of rats. With equal molars of test compound administered, the activity of DPhG-L-Dopa in reducing the rotation of rats was significantly higher than that of L-Dopa ( p < 0.05, Table 3).
4.
Discussion
This study aimed to compare the comparative systemic and brain bioavailability of D-PhG-L-Dopa and L-Dopa, the degree of their penetration through the bloodebrain barrier, and dopamine in the brain. Systemic exposures upon i.v. injection of the prodrugs were investigated first. D-PhG-L-Dopa demonstrated 1.97-fold higher systemic bioavailability than L-Dopa (AUCplasma 23.79 mmol*min/mL vs. 12.09 mmol*min/mL), probably due to its lower clearance rate and larger volume of distribution. The biological half-life of this dipeptide was longer
Anti-Parkinsonism effect in rats
The in vivo anti-Parkinsonism effect was determined, in crossover design experiments, with a conventional rotation
Table 1 e Comparison of pharmacokinetic parameters of D-PhG-L-Dopa and L-Dopa.
AUCbrain (mmol*min/mL) (a) AUCplasma (mmol*min/mL) (b) AUCbrain/AUCplasma (a/b) AUMCbrain (mmol*min2/mL) (c) AUMCplasma (mmol*min2/mL) (d) AUMCbrain/AUMCplasma (c/d) Tmax, brain (min) Cmax, plasma (mmol/mL) Cmax, brain (mmol/mL) T1/2, plasma (min) MRTplasma (min) CLp, plasma (L/kg/min) Vdssplasma (L/kg)
Fig. 5 e Brain dopamine after i.v. administration of D-PhG-LDopa (n [ 6) or L-Dopa (n [ 4).
D-PhG-L-Dopa
L-Dopa
(I) (n ¼ 6)
(II) (n ¼ 4)
2.10 23.79 8.83% 85.22 454.72 18.74% 1.0 3.34 0.12 13.25 19.11 0.013 0.243
0.92 12.09 7.61% 38.06 190.21 20.01% 1.0 1.88 0.02 10.90 15.73 0.042 0.066
Ratio (I/II) 2.28 1.97 1.16 2.26 2.39 0.94 d 1.78 5.90 1.22 1.21 0.31 3.68
AUCbrain/AUCplasma (a/b) ¼ the fraction of prodrug that penetrated into the brain.
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Table 2 e Pharmacokinetic parameters of brain dopamine after i.v. administration of D-PhG-L-Dopa and L-Dopa. From Ratio From D-PhGL-Dopa L-Dopa (I/II) (n ¼ 6) (I) (n ¼ 4) (II) AUCbrain dopamine (mmol*min/mL) AUMCbrain dopamine (mmol*min2/mL) MRT (min) Cmax (mmol/mL) Cmin (mmol/mL) Cmax/Cmin Tmax (min) T1=2 Tmax to end ðminÞ T1/2, 30 min to end (min)
0.42 32.77
1.75 64.28
0.24 0.51
172.15 2.32 1.43 1.62 30.0 112.51 112.51
53.78 60.58 6.15 9.85 3.0 13.46 44.85
3.20 0.38 0.23 0.16 10.0 8.36 2.51
than that of L-Dopa (13.25 minutes vs. 10.90 minutes). As a consequence, the AUMCplasma of the D-PhG-L-Dopa dipeptide was 2.39 times higher than that of L-Dopa (454.72 mmol*min2/ mL vs. 190.21 mmol*min2/mL) (Table 1). Both D-PhG-L-Dopa and L-Dopa penetrated to the brain rapidly (Tmax brain 1 minute). The AUCbrain of D-PhG-L-Dopa was 2.28-fold larger than that of L-Dopa (2.10 mmol*min/mL vs. 0.92 mmol*min/mL). The Cmax-brain of D-PhG-L-Dopa was 5.90fold higher than that of L-Dopa (0.12 mmol/mL vs. 0.02 mmol/ mL). As the fractions of systemic D-PhG-L-Dopa and L-Dopa that penetrated to the brain (AUCbrain/AUCplasma) were similar (8.83% vs. 7.61%), the higher AUCbrain and Cmax-brain of D-PhG-LDopa compared to that of L-Dopa