Lead Detoxification Activity and ADMET Hepatotoxicity of N-(α-l

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Lead Detoxification Activity and ADMET Hepatotoxicity of N-(r-LArabino-furanos-1-yl)-L-cysteine Ming Zhao, Yuji Wang, Caixia Huo, Jiawang Liu, Chunyu Li, Xiaoyi Zhang, Li Peng, and Shiqi Peng* College of Pharmaceutical Sciences, Capital Medical UniVersity, Beijing 100069, PR China ReceiVed May 2, 2010

N-(R-L-Arabinofuranos-1-yl)-L-cysteine was stereoselectively prepared from L-arabinose and L-cysteine. Its in ViVo detoxification action was evaluated on lead loaded mice at the doses of 0.1, 0.2, and 0.4 mmol/kg. The results show that lead accumulation in the livers, kidneys, brains, and femurs of the treated mice could be efficiently decreased by N-(R-L-arabinofuranos-1-yl)-L-cysteine, even at the dose of 0.1 mmol/kg. Compared with the lead detoxification efficacy, 0.4 mmol/kg of N-(R-L-arabinofuranos-1-yl)L-cysteine did not affect the essential metals in the treated mice, such as Fe, Cu, Zn, and Ca. In the apparent permeability coefficient test, the values of Papp(AfB), Papp(BfA), and Papp(AfB)/Papp(BfA) indicated that N-(R-L-arabinofuranos-1-yl)-L-cysteine was transported actively across the Caco-2 cell monolayer. Silico molecular modeling results predicted that N-(R-L-arabinofuranos-1-yl)-L-cysteine had no hepatotoxicity. Introduction The introduction of unleaded gasoline has significantly reduced environmental exposure to lead (Pb); however, during the past two decades the levels of lead in air, water, and soils have increased in urban centers of both developed and developing countries (1-5). In terms of potential adverse effects on animal and human health, lead is a widespread toxic metal and accumulates in the human body with a half-life time ranging from 6 to 10 years (4, 6, 7). It has been established that about 95% of the whole body burden of lead is comprised in bone (6), and bone stores of Pb accrued from environmental exposure have been associated with a series of diseases such as hypertension, cognitive deficits, hyperthyroidism, osteoporosis, and skeletal disorders (3-5, 8). Lead exposure will continue to be a major public health issue around the world for the foreseeable future, and there is an urgent need to find novel agents for the treatment of lead poisoning (9, 13). It has been established that relying on chelation, glycosylamino acids were able to efficiently remove the cadmium and/ or lead accumulated in the tissues of mice, and this detoxification did not affect the essential metals. In the course of the development of N-(1-deoxy-D-fructos-1-yl)-L-amino acids, we found that the efficacy of lead detoxification depended on anomeric configuration and that the R-anomer had higher efficacy than the β-anomer; however, the R-anomers could be prepared only as a minor component (14-16). Therefore, it is a necessary to establish a stereoselective route for the preparation of R-glycosylamino acids. L-Arabinose has been used as the pharmacophore of bioactive agents (17, 18), the moiety of the polymer chain of a drug carrier (19), and the functional group to increase the transmembrane permeability of bioactive agents. In addition, a stereo-selective synthesis of N-(R-L-arabinose-1-yl)-L-amino acids was also reported in our previous publication (20). * To whom correspondence should be addressed. Tel: +86-10-83911528. Fax: +86-10-8391-1528. E-mail: [email protected] or [email protected].

In this work, the stereoselective synthesis, lead-detoxificating evaluation, apparent permeability coefficient measurement, and hepatotoxicity prediction of N-(R-L-arabinofuranos-1-yl)-Lcysteine are reported.

Materials and Methods Chemicals. All chemicals were purchased from Sigma Aldrich Co. (Milwaukee, WI, USA). N-(R-L-Arabinofuranos-1-yl)-L-cysteine was obtained as a major anomer from 2,3,5-tri-O-acetyl-L-arabinose and N-(2-nitrophenylsulfonyl)-L-cysteine t-butylester through a seven-step-procedure described previously (20). Mp 243 °C (decomp.), [a] D25 ) 38.1 (c ) 1.0, H2O). 1H NMR (500 MHz, D2O) δ ) 4.32 (m, J ) 5.4 Hz, 1H), 4.17 (d, J ) 4.0 Hz, 1H), 4.05 (dd, J ) 12.0 Hz, J ) 4.0 Hz, 1H), 3.82 (dd, J ) 12.5 Hz, J ) 3.0 Hz, 1H), 3.81 (m, J ) 4.7 Hz, 1H), 3.28 (d, J ) 10.0 Hz, 1H), 3.08(d, J ) 10.0 Hz, 1H), 2.89 (m, J ) 5.0 Hz, 2H). FAB/MS (m/e): 254 [M + H]+. Animals. Male imprinting control region (ICR1) mice (weighting 23 ( 2 g) were purchased from Experimental Animal Center of Peking University. The chemicals used in the in ViVo assays were purchased from Sigma Aldrich Chemical Co. (Milwaukee, WI, USA). The study described herein was performed according to a protocol reviewed and approved by the ethics committee of Capital Medical University. The committee assures that the welfare of the animals was maintained in accordance with the requirements of the animal welfare act and according to the guide for care and use of laboratory animals. Lead Detoxification Assay (21). Five groups (each 10) of mice were loaded with lead via i.p. injection of 8.2 mg/kg of Pb(C2H3O2)2·3H2O in 0.5 mL of water per day for seven consecutive days. After a 2-day interval, one group served as the control and was given a daily i.p. injection of 0.2 mL of 0.9% normal saline (NS), another three groups served as the positive controls and were 1 Abbreviations: Papp(AfB), apparent permeability coefficient from the apical side to the basolateral side; Papp(BfA), apparent permeability coefficient from the basolateral side to the apical side; Pb, lead; ICR, imprinting control region; NS, normal saline; HBSS, Hank’s balanced salt solution; DL-PA, DL-penicillamine; L-Arab, L-arabinose; L-Cys, L-cysteine; AP side, apical side; BL side, basolateral side; ADMET, absorption, distribution, metabolism, elimination, and toxicity.

