CNS delivery of l-dopa by a new hybrid glutathione–methionine peptidomimetic prodrug
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- Pinnen, F., Cacciatore, I., Cornacchia, C. et al. Amino Acids (2012) 42: 261. doi:10.1007/s00726-010-0804-z
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Parkinson’s disease (PD) is a neurodegenerative disorder associated primarily with loss of dopamine (DA) neurons in the nigrostriatal system. With the aim of increasing the bioavailability of l-dopa (LD) after oral administration and of overcoming the pro-oxidant effect associated with LD therapy, we designed a peptidomimetic LD prodrug (1) able to release the active agent by enzyme catalyzed hydrolysis. The physicochemical properties, as well as the chemical and enzymatic stabilities of the new compound, were evaluated in order to check both its stability in aqueous medium and its sensitivity towards enzymatic cleavage, providing the parent LD drug, in rat and human plasma. The radical scavenging activities of prodrug 1 was tested by using both the DPPH–HPLC and the DMSO competition methods. The results indicate that the replacement of cysteine GSH portion by methionine confers resistance to oxidative degradation in gastric fluid. Prodrug 1 demonstrated to induce sustained delivery of DA in rat striatal tissue with respect to equimolar LD dosages. These results are of significance for prospective therapeutic application of prodrug 1 in pathological events associated with free radical damage and decreasing DA concentration in the brain.
Parkinson’s disease (PD) and Alzheimer’s disease (AD) are progressive neurodegenerative disorders that affect an increasing number of the elderly population. Although current drug therapy of PD turned out to be more successful compared to that of AD, it does not stop the degenerative process, and pharmacological treatments lose effectiveness with progression of the disease (Everts et al. 2001). Dopamine (DA) shortage is mainly responsible for the motor deficits of the disorder and several drugs, that boost the levels of DA or mimic its effects, are available for treating PD; nevertheless, none of them exceeded the clinical efficacy of their biological precursor l-dopa (LD) (LeWitt 2008). Since its introduction in the late 1960s, LD, which counteracts parkinsonian motor symptoms by restoring the nigrostriatal DA deficiency, still remains the key compound of pharmacotherapy for PD to which all other therapies are compared. Up to now, several compounds have been studied with the aim of enhancing LD chemical stability and water or lipid solubility, and diminishing its susceptibility to enzymatic degradation. During the last two decades the prodrug approach was demonstrated to be quite promising in addressing the problem of the rapid in vivo degradation of LD, and consequently, in improving its bioavailability (Wang et al. 1995; Marrel et al. 1985; Cooper et al. 1984; Garzon-Aburbeh et al. 1986; Cooper et al. 1987; Ihara et al. 1989; Cingolani et al. 2000; Giorgioni et al. 2010). In recent years an ameliorative version of the prodrug approach, developed for favoring the delivery of LD to the central nervous system (CNS), has involved the development of dual-acting prodrugs. A few papers reported on the efficacy of LD, covalently linked to different antioxidant molecules, to induce sustained release of drug in rat striatum and, at the same time, to protect against the oxidative stress deriving from autoxidation of DA (More and Vince 2008; Sozio et al. 2008; Di Stefano et al. 2006; Pinnen et al. 2007; Pinnen et al. 2009). In order to efficiently deliver small, hydrophilic molecules across the BBB two different strategies are currently used: (1) the chemical modification of hydrophilic molecules to more lipophilic derivatives, named lipidization; and (2) the transporter-mediated delivery, in which the substrate of one of the numerous transporters located within the BBB is exploited as the carrier moiety of the drug (Pardridge 2009).
Microanalyses were performed on a 1106 Carlo Erba CHN analyzer. 1H- and 13C-NMR spectra were recorded on a Varian VXR 300-MHz spectrometer. Chemical shifts are reported in parts per million (δ) downfield from the internal standard tetramethylsilane (Me4Si). The LC–MS/MS system consisted of an LCQ (Thermo Finnigan) ion trap mass spectrometer (San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. The capillary temperature was set at 300°C and the spray voltage at 4.25 kV. The fluid was nebulized using nitrogen (N2) as both the sheath gas and the auxiliary gas. The identity of the new prodrug was confirmed by elemental analysis, NMR data and LC–MS/MS measurements; homogeneity was confirmed by TLC on silica gel Merck 60 F254. Solutions were routinely dried over anhydrous sodium sulphate prior to evaporation. Chromatographic purifications were performed by Merck 60 70–230 mesh ASTM silica gel column.