might come from its higher systemic exposure (Table 1). As brain dopamine exposures were compared, AUCbrain-dopamine released from D-PhG-L-Dopa was only 24% of that released from L-Dopa (0.42 mmol*min/mL vs. 1.75 mmol*min/mL) (Table 2). However, the mean residual time (MRTbrain dopamine) of the dipeptide was 3.20 times longer than that of L-Dopa (172.15 minutes vs. 53.78 minutes). It also exhibited a 10 times longer Tmax brain dopamine (30.0 minutes vs. 3.0 minutes) and 8.36 times longer T1=2 Tmax to end (112.51 minutes vs. 13.46 minutes) than L-Dopa. The terminal half-life (T1/2 30 min to end) of brain dopamine upon D-PhG-L-Dopa administration was 2.51 times longer than that upon L-Dopa administration (112.51 minutes vs. 44.85 minutes). This supported our design of this dipeptide as a dopamine prodrug for preventing or prolonging the fast decarboxylation
Table 3 e Equal molars of D-PhG-L-Dopa and L-Dopa were administered i.p. and anti-Parkinsonism activities were determined by rotational behavior assay in crossover experiments. Compound
D-PhG-L-Dopa L-Dopa
No. of Dose (mg/kg)a experiments 16.7 10.0
10 8
Reduction Paired in rotation t-test (%, mean SD) 47.9 5.5 27.3 4.8
p < 0.05 Control
a The dose used in the experiments was 0.051 mmol/kg for each compound.
Fig. 6 e Proposed biological transformation of D-PhG-LDopa and L-Dopa prodrugs to dopamine.
commonly encountered with L-Dopa administration (Fig. 6). The pharmacokinetic profile of brain dopamine also confirmed the finding from our previous study that oral administration of D-PhG-L-Dopa led to an intrinsic sustained dopamine-releasing profile in rats [16]. Although brain dopamine exposure (AUCbrain dopamine) on i.v. administration of D-PhG-L-Dopa was less than with L-Dopa, this dipeptide demonstrated higher activity in reducing the rotation of Parkinsonism rats (47.9 5.5% vs. 27.3 4.8%, p < 0.001, i.p.). The sustained dopamine-releasing might explain its effective anti-Parkinsonism activity. Reports have indicated that pulsatile administration of dopamine agonist induced dopamine-related dyskinesia, and repeated administration will result in wearing-off phenomenon which is generally observed in chronic Parkinsonism patients [13,21,22]. Long-term use of L-Dopa may lead to a decreased therapeutic window for individual patients; therefore, monitoring of blood L-Dopa level is important in the clinical management of Parkinson’s disease. Various approaches including new formulation and new molecular entity design were thus undertaken to achieve sustained-dopamine releasing properties which maintained a stable concentration profile of dopamine [15,23]. In our case, although brain dopamine (Cmax brain dopamine) on D-PhG-L-Dopa administration was much lower than from L-Dopa administration (2.32 nmol/mL vs. 60.58 nmol/mL), the concentration range (Cmax brain dopamine/ Cmin brain dopamine) was six times narrower (1.62 vs. 9.85), which greatly reduced the risk from fluctuations in concentration.
5.
