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Full Paper New L-Dopa Codrugs as Potential Antiparkinson Agents Piera Sozio1, Antonio Iannitelli1, Laura Serafina Cerasa1, Ivana Cacciatore1, Catia Cornacchia1, Gianfabio Giorgioni2, Massimo Ricciutelli2, Cinzia Nasuti3, Franco Cantalamessa3, and Antonio Di Stefano1 1
Dipartimento di Scienze del Farmaco, Universit ”G. D'Annunzio”, Chieti, Italy Dipartimento di Scienze Chimiche, Universit di Camerino, Camerino (MC), Italy 3 Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Universit di Camerino, Camerino (MC), Italy 2
This paper reports the synthesis and preliminary evaluation of new L-dopa (LD) conjugates (1 and 2) obtained by joining LD with two different natural antioxidants, caffeic acid and carnosine, respectively. The antioxidant efficacy of compounds 1 and 2 was assessed by evaluating plasmatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the rat. Rat striatal concentration of LD and dopamine (DA), and central nervous effects were evaluated after oral administration of the codrugs 1 and 2. The results suggest that, though our codrugs are devoid of significant antioxidant activity, they are able to induce sustained delivery of DA in rat striatum and can improve LD and DA release in the brain. Keywords: Caffeic acid / Carnosine / L-dopa / L-dopa codrugs / Parkinson's disease / Received: November 8, 2007; accepted: February 1, 2008 DOI 10.1002/ardp.200700228
Introduction L-Dopa (LD) is a direct precursor of dopamine (DA), which is deficient in the brains of patients suffering from Parkinson's disease (PD). Although LD is the treatment of choice, its metabolism generates a variety of cytotoxic reactive oxygen species (ROS) that contribute to the progression of the disease, increasing the loss of nigrostriatal dopaminergic neurons [1]. Therefore, employing antioxidant molecules to scavenge ROS is an important strategy for preventing or slowing PD. We have recently demonstrated that multifunctional codrugs containing LD and (R)-a-lipoic acid or glutathione seem to protect partially against the oxidative stress deriving from auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA [2, 3]. In order to further explore the radicalCorrespondence: Antonio Di Stefano, Dipartimento di Scienze del Farmaco, Universit ”G. D'Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy. E-mail:
[email protected] Fax: +39 871 355-4706 Abbreviations: dopamine (DA); L-dopa (LD); glutathione peroxidase (GPx); monoamine oxidase (MAO); Parkinson's disease (PD); reactive oxygen species (ROS); superoxide dismutase (SOD)
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scavenging activity of antioxidant molecules covalently linked with LD, we have focused our attention on caffeic acid and carnosine. Caffeic acid and its derivative clovamide (N-caffeoyl-L-dopa) have been reported to completely block the production of ROS [4]. Their radical scavenging and antioxidative activities are mainly due to the presence of two phenolic alcohol groups at ortho positions. Carnosine, synthesized in the skeletal muscle or brain tissue by carnosine synthetases, prevents ROSinduced peroxidation of lipids and cell death [5, 6]. Recent reports suggest that carnosine has potential therapeutic applications in many diseases of the central nervous system such as Alzheimer's disease, PD, and cerebral ischemic diseases [7 – 10]. Our studies focused on providing two novel codrugs (1 and 2) obtained by joining the 3,4-diacetyloxy-L-dopa methyl ester with caffeic acid or carnosine (Fig. 1). These compounds were evaluated as potential antiparkinson drugs with antioxidant properties. The present paper reports the physicochemical (lipophilicity and solubility), pharmacokinetic (rates of chemical and enzymatic hydrolysis), antioxidant (activities of SOD and GPx), and dopaminergic (effects on spontaneous locomotor activity of rats) properties of both codrugs.
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Reagents: a) DMF, DCM, Et3N, PyBOP.
Scheme 1. Synthesis of codrug 1.
Figure 1. Chemical structures of multifunctional codrugs 1 and 2.