Ac-Glu-OMe was obtained from Bachem. All other chemicals were of the highest commercially available purity.
To a solution of the N-protected dipeptide t-butyl ester 2 (5 g, 10.3 mmol) in CH2Cl2 (70 mL) DBU (1.57 g, 10.3 mmol) was added at room temperature. After 20 min the solution was evaporated to dryness and the residue chromatographed on silica gel using CHCl3:MeOH (95:5) as eluant to yield pure N-deprotected dipeptide t-butyl ester 3 (1.68 g, 62%). Rf = 0.43, CHCl3:MeOH (95:5); 1H-NMR (CDCl3) δ: 1.44 (9H, s, OBut), 2.45 (2H, s br, NH2), 1.65–1.80 (1H, m, Met β-CHA), 2.15 (3H, s, Met CH3), 2.16–2.20 (1H, m, Met β-CB), 2.45–2.55 (2H, m, Met γ-CH2), 3.50 (1H, m, Met α-CH), 3.85–3.90 (2H, m, Gly α-CH), 7.84 (1H, s br, Gly NH). 13C-NMR (CDCl3) δ: 15.48 (Met CH3), 28.24 (OBut), 30.77 (Met β-CH2), 34.19 (Met γ-CH2), 41.88 (Gly α-CH), 54.31 (Met α-CH), 82.33 (OBut), 169.28 and 175.02 (2 × CO).
To a stirred solution of N-deprotected dipeptide t-butyl ester 3 (1.5 g, 5.7 mmol) in dry DMF (7 mL) Ac-Glu-OMe (1.16 g, 5.7 mmol) in dry DMF (7 mL) was added at 0°C followed by portion-wise addition of HOBt (0.77 g, 5.7 mmol) and DCC (1,10 g, 5.7 mmol) in dry DMF (8 mL). After 3 h at 0°C and 16 h at 5°C, the reaction mixture was filtered and the resulting solution was evaporated under vacuum. The residue was taken up in EtOAc and the organic layer washed with 1 N KHSO4, saturated aqueous NaHCO3 and brine. The residue obtained after drying and evaporation was chromatographed on silica gel by using CHCl3:MeOH (95:5) as the eluant to give the corresponding pure tripeptide t-butyl ester 4 (1.97 g, 77%). Rf = 0.30, CHCl3:MeOH (95:5); 1H-NMR (CDCl3) δ: 1.44 (9H, s, OBut), 1.96–2.01 (2H, m, Met β-CH2), 2.02 (3H, s, Met CH3), 2.11 (3H, s, Glu Ac), 2.16–2.20 (2H, m, Glu β-CH2), 2.33-2.38 (2H, m, Glu γ-CH2), 2.55–2.63 (2H, m, Met γ-CH2), 3.74 (3H, s, OMe), 3.91–3.93 (2H, m, Gly α-CH), 4.56 (1H, m, Glu α-CH), 4.65 (1H, m, Met α-CH), 6.65 (1H, d, J = 7.2 Hz, Glu NH), 6.89 (2H, m br, Mat and Gly NH); 13C-NMR (CDCl3) δ: 15.41 (Met CH3), 23.25 (Glu Ac), 28.25 (OBut), 29.89 (Glu β-CH2), 30.36 (Met β-CH2), 31.38 (Glu γ-CH2), 32.45 (Met γ-CH2), 42.21 (Gly α-CH), 52.02 (OCH3), 52.33 (Glu α-CH), 52.77 (Met α-CH), 82.57 (OBut), 168.92, 170.81, 171.47, 172.53 and 172.75 (5 × CO).
The above reported tripeptide t-butyl ester 4 (1.80 g, 4.02 mmol) was dissolved in TFA (5.1 mL). After 2 h at room temperature the solution was evaporated to dryness and the residue repeatedly evaporated with ether to give 5 in quantitative yield. This product was used without further purification (1.54 g, 98%).