Conclusion
The CNS bioavailability of D-PhG-L-Dopa and L-Dopa, and the brain dopamine level after i.v. injection of both compounds were investigated. D-PhG-L-Dopa exhibited lower CNS dopamine exposure than L-Dopa. However, it exhibited an intrinsic sustained dopamine-releasing profile with longer CNS dopamine residual properties and more stable dopamine concentration profile. The sustained dopamine-releasing property of D-PhG-L-Dopa might be beneficial for clinical management, indicating its usefulness as a potential agent for treating Parkinson’s disease.
j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 1 ( 2 0 1 3 ) 1 3 6 e1 4 1
Acknowledgments This work was supported by the National Science Council and the Ministry of Economics of the Republic of China (grant numbers NSC97-2323-B-038-002 and MOE100-EC-17-A20-S1-196). Professor Kwan-Yang Hsu of Taipei Medical University is appreciated for the discussion on pharmacokinetic data.
references
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Systemic and brain bioavailabilities of D-Phenylglycine-LDopa, a sustained dopamine-releasing prodrug Chun-Li Wang a, Yang-Bin Fan b, Shi-En Lee b, Jang-Feng Lian a, Jing-Ping Liou a, Hui-Po Wang a,* a b
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC
article info
abstract
Article history:
D-Phenylglycine-L-Dopa
Received 18 October 2012
dopamine, has been proven to have 31-fold higher oral bioavailability than L-Dopa in rats.
Received in revised form
To further investigate if this dipeptide enters the brain, a single-dose pharmacokinetic
(D-PhG-L-Dopa), designed as a dipeptide mimetic prodrug of
26 February 2013
study by i.v. administration, in comparison with L-Dopa, was conducted to monitor brain
Accepted 5 March 2013
availability. The dopamine level in the brain was also determined. The results indicated
Available online 1 June 2013
that both D-PhG-L-Dopa and L-Dopa entered the brain rapidly (Tmax 1 minute) with similar
Keywords:
systemic exposure than L-Dopa (AUCplasma 23.79 mmol*min/mL vs. 12.09 mmol*min/mL), it
Anti-Parkinsonism activity
thus had higher brain availability (AUCbrain 2.0 mmol*min/mL vs. 0.92 mmol*min/mL).
Brain homogenate
Although the AUCbrain
CNS bioavailability
Dopa treatment, it however exhibited higher anti-Parkinsonism activity than
D-Phenylglycine-L-Dopa
(reduction in rotation number 47.9 5.5% vs. 27.3 4.8%). Higher brain dopamine residual
degrees of penetration (AUCbrain/AUCplasma 8.83% vs. 7.61%). As D-PhG-L-Dopa had higher
dopamine
after D-PhG-L-Dopa treatment was only 24% of that from LL-Dopa
properties of D-PhG-L-Dopa treatment compared to L-Dopa treatment, namely 3.2 times
PepT1
longer MRTbrain dopamine (172.15 vs. 53.78 minutes), 10 times longer Tmax brain dopamine (30 vs. 3 minutes) and 8.36 times longer half-life (T1=2
Tmax
to
end
ðminÞ 112.51 vs. 13.46 minutes),
may explain the result. Moreover, the long terminal half-life of brain dopamine upon D-PhG-L-Dopa L-Dopa
treatment indicated its slow dopamine-releasing property compared with
treatment (112.51 vs. 44.85 minutes). It is also important to note that brain dopa-
mine level was better controlled in the D-PhG-L-Dopa group than in the L-Dopa group (Cmax/ Cmin 1.62 vs. 9.85). In conclusion, this study demonstrated that D-PhG-L-Dopa is an intrinsic dopamine-sustained-releasing prodrug. The limited concentration fluctuation of released dopamine in the brain is beneficial for the clinical management of Parkinson’s disease. Copyright ª 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.
1. L-Dopa
Introduction
(Fig. 1), used for treating Parkinson’s disease, has a wide range of inter- and intra-individual variations in the
rate and extent of absorption [1e7]. The variation is primarily from foodedrug interactions in intestinal absorption via amino acid transporters [8e10]. Many studies have been undertaken to overcome the unsatisfactory clinical outcome
* Corresponding author. School of Pharmacy, College of Pharmacy, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 11031, Taiwan, ROC. E-mail address: [email protected] (H.-P. Wang). 1021-9498/$ e see front matter Copyright ª 2013, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jfda.2013.05.001
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Fig. 1 e Structures of dopamine, L-Dopa and D-Phenylglycine-L-Dopa.