10.1021/tx100155r  2010 American Chemical Society Published on Web 06/14/2010

Lead Detoxification ActiVity given a daily i.p. injection of 0.4 mmol/kg of DL-penicillamine, L-arabinose, and L-Cys in 0.2 mL of NS, and the remaining group was given a daily i.p. injection (0.4 mmol/kg in 0.2 mL of NS) of N-(R-L-arabinifuranos-1-yl)-L-cysteine for five consecutive days. On each day, 2 h after i.p. injection, the urine samples of each group were continually collected for 5 h, and the feces samples of each group were continually collected for 24 h. Twenty-four hours after the last i.p. injection, all of the mice were sacrificed by diethyl ether anesthesia and dissected immediately to obtain the livers, kidneys, brains, and left femurs. The urine, fecal, liver, kidney, brain, and left femur samples were digested in a 1/3 mixture of HClO4 and HNO3 on a heating block, dried at 80 °C, and dissolved in 1% nitric acid to measure the lead content on a Varian Spectr AA-40 atomic absorption spectrometer in the graphite furnace. The variety of the essential metals was measured by synchrotron X-ray fluorescence. All data are expressed as means ( SD. The statistical analysis of the data was carried out using an ANOVA test; p < 0.05 is considered significant. Apparent Permeability Coefficient Test. Caco-2 cells (from the American Type Culture Collection, Rockville, MD, USA) were cultivated on polycarbonate filters (traswell cell culture inserts, 12 mm in diameter, 3.0 µM in mean pore size) as described elsewhere (22). Caco-2 cells grown on filter supports for 21 days were used for all transport tests, and the integrity of the monolayer was routinely checked by the measurements of the transepithelial electrical resistance (approximately 700 Ω·cm2). All absorption tests were performed in Hank’s balanced salt solution (HBSS). DLPenicillamine and N-(R-L-arabinofuranos-1-yl)- L-cysteine were dissolved in HBSS to prepare a test solution at a final concentration of 4 mM. In the apical to the basolateral direction, transport was initiated by the addition of the test solution (total AP volume, 0.5 mL) to the apical compartment of the inserts held in the transwell containing 1.5 mL of HBSS (basolateral compartment). In the basolateral to the apical direction, transport was initiated by the addition of 1.5 mL of the test solution to the basolateral compartment and 0.5 mL of HBSS as receiving solution to the apical side of the monolayer. The monolayer was incubated in air at 37 °C and 95% relative humidity. At 30, 60, 90, and 120 min, the samples were withdrawn from the receiving side, and the concentration of the sample was measured with HPLC. The resistance of the monolayer was checked at the end of each experiment. Apparent permeability coefficient (Papp) was calculated according to Papp ) dQ/dt·1/(A·C0), wherein dQ/dt is the permeability rate, C0 is the initial concentration in the donor chamber, and A is the surface area of the monolayer (1 cm2).

Results and Discussion Lead Detoxification Activity of N-(r-L-Arabinofuranos-1yl)-L-cysteine. In the lead detoxification assay, the lead-loaded mice were treated with N-(R-L-arabinofuranos-1-yl)-L-cysteine, DL-penicillamine (positive control), NS (negative control), L-arabinose, and L-Cys (reference compounds), and the lead in the livers, kidneys, brains, and femurs of the mice were measured and are shown in Figure 1. The results illustrate that the lead accumulated in the livers, kidneys, left femurs, and brains of the mice receiving N-(R-L-arabinofuranos-1-yl)-Lcysteine are significantly less than the lead in the corresponding organs of the mice receiving DL-penicillamine and NS. The results also indicate that the reference compounds, L-arabinose and L-cysteine, have no lead detoxification activity. It implies that the lead detoxification activity of N-(R-L-arabinofuranos1-yl)-L-cysteine does not depend on the possible decomposition products, L-arabinose and L-cysteine. Membrane Permeability of N-(r-L-Arabinofuranos-1-yl)-Lcysteine. To explore the relationship between the membrane permeability and lead detoxification activity of N-(R-L-arabinofuranos-1-yl)-L-cysteine, the active transport experiments were carried out on a confluent monolayer of Caco-22 cells. As one

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Figure 1. Lead in the tissues of the treated mice. (a) Data are represented with mg of lead per g of tissue (mean ( SE). DL-PA ) DL-penicillamine, L-Arab ) L-arabinose, NS ) vehicle, 6 ) N-(R-Larabinofuranos-1-yl)-L-cysteine, n ) 10, dose ) 0.4 mmol/kg, Larabinose, and L-Cys ) reference compounds. (b) Compared to NS and the reference compounds, p < 0.001. (c) Compared to NS, the reference compounds, and DL-penicillamine, p < 0.01. (d) Compared to NS and the reference compounds, p < 0.05. (e) Compared to NS, the reference compounds and DL-penicillamine, p < 0.001. (f) Compared to NS and the reference compounds, p < 0.001. (g) Compared to NS, the reference compounds and DL-penicillamine, p < 0.001. (h) Compared to