To an ice-cold solution of deprotected tripeptide 5 (1.40 g, 3.58 mmol) in dry DMF (5 mL) TEA (0.50 mL, 3.58 mmol) and isobutyl chloroformate (IBCF) (0.47 mL, 3.58 mmol) were added under stirring. After 15 min at −15°C, H-LD(Ac)2-OMe.HCl (1.19 g, 3.58 mmol) in TEA (0.50 mL, 3.58 mmol) and dry DMF (5 mL) were added to the mixture at −15°C with stirring. After 3 h at 0°C and 16 h at 5°C, the reaction mixture was filtered and the resulting solution was evaporated under vacuum. The residue was taken up in AcOEt and the organic layer washed with 1 N KHSO4, saturated aqueous NaHCO3 and brine. The residue obtained after drying and evaporation was crystallized from AcOEt (0.96 g, 40%). Rf = 0.41, CHCl3:MeOH (95:5); 1H-NMR (d6-DMSO) δ: 1.77–1.94 (4H, m, Glu and Met β-CH2), 1.90 (3H, s, Met CH3), 2.01 (3H, s, Glu Ac), 2.09–2.55 (4H, m, Glu and Met γ-CH2), 2.23 and 2.25 (6H, 2 × s, LD Ac), 2.95–3.10 (2H, m, LD β-CH2), 3.65 (3H, s, OMe), 3.70 (3H, s, OMe), 3.72–3.80 (2H, m, Gly α-CH2), 4.10–4.25 (2H, m, Glu and Met α-CH), 4.45 (1H, m, LD α-CH), 7.10–7.20 (3H, m, Ar), 8.05 (1H, d, J = 7.2 Hz, Glu NH), 8.10 (1H, t, J = 5.8 Hz, Gly NH), 8.15 (1H, d, J = 6.89 Hz, Met NH), 8.25 (1H, d, J = 7.1 Hz, LD NH); 13C-NMR ((d6-DMSO) δ: 15.21 (Met CH3), 21.03 and 21.05 (LD 2 × Ac), 22.94 (Glu Ac), 27.36 (Glu β-CH2), 30.21 (Met β-CH2), 31.90 (Glu γ-CH2), 32.17 (Met γ-CH2), 36.53 (LD β-CH2), 42.19 (Gly α-CH), 52.14 (OCH3), 52.47 (Glu α-CH), 52.57 (OCH3), 52.76 (Met α-CH), 54.09 (LD α-CH), 123.97, 124.83, 127.89, 136.50, 141.34 and 142.31 (Ar), 168.87, 168.92, 169.57, 170.13, 172.23, 172.32 and 173.22 (6 × CO). Anal. calculated for (C29H40N4O12S): C, 52.09; H, 6.03; N, 8.38; S, 4.80; found: C, 52.29; H, 6.02; N, 8.40; S, 4.65. MS (ESI) m/z 669 (M–H)−.
Analytical HPLC measurements were run on a Waters 1525 Binary HPLC pump, equipped with a Waters 2996 photodiode array detector, a 20-μL Rheodyne injector and a computer integrating apparatus. The column was a Waters Symmetry RP-C18 column (4.6 × 150 mm, 5 μm), the mobile phase was a mixture of water/methanol (10:90) at a flow rate of 1 mL/min, the UV-detector was set at a length of 264 nm.
Pharmacokinetic analysis were run on the HPLC system consisted of a Waters 600 controller pump, a Rheodyne 7295 injector with a 10-μL loop and an Antec Leyden Decade II detector; the operating potential was 0.75 V. Separation was achieved on a Waters Symmetry RP-C18 column (4.6 × 150 mm, 5 µm). The mobile phase consisted of 0.045 M monobasic sodium phosphate, 0.001 M 1-octanesulphonic acid sodium salt, 0.006% triethylamine, 0.015% 100 μM sodium EDTA and 6% acetonitrile. The pH of the mobile phase was adjusted to 3.0 by o-phosphoric acid. The mobile phase was filtered and degassed by vacuum. A flow rate of 1 mL/min was used in all experiments. Monoamine stock solutions were prepared at a concentration of 1 mg/mL (as a free base) in 0.05 N perchloric acid containing 0.064% 1-octanesulphonic acid sodium salt, 0.060% heptanesulphonic acid sodium salt, 0.004% sodium EDTA, and 0.010% sodium metabisulphite. These standard solutions were freshly prepared every week and stored at 4°C for use right away. The monoamine and their metabolites were identified on the basis of retention time. Measurements were performed in triplicate for each original sample.