[6,11e15]. A rational approach in improving the oral bioavailability of L-Dopa is thus to prevent the drug from competing with amino acids for absorption via intestinal amino acid transporters [15]. We reported the discovery of D-Phenylglycine-L-Dopa (D-PhG-L-Dopa; Fig. 1) using D-Phenylglycine as a delivery tool for guiding L-Dopa to transport via intestinal oligopeptide transporter (PepT1). The dipeptide showed an oral absorption that was 32-fold higher than that of L-Dopa. The higher antiParkinsonism activity of this dipeptide compared to that of L-Dopa might come from its improved systemic bioavailability after oral administration [16]. To investigate if D-PhG-L-Dopa enters the brain, pharmacokinetic studies for determining the systemic and CNS bioavailability of this dipeptide, compared with L-Dopa, were conducted in rats. Brain availability of dopamine upon i.v. administration of D-PhG-L-Dopa and L-Dopa were determined. Pharmacokinetic properties of both prodrugs and in vivo antiParkinsonism activity were also analyzed.
2.2.
Pharmacokinetic studies
2.2.1.
Animal experiments
Male Wistar rats (200e250 g) were fasted for at least 18 hours prior to the study. The rats were put under a heating lamp to maintain their body temperatures at 37 C throughout the experiment. A single dose of D-PhG-L-Dopa (100 mg/kg equivalent to 0.3 mmol/kg) or L-Dopa (59.74 mg/kg equivalent to 0.3 mmol/kg) was dissolved in 2.5 mL of normal saline and administered i.v. into the tail vein. Blood samples were withdrawn from the carotid artery at time intervals of 1, 3, 5, 10, 15, 30, 45, 60 and 90 minutes before the rats were sacrificed for the determination of systemic bioavailability. The whole brain was harvested for the determination of CNS bioavailability. All procedures involving the use of animals were in compliance with the guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of Taipei Medical University.
2.2.2.
2.
Methods
2.1.
Materials
D-PhG-L-Dopa was synthesized in our laboratory [16]. Analytical grade chemicals for biological studies were from Sigma-Aldrich (St. Louis, MO, USA), E. Merck KG (Darmstadt, Germany), Fluka Chemika (Buchs, Switzerland), Acros (Morris Plains, NJ, USA) and Wako (Richmond, VA, USA). Acidwashed alumina was purchased from RiedaL-de Haen Company (Spring Valley, CA, USA). High-performance liquid chromatography (HPLC) grade acetonitrile and methanol were purchased from Alpus Pharmaceutical Industries Co. (Gifu, Japan). Branson Sonifier 450 sonicator (Danbury, CT, USA), Kubota 2010 (Tokyo, Japan), Eppendorf AG 5415C centrifuge (Hamburg, Germany), Model 905 incubator and Ystral Laboratory series 10/20 homogenizer (BallrechtenDottingen, Germany) were used in the preparation of biological samples. Male Wistar rats (200e250 g) for pharmacokinetic studies and rotational behavior studies were purchased from the Laboratory Animal Center of National Taiwan University (Taipei, Taiwan). The animals were pathogen-free and allowed to acclimate to the environmentally-controlled quarters (24 1 C and 12:12 hour lightedark cycle) for at least 5 days before the experiments. The animal studies were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Analytical sample preparation
Heparin sodium (25 IU/mL in 0.3 mL of saline) was added to the blood samples and centrifuged (5585g) at 4 C for 8 minutes. The plasma was frozen immediately and kept at e78 C until they were analyzed, whereupon 200 mL of the plasma sample in a 10-mL test tube was mixed with 500 mL of 1 M Tris-HCl buffer (adjusted to pH 8.6 by adding disodium EDTA), 10 mL of dihydroxybenzylamine (DHBA, 2 mg/mL, as internal standard) and 100 mg of alumina. The tube was shaken for 15 seconds and the supernatant was decanted. The alumina was washed four times with 5 mL of water. The compounds adsorbed on the alumina were eluted with 200 mL of acid buffer comprising 0.9 mL of glacial acetic acid in 4 mL of 1.0 M phosphate buffer; 30 mL of the solution was then analyzed with HPLC. The isolated brain was immersed in Ringer’s solution, cleaned and homogenized. After centrifugation (at 5585g and 4 C for 8 minutes), the clear supernatant from the brain homogenate was subjected to alumina extraction using the same procedure as that used for preparing the plasma sample.