Chemistry/Synthesis Codrug 1 was synthesized through the interaction of 3,4diacetyloxy-L-dopa methyl ester hydrochloride 3 with caffeic acid 4 (Scheme 1) [11 – 13]. Compound 2 was prepared by condensation of N-acetyl-3,4-diacetyloxy-L-dopa 5 and carnosine methyl ester hydrochloride 6 (Scheme 2 and Section 4, Experimental). Coefficient partition (log P) of conjugates 1 and 2 was determined in n-octanol/phosphate buffer, pH 7.4, by saturation of the shake-flask method [13, 14]. Solubility in
aqueous solutions was also evaluated, and the results are listed in Table 1. Codrug 2 showed good water solubility (more than 9 mg/mL at pH 7.4) necessary for good oral absorption, while the log P was high only for compound 1 (more than 2). Biological testing The new compounds were also investigated in vitro to evaluate their chemical and enzymatic stability. The reactivities to chemical hydrolysis in buffer solutions at pH 1.3, 5.0, and 7.4, were evaluated by pseudo-first-order rate constants. The rate data showed that the two codrugs are stable in aqueous solutions; in particular, at pH 1.3 all compounds are stable (t A 2.94 h) and at pH 5.0 and 7.4 they are stable enough (t A 0.44 h) to be absorbed intact from the intestine (see Table 2). In rat plasma, catechol esters and amide bonds of the studied
Table 1. Physicochemical properties of codrugs 1 and 2. Compound
logPa)
1
2.29 (l 0.11)
2
– 1.12 (l 0.06)
a)
Solubility in watera) (mg/mL) 0.26610 – 3 (l 1610 – 4) 29.00 (l 1.45)
Solubility at pH 1.3a) (mg/mL)
Solubility at pH 7.4a) (mg/mL)
Solubility at pH 5.0a) (mg/mL)
2.08610 – 3 (l 0.10610 – 3) 35.53 (l 1.68)
0.10610 – 3 (l 0.01610 – 3) 9.10 (l 0.04)
1.17610 – 3 (l 0.06610 – 3) 27.55 (l 1.38)
Values are means of three experiments; standard deviation is given in parentheses.
Table 2. Kinetic data for chemical hydrolysis of codrugs 1 and 2 at 378C. pH 1.3a)
Compound
1 2 a)
i
pH 5.0a)
pH 7.4a)
t (h)
Kobs (h – 1)
t (h)
Kobs (h – 1)
t (h)
Kobs (h – 1)
2.94 (l 0.10) 147.00 (l 7.39)
0.24 (l 0.01) 0.005 (l 0.2610 – 3)
1.77 (l 0.07) 18.81 (l 0.94)
0.40 (l 0.02) 0.037 (l 0.2610 – 3)
0.44 (l 0.02) 10.15 (l 0.50)
1.57 (l 0.07) 0.068 (l 3.3610 – 3)
Values are means of three experiments; standard deviation is given in parentheses. 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Table 3. Rate constants for the hydrolysis of codrugs 1 and 2 in 80% rat plasma and 80% human plasma at 378C. Rat plasmaa)
Compound
1 2 a)
Reagents: a) DMF, Et3N, DCC.
Scheme 2. Synthesis of codrug 2.
derivatives were cleaved and LD was released in one step with an observed immediate conversion. Hydrolysis in human plasma proceeds more slowly with formation of LD. These derivatives were identified by LC/MS and NMR analysis and the data are listed in Table 3. The degradation process was found to be correlated with first-order kinetics, and LD was released in quantitative amounts. The high water solubility of codrug 2 meets the requirements for gastro-intestinal absorption, while the low lipophilicity (log P = – 1.12) probably prevents the cross of the blood-brain barrier (BBB). Codrug 2, by peripheral hydrolysis, produces LD and carnosine which
Human plasmaa)
t
Kobs
t (min)
Kobs (min – 1)
immediate hydrolysis immediate hydrolysis
/ /
6.53 (l 0.23) 22.29 (l 1.07)
0.11 (l 0.4610 – 2) 0.031 (l 0.5610 – 3)
Values are means of three experiments; standard deviation is given in parentheses.
can use specific transporters to enter the CNS and act as antioxidant agent [15, 16]. Figure 2 shows LD plasma concentration trends obtained in rats over time following administration of codrugs 1, 2, and LD. The values of the LD-plasma concentration at 3 h post-dose were about 85 lg/mL for compound 2 and 80 lg/mL for compound 1. A rapid decrease of concentration levels was observed 2 h after LD administration. The results indicate that codrugs 1 and 2 are able to prolong plasma LD levels and could be beneficial in the treatment of motor dysfunctions directly related to LD plasma-level fluctuations [17, 18]. The new LD codrugs were also evaluated comparing neostriatal LD and DA levels after administration of compounds 1, 2, and LD. A previously reported high performance liquid chromatography (HPLC) method with electrochemical detection (EC) was utilized [19]. The changes in striatal levels of LD and DA are shown in Fig. 3. The studied compounds seem able to induce sustained delivery of both LD and DA in rat striatum with respect to an equimolar dose of LD; the striatal levels of DA were still elevated after 10 h (about 50 pmol/mg for compound 1).