Compound 1 (50 mg) was placed in a microtube containing 1 mL of deionized water or buffer solutions (at pH 7.4, 5.0 and 1.3 by using 0.02 M phosphate buffer, 0.02 M acetate buffer or 0.02 M hydrochloridic acid buffer, respectively) and shaken at 25°C for 1 h to ensure the solubility equilibrium. After centrifugation, a 20-μL portion of the supernatant was analyzed by HPLC (Pinnen et al. 2009).
The calculated clog P was determined by using ACD Log P software package, version 4.55 (Advanced Chemistry Development Inc., Toronto, Canada).
Octanol/water partition coefficient
Octanol/water partition coefficient (log P) was determined by placing approximately 5 mg of compound 1 in 1 mL of aqueous saturated n-octanol, shaking vigorously for about 2 min and filtering. An equal volume of phosphate buffer pH 7.4 was added and the mixture was equilibrated by repeated inversions of up to 200 times for 5 min and then allowed to stand for 30 min for the phases to fully separate. Thereafter the respective phases were analyzed by HPLC.
Solute hydrophobicity of different compounds can be estimated from the corresponding retention times (RTs) due to the good relationship between log octanol/water partition coefficient and logarithmic retention factors in reverse-phase chromatography (log k value) determined by using octadecyl silica columns. (Vailaya and Horváth 1998; Angelini et al. 2005). Retention in reverse-phase chromatography increases with solute lipophilic character and, for a given combination of solute and stationary phase, with the water content of the mobile phase.
Compound 1 was dissolved in methanol (concentration 1 mg/mL). Aliquots of this solution were filtered and analyzed by HPLC. The mobile phase consisted of acetonitrile and water with acetonitrile content varying in the range 90–55% (v/v) with 5% increments (Bajda et al. 2007).
By plotting log k values against the acetonitrile content it is possible to calculate, by extrapolation to zero acetonitrile percentage, the log k0 value, that is the solute retention with pure water as the eluent.
Kinetics of chemical hydrolysis
A 0.02 M hydrochloridic acid buffer of pH 1.3 as non-enzymatic simulated gastric fluid (SGF) and a 0.02 M phosphate buffer of pH 7.4 were used in this study. Reactions were initiated by adding 1 mL of 10−4 M stock solution (in acetonitrile) of prodrug 1 to 10 mL of the appropriate thermostatted (37 ± 0.5°C) aqueous buffer solution, containing 20% acetonitrile. At appropriate time intervals, samples of 20 μL were withdrawn and analyzed by HPLC. Pseudo-first-order rate constants (kobs) for the hydrolysis of the prodrug were then calculated from the slopes of the linear plots of log (% residual prodrug) against time. The experiments were run in triplicate and the mean values of the rate constants were calculated.
Kinetics of enzymatic hydrolysis
Plasma from rats and human was obtained by centrifugation of blood samples containing 0.3% citric acid at 3,000×g for 15–20 min. Plasma fractions (4 mL) were diluted with 0.02 M phosphate buffer (pH 7.4) to give a final volume of 5 mL (80% plasma). Incubations were performed at 37 ± 0.5°C using a shaking water bath. The reactions were initiated by adding 100 μL of a stock solution of drug (1 mg/mL in acetonitrile) to 5 mL of preheated plasma. Aliquots (100 μL) were taken at various times and deproteinized by mixing with 200 μL of 0.01 M HCl in methanol. After centrifugation for 5 min at 5,000×g, 10 μL of the layer supernatant was chromatographed as described above. The logarithms of the remaining original prodrug were plotted as a function of incubation time in order to give the corresponding kobs.
Radical scavenging activity
DMSO competition method
Male Wistar rats (n = 75) (Harlan, UD, Italy) weighing 250–300 g were employed. Five rats were assigned to each treatment group. The animals were housed in plastic (Makrolon) cages in a temperature-controlled room (21 ± 5°C) and maintained on a laboratory diet and water ad libitum. The light/dark cycle was from 7 a.m. to 7 p.m.