2.2.3. Chromatography 2.2.3.1. Assay methods. Assays were performed using a HPLC system with an ion exchange column coupled with an electrochemical detector. The HPLC system consisted of an autosampler (AS950, Jasco, Tokyo, Japan), a Waters Model 600E solvent delivery pump (Millipore, Milford, MA, USA), a Model LC-4C electrochemical detector with a glassy-carbon electrode (Bioanalytical Systems, Inc., West Lafayette, IN, USA), and an integrator (Macintosh LC II with Macintegrator I). A Nucleosil 10 SA cationic ion-exchange column (10 mm, 300 4.0 mm; Macherey-Nagel, Du¨ren, Germany) with a mobile phase
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comprising NaCl (50 mM) and Na2-EDTA (1 mM) in 0.1 M phosphate buffer (pH 2.0) at a flow rate of 2.0 mL/min was used for the analysis of D-PhG-L-Dopa and L-Dopa. A C18 reversed phase microbore column (particle size 5 mm, 150 1 mm I.D.; Bioanalytical Systems, West Lafayette, IN, USA) was used for the analysis of dopamine in brain homogenate. The eluents were filtered through a Millipore 0.22-mm filter and degassed prior to analysis. The flow rate was set at 0.05 mL/min.
2.2.3.2. Validation of assay methods. The lower limit of detection (LOD) and limit of quantification (LOQ) of D-PhG-LDopa and L-Dopa were determined. Assay methods were validated by determining the precision and accuracy of intraday and inter-day analyses of serum standards over a period of 6 days. The coefficients of variation for inter- and intra-day assays were less than 18%. 2.2.3.3. Data treatment. Pharmacokinetic parameters (Cmax, Tmax, AUC, AUMC, elimination rate constant, Vdss, T1/2) were calculated using the logelinear trapezoidal rule. Plasma concentrations after i.v. administration of drugs were calculated using WINNONLIN software by non-compartment model. Data analyses were performed on Microsoft Excel and represented as mean SD for n experiments. Treatment differences were evaluated by paired t-test. 2.2.4.
Rotational behavior of rats [17e20]
Male Wistar rats (180e200 g) were anesthetized with pentobarbital sodium (30 mg/kg body weight, i.p.) and the heads were fixed in a David-Kopf steric taxic frame. A solution of 6hydroxydopamine (6-OHDA, 2.0 mg/mL 8 mL) in saline was infused using Paxinos and Watson coordinates (AP 5.3, L 2.0, H 7.8 mm [17]) into the unilateral substantia nigra compacta (SNc) of the brain with a syringe pump through a 30-gauge stainless steel needle at a flow rate of 2 mL/min. After 2 weeks’ recovery period, the 6-OHDA treated rats were placed in a spherical bowl (radius 20 cm) and secured by a thoracic harness which was connected to a 486 PC computer for automatic recording of rotation induced by (þ)-methamphetamine (MA; from SigmaAldrich, St. Louis, MO, USA). The rotational behavior of rats was recorded 10 minutes after MA treatment (MA in saline, 4 mg/kg body weight of rat, s.c.). The number of turns recorded was defined as the control value (T0) for each individual animal. Only animals showing a T0 greater than 400 were chosen for further experiments. After 2 weeks’ wash-out period, the animals were subjected to drug treatment. A single dose (0.051 mmol/kg) of each test compound was administered i.p. to rats 5 minutes prior to MA treatment (4.0 mg/kg body weight, s.c.). The rotation counted for a period of 110 minutes starting 10 minutes after MA treatment was recorded as Td for each tested rat. The percentage of reduction in rotation for each animal was calculated and presented as (Td T0)/T0 100%.