Figure 2. Plasma-concentration profile of LD after administration of LD, 1, and 2 in rats. Data are expressed as mean l SE. Each experiment was performed in triplicate.
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Figure 3. Rat striatal levels of LD and DA after administration of LD, 1 and 2. Data are expressed as mean l SE. Each experiment was performed in triplicate.
Figure 4. Locomotion, rearing, and grooming, 1.5 h after administration of LD, 1, and 2 in rats. Data are expressed as mean l SE of five experiments. * P a 0.05 compared to control group; a P a 0.05 compared to LD-treated group.
Conclusion In this study, we assessed the antioxidant efficacy of the codrugs 1 and 2, evaluating, in the plasma of rats, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), two enzymes critical for protection against oxidative stress, produced by LD [20, 21]. Table 4 shows the activities of these antioxidant enzymes that form the primary defense system against ROS in the plasma 1.5 h after drug administration in the rats. No significant modifications were revealed in the SOD and GPx activities among the four groups treated with vehicle, LD, 1, and 2. For these reasons, the studied codrugs do not seem to protect against the oxidative stress deriving from the MAO-mediated metabolism of DA. From the lit-
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erature, caffeic acid and carnosine act as natural antioxidants with hydroxyl-radical-scavenging and lipid-peroxidase activities and can modulate the SOD and GPx activities when there are strong oxidative stress conditions. In our experimental conditions (after only one drug administration), we suppose that the antioxidant efficacy does not appear because the ROS levels are too low [9, 22, 23]. To evaluate the dopaminergic activity, we studied the effects of 1 and 2 on the spontaneous locomotor activity of rats in comparison with LD-treated animals. As can be observed in Fig. 4, drug administration 1.5 h after gavage led to a pattern of behavioral depression in the treated groups compared to the control group, which received vehicle only. The results obtained after treatment with 2 showed less behavioral depression on locomotion www.archpharm.com
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Arch. Pharm. Chem. Life Sci. 2008, 341, 412 – 417
Table 4. Values of superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, 1.5 h after administration of LD, 1, and 2 in rats.
Control LD 1 2 a)
SOD (U/g N Hb)a)
GPx (U/g N Hb)a)
143 (l12) 171 (l16) 149 (l3) 167 (l14)
821 (l25) 760 (l11) 795 (l3) 801 (l16)
Data are expressed as mean l SE of five experiments.
group. As observed for locomotor activity, treatment with LD, 1, and 2 also led to a pattern of behavioral depression in anxiety-like behavior (Fig. 5). In addition, treatment with 2 induced a significant (P a 0.05) increase of entries and time spent in the center of the open field compared with the LD-treated group, as shown in Fig. 5. The data obtained allow us to distinguish the different pharmacokinetics of the codrugs used at the same molar doses as LD. It seems that, compared to LD treatment, the two codrugs induce fewer sedative effects impairing locomotion and anxiety-related behavior. This may be due to the slower biodistribution of codrug 2 in the CNS: the lower values of plasma concentration of LD, 1.5 h after gavage, could induce a slower DA delivery to the CNS and, thus, there would be fewer effects on behavioral activities. On the whole, these results suggest that our codrugs are devoid of significant antioxidant activity but can improve the release of LD and DA in the brain. Nevertheless, to better investigate the bio-availability of compounds 1 and 2, ”in-vivo” microdialysis studies in rat striatum are in progress. Financial support from the Ministero dell' Istruzione, dell'Universit e della Ricerca (MIUR) is gratefully acknowledged. The authors have declared no conflict of interest.
Figure 5. Anxiety test 1.5 h after administration of LD, 1, and 2 in rats. Data are expressed as mean l SE of five experiments. * P a 0.05 compared to control group; a P a 0.05 compared to LDtreated group.
(975.56 l 2.85) compared to the LD-treated group (555.5 l 97.92) (P a 0.05). A similar pattern can be observed in the anxiety-like behavior 1.5 h after drug administration (Fig. 5). As shown, treatment with LD and codrugs 1, 2 induced a significant decrease of entries (P a 0.05) and time spent in the center of the open field compared with the control
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Experimental The purity of all new compounds was checked by HPLC using the column Waters X-Terra RP18 (5 lm, 3.0615 mm) with MeOH / H2O (60 : 40) as eluent. The microanalyses results were within l 0.4%. 1H-NMR spectra were recorded at 300 MHz in DMSO-d6 as solvent. Compound 1: d ppm 9.41 (s, 1H, Ar-OH), 9.15 (s, 1H, Ar-OH), 8.48 (d, 1H, J = 270 Hz, NHCO), 7.34 – 6.60 (m, 6H, ArH), 6.52 (d, 1H, J = 270 Hz, =CH-Ar), 6.34 (d, 1H, J = 360 Hz, CHCO), 4.55 (q, 1H, aCH), 3.58 (s, 3H, COOCH3), 2.85 (m, 2H, CH2), 2.23 (s, 6H, 26CH3CO). Compound 2: d ppm 1.91 (s, 3H, CH3CON), 2.1 (s, 6H, CH3CO), 2.21 – 2.41 (m, 2H, bCH2), 2.60 – 2.79 (m, 2H, aCH2), 2.8 – 3.19 (m, www.archpharm.com
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4H, 26CH2), 3.66 (s, 3H, OCH3), 4.38 – 4.44 (t, 1H, aCH), 4.6 – 4.72 (t, 1H, aCH), 6.42 – 6.71 (m, 3H, ArH), 6.87 (s, 1H, CH), 7.62 (s, 1H, CH).