Benserazide hydrochloride, a peripheral dopa-decarboxylase inhibitor, was dissolved in water whereas the prodrug 1, LD and tripeptide were dissolved in dimethyl sulfoxide. All animals received a dose of benserazide (0.083 mmol/kg) combined with 1, LD or tripeptide in equimolar doses (0.332 mmol/kg). The drugs were given at a volume of 5 ml/kg in a single oral administration by intragastric tube. This study was carried out in accordance with the Italian government’s guidelines for the care and use of laboratory animals (D.L. n. 116 of January 27, 1992).
After slight anesthesia with carbon monoxide, the blood of rats, was collected, by cardiac puncture, in vials containing heparin (250 IU), centrifuged at 2,000×g for 10 min and kept at −80°C until analysis. Aliquots (400 μL) were taken at various times and deproteinized by mixing with 40 μL of 4 M perchloric acid. After centrifugation for 5 min at 5,000×g and filtration (Millipore 0.45 μm), 10 μL of the layer supernatant was chromatographed as described below. The amounts of LD were plotted as a function of incubation time.
The striatum tissue of the rats were dissected out, frozen as well in liquid nitrogen and stored at −80°C until use. The sampling schedules were 1, 2, 4 and 6 h after treatment with drugs. All striatal tissues were individually homogenized for 2 min with a Dyna-Mix homogenizer (Fisher Scientific) in 500 μL of 0.05 N perchloric acid solution containing (by weight/volume) 0.064% 1-octanesulphonic acid sodium salt, 0.060% heptanesulphonic acid sodium salt, 0.004% sodium EDTA, 0.010% sodium metabisulfite and 25 ng/mL dihydroxybenzylamine (DHBA) as an internal standard. The whole procedure was carried out on ice. The resulting homogenate was then centrifuged at 4,500 g for 10 min and the supernatant was filtered by using 0.45 μm Millipore filters. For each measurement, 10 μL of the obtained filtrate were injected into the liquid chromatography equipment (Giorgioni et al. 2010).
DA concentrations in rat striatal tissue are given as percentages of mean values with respect to basal levels. Basal concentrations were determined as the mean of at least three measurements with ≤5% variation obtained at the beginning of the experiment. Data are expressed as means with error bars for standard deviations of five rats. One-way ANOVA was used to evaluate the effect of procedures on each group of animals. If a general effect was determined by ANOVA, post hoc analysis was performed with the Scheffe or Student’s test with P ≤ 0.05 used as the level of significance.
Results and discussion
Physicochemical properties of prodrug 1
In order to evaluate the penetration of a molecule through the BBB, the lipophilicity was estimated by using the apparent partition coefficient (log Papp) measured in n-octanol/phosphate buffer at pH 7.4 and the log capacity factor (log k0) calculated from reverse-phase chromatographic retention times with acetonitrile and water as the eluents (Bonina et al. 1996). For the sake of comparison, the lipophilicity of prodrug 1 was also calculated using the ACD Log P software package, version 4.55 (Advanced Chemistry Development Inc., Toronto, Canada). Data reported in Table 1 point out that compound 1 is quite hydrophilic as evidenced by negative log P and clog P values. The value of log k0 = 1.21 is in agreement with a low retention in reverse-phase chromatography as expected for not very lipophilic molecules (Di Stefano et al. 2008).
Rate constants for the chemical and enzymatic hydrolysis of prodrug 1 at 37°C
5.67 (±0.17) 10−4
1.07 (±0.05) 10−3
In conclusion, we have synthesized a new peptidomimetic prodrug constituted by a modified GSH, as the carrier towards CNS, and LD methylester, as the active antiparkinson agent. This compound demonstrated to cross unaltered the acidic environment of the stomach, to be stable enough to be absorbed from the intestine, to have radical scavenging activity and to release LD in human plasma after enzymatic hydrolysis. Compared to other drugs intended for PD therapy, prodrug 1 appears to be particularly promising because it showed to induce sustained delivery of DA in rat striatal tissue with respect to equimolar administration of LD itself. Taken together, these results are of significance for prospective therapeutic application of prodrug 1 in pathological events associated with free radical damage and decreasing DA concentration in the brain.
Financial support from Ministero dell’ Istruzione, dell’Università e della Ricerca (MIUR) is gratefully acknowledged.
Conflict of interest
The authors declare that they have no conflict of interest.