3.
Results
3.1.
Chromatography and validation of assay methods
The HPLC chromatogram for the analysis of D-PhG-L-Dopa, L-Dopa and dopamine is depicted in Fig. 2. The LODs of
Fig. 2 e Chromatogram of D-PhG-L-Dopa, L-Dopa, internal standard dihydroxybenzylamine (DHBA) and dopamine in brain sample.
D-PhG-L-Dopa in plasma samples and brain homogenates were 0.1 mg/mL and 0.25 mg/mL, respectively. The LOQs were in the range of 0.5 mg/mL and 12.5 mg/mL for plasma samples (R2 ¼ 0.9999 intra-day; R2 ¼ 0.9947 inter-day) and brain homogenates (R2 ¼ 0.9973 intra-day; R2 ¼ 0.9999 inter-day), respectively. The LOD of L-Dopa was 0.05 mg/mL in plasma samples and brain homogenates. LOQ was in the range of 0.05 m/mL for plasma samples (R2 ¼ 0.9994 intra-day; R2 ¼ 0.9999 inter-day) and 2.5 mg/mL for brain homogenates (R2 ¼ 0.9996 intra-day; R2 ¼ 0.9987 inter-day). The LOQ values of dopamine for brain homogenates were in the range of 0.05 mg/mL and 1.25 mg/mL (R2 ¼ 0.9950 intra-day; R2 ¼ 0.9990 inter-day), respectively. The coefficients of variation of all assays were less than 18%.
3.2. Systemic and brain concentrationetime profile upon i.v. administration of D-PhG-L-Dopa The plasma and brain concentrationetime profile of D-PhG-LDopa after i.v. administration (50 mg/kg) is depicted in Fig. 3.
Fig. 3 e The concentration-time curves of D-PhG-L-Dopa in rat blood and brain after i.v. administration (50 mg/kg). The values represent the group mean ± SD (n [ 4).
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Fig. 4 e Concentrationetime curve of (A) plasma and (B) brain homogenate after i.v. administration of D-PhG-L-Dopa (n [ 6) or L-Dopa (n [ 4) in rats.
3.3. Comparison of CNS bioavailability of D-PhG-L-Dopa and L-Dopa Equal molar (0.3 mmol/kg) of D-PhG-L-Dopa or L-Dopa was administered to the rats by i.v. injection. For comparison, the concentrationetime curves in plasma (Fig. 4A) and in brain homogenate (Fig. 4B) are depicted. The dopamine profile in brain homogenate upon administration of D-PhG-L-Dopa or LDopa is depicted in Fig. 5. The pharmacokinetic parameters of D-PhG-L-Dopa and LDopa are summarized in Table 1. AUCbrain/AUCplasma was calculated as an indicator of the portion of prodrugs that penetrated into the brain. As time factor is important for the biotransformation of the prodrugs to parent dopamine, AUMCbrain/AUMCplasma of the prodrugs was also calculated for comparison. The key pharmacokinetic parameters of dopamine in brain upon i.v. administration of D-PhG-L-Dopa and L-Dopa are summarized in Table 2. The CNS bioavailability of dopamine released from both prodrugs was compared. MRTbrain dopamine, T1/2 and Tmax are also listed for the comparison of dopaminereleasing properties.
3.4.
model measured in 6-OHDA-treated unilateral striatallesioned rats in which rotations were elicited with (þ)-MA. The reduction in rotational counts was used as an indicator of Parkinsonism activity. Both D-PhG-L-Dopa and L-Dopa demonstrated inhibition of MA-induced rotation of rats. With equal molars of test compound administered, the activity of DPhG-L-Dopa in reducing the rotation of rats was significantly higher than that of L-Dopa ( p < 0.05, Table 3).
4.