References [1] D. G. Graham, Mol. Pharmacol. 1978, 14, 633 – 643. [2] A. Di Stefano, P. Sozio, A. Cocco, A. Iannitelli, et al., J. Med. Chem. 2006, 49, 1486 – 1493. [3] F. Pinnen, I. Cacciatore, C. Cornacchia, P. Sozio, et al., J. Med. Chem. 2007, 50, 2506 – 2515. [4] J. P. Ley, H. J. Bertram, J. Agric. Food Chem. 2003, 51, 4596 – 4602. [5] R. Kohen, Y. Yamamoto, K. C. Cundy, B. N. Ames, Proc. Natl. Acad. Sci. USA 1988, 85, 31753179. [6] S. D. Marchis, C. Modena, P. Peretto, A. Migheli, et al., Biochemistry 2000, 65, 824 – 833. [7] A. N. Fonteh, R. J. Harrington, A. Tsai, P. Liao, M. G. Harrington, Amino Acids 2007, 32, 213 – 224. [8] J. E. Preston, A. R. Hipkiss, D. T. Himsworth, I. A. Romero, J. N. Abbott, Neurosci. Lett. 1998, 242, 105 – 108. [9] J. H. Kang, K. S. Kim, S. Y. Choi, H. Y. Kwon, et al., Biochim. Biophys. Acta 2002, 1570, 89 – 96. [10] A. Boldyrev, R. Song, D. Lawrence, D. O. Carpenter, Neuroscience 1999, 94, 571 – 577. [11] N. Bodor, K. B. Sloan, T. Higuchi, K. Sasahara, J. Med. Chem. 1977, 20, 1435 – 1445.
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[12] S. H. Park, S. H. Kang, S. H. Lim, H. S. Oh, K. H. Lee, Bioorg. Med. Chem. Lett. 2003, 13, 3455 – 3459. [13] C. Chavis, F. Grodenic, J.-L. Imbach, Eur. J. Med. Chem. 1981, 16, 219 – 227. [14] D. J. W. Grant, T. Higuchi in Solubility behaviour of organic compounds, Wiley, New York, 1990, pp. 164 – 175. [15] W. H. Streng in Characterization of Compounds in Solution – Theory and practice, Kluver Academic Plenum Publisher, New York, 2001, pp.161 – 180. [16] N. S. Teuscher, R. F. Keep, D. E. Smith, Pharmaceut. Res. 2001, 18, 807 – 813. [17] R. Kohen, Y. Yamamoto, K. C. Cundy, B. N. Ames, Proc. Natl. Acad. Sci. USA 1988, 85, 3175 – 3179. [18] U. Gundert-Remy, R. Hildebrand, A. Stiehl, E. Weber, et al., Eur. J. Clin. Pharmacol. 1983, 25, 69 – 72. [19] M. Da Prada, H. H. Keller, L. Pieri, L. Kettler, W. E. Haefely, Experientia 1984, 40, 1165 – 1172. [20] C. Nasuti, R. Gabbianelli, M. L. Falcioni, A. Di Stefano, et al., Toxycology 2007, 229, 194 – 205. [21] C. Michiels, M. Raes, O. Topussaint, J. Remacle, Free Radic. Biol. Med. 1994, 17, 235 – 248. [22] A. J. Nappi, E. Vass, Biochim. Biophys. Acta 1997, 1336, 295 – 301. [23] U. J. Jung, M. K. Lee, Y. B. Park, S. M. Jeon, M. S. Choi, J. Pharmacol. Exp. Ther. 2006, 318, 476 – 483. [24] M. A. Babizhayev, M. C. Seguin, J. Gueyne, R. P. Evstigneeva, et al., Biochem. J. 1994, 304, 509 – 516.
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