Discussion
This study aimed to compare the comparative systemic and brain bioavailability of D-PhG-L-Dopa and L-Dopa, the degree of their penetration through the bloodebrain barrier, and dopamine in the brain. Systemic exposures upon i.v. injection of the prodrugs were investigated first. D-PhG-L-Dopa demonstrated 1.97-fold higher systemic bioavailability than L-Dopa (AUCplasma 23.79 mmol*min/mL vs. 12.09 mmol*min/mL), probably due to its lower clearance rate and larger volume of distribution. The biological half-life of this dipeptide was longer
Anti-Parkinsonism effect in rats
The in vivo anti-Parkinsonism effect was determined, in crossover design experiments, with a conventional rotation
Table 1 e Comparison of pharmacokinetic parameters of D-PhG-L-Dopa and L-Dopa.
AUCbrain (mmol*min/mL) (a) AUCplasma (mmol*min/mL) (b) AUCbrain/AUCplasma (a/b) AUMCbrain (mmol*min2/mL) (c) AUMCplasma (mmol*min2/mL) (d) AUMCbrain/AUMCplasma (c/d) Tmax, brain (min) Cmax, plasma (mmol/mL) Cmax, brain (mmol/mL) T1/2, plasma (min) MRTplasma (min) CLp, plasma (L/kg/min) Vdssplasma (L/kg)
Fig. 5 e Brain dopamine after i.v. administration of D-PhG-LDopa (n [ 6) or L-Dopa (n [ 4).
D-PhG-L-Dopa
L-Dopa
(I) (n ¼ 6)
(II) (n ¼ 4)
2.10 23.79 8.83% 85.22 454.72 18.74% 1.0 3.34 0.12 13.25 19.11 0.013 0.243
0.92 12.09 7.61% 38.06 190.21 20.01% 1.0 1.88 0.02 10.90 15.73 0.042 0.066
Ratio (I/II) 2.28 1.97 1.16 2.26 2.39 0.94 d 1.78 5.90 1.22 1.21 0.31 3.68
AUCbrain/AUCplasma (a/b) ¼ the fraction of prodrug that penetrated into the brain.
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Table 2 e Pharmacokinetic parameters of brain dopamine after i.v. administration of D-PhG-L-Dopa and L-Dopa. From Ratio From D-PhGL-Dopa L-Dopa (I/II) (n ¼ 6) (I) (n ¼ 4) (II) AUCbrain dopamine (mmol*min/mL) AUMCbrain dopamine (mmol*min2/mL) MRT (min) Cmax (mmol/mL) Cmin (mmol/mL) Cmax/Cmin Tmax (min) T1=2 Tmax to end ðminÞ T1/2, 30 min to end (min)
0.42 32.77
1.75 64.28
0.24 0.51
172.15 2.32 1.43 1.62 30.0 112.51 112.51
53.78 60.58 6.15 9.85 3.0 13.46 44.85
3.20 0.38 0.23 0.16 10.0 8.36 2.51
than that of L-Dopa (13.25 minutes vs. 10.90 minutes). As a consequence, the AUMCplasma of the D-PhG-L-Dopa dipeptide was 2.39 times higher than that of L-Dopa (454.72 mmol*min2/ mL vs. 190.21 mmol*min2/mL) (Table 1). Both D-PhG-L-Dopa and L-Dopa penetrated to the brain rapidly (Tmax brain 1 minute). The AUCbrain of D-PhG-L-Dopa was 2.28-fold larger than that of L-Dopa (2.10 mmol*min/mL vs. 0.92 mmol*min/mL). The Cmax-brain of D-PhG-L-Dopa was 5.90fold higher than that of L-Dopa (0.12 mmol/mL vs. 0.02 mmol/ mL). As the fractions of systemic D-PhG-L-Dopa and L-Dopa that penetrated to the brain (AUCbrain/AUCplasma) were similar (8.83% vs. 7.61%), the higher AUCbrain and Cmax-brain of D-PhG-LDopa compared to that of L-Dopa might come from its higher systemic exposure (Table 1). As brain dopamine exposures were compared, AUCbrain-dopamine released from D-PhG-L-Dopa was only 24% of that released from L-Dopa (0.42 mmol*min/mL vs. 1.75 mmol*min/mL) (Table 2). However, the mean residual time (MRTbrain dopamine) of the dipeptide was 3.20 times longer than that of L-Dopa (172.15 minutes vs. 53.78 minutes). It also exhibited a 10 times longer Tmax brain dopamine (30.0 minutes vs. 3.0 minutes) and 8.36 times longer T1=2 Tmax to end (112.51 minutes vs. 13.46 minutes) than L-Dopa. The terminal half-life (T1/2 30 min to end) of brain dopamine upon D-PhG-L-Dopa administration was 2.51 times longer than that upon L-Dopa administration (112.51 minutes vs. 44.85 minutes). This supported our design of this dipeptide as a dopamine prodrug for preventing or prolonging the fast decarboxylation
Table 3 e Equal molars of D-PhG-L-Dopa and L-Dopa were administered i.p. and anti-Parkinsonism activities were determined by rotational behavior assay in crossover experiments. Compound
D-PhG-L-Dopa L-Dopa
No. of Dose (mg/kg)a experiments 16.7 10.0
10 8
Reduction Paired in rotation t-test (%, mean SD) 47.9 5.5 27.3 4.8
p < 0.05 Control
a The dose used in the experiments was 0.051 mmol/kg for each compound.
Fig. 6 e Proposed biological transformation of D-PhG-LDopa and L-Dopa prodrugs to dopamine.
commonly encountered with L-Dopa administration (Fig. 6). The pharmacokinetic profile of brain dopamine also confirmed the finding from our previous study that oral administration of D-PhG-L-Dopa led to an intrinsic sustained dopamine-releasing profile in rats [16]. Although brain dopamine exposure (AUCbrain dopamine) on i.v. administration of D-PhG-L-Dopa was less than with L-Dopa, this dipeptide demonstrated higher activity in reducing the rotation of Parkinsonism rats (47.9 5.5% vs. 27.3 4.8%, p < 0.001, i.p.). The sustained dopamine-releasing might explain its effective anti-Parkinsonism activity. Reports have indicated that pulsatile administration of dopamine agonist induced dopamine-related dyskinesia, and repeated administration will result in wearing-off phenomenon which is generally observed in chronic Parkinsonism patients [13,21,22]. Long-term use of L-Dopa may lead to a decreased therapeutic window for individual patients; therefore, monitoring of blood L-Dopa level is important in the clinical management of Parkinson’s disease. Various approaches including new formulation and new molecular entity design were thus undertaken to achieve sustained-dopamine releasing properties which maintained a stable concentration profile of dopamine [15,23]. In our case, although brain dopamine (Cmax brain dopamine) on D-PhG-L-Dopa administration was much lower than from L-Dopa administration (2.32 nmol/mL vs. 60.58 nmol/mL), the concentration range (Cmax brain dopamine/ Cmin brain dopamine) was six times narrower (1.62 vs. 9.85), which greatly reduced the risk from fluctuations in concentration.
5.
Conclusion
The CNS bioavailability of D-PhG-L-Dopa and L-Dopa, and the brain dopamine level after i.v. injection of both compounds were investigated. D-PhG-L-Dopa exhibited lower CNS dopamine exposure than L-Dopa. However, it exhibited an intrinsic sustained dopamine-releasing profile with longer CNS dopamine residual properties and more stable dopamine concentration profile. The sustained dopamine-releasing property of D-PhG-L-Dopa might be beneficial for clinical management, indicating its usefulness as a potential agent for treating Parkinson’s disease.
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Acknowledgments This work was supported by the National Science Council and the Ministry of Economics of the Republic of China (grant numbers NSC97-2323-B-038-002 and MOE100-EC-17-A20-S1-196). Professor Kwan-Yang Hsu of Taipei Medical University is appreciated for the discussion on pharmacokinetic data.
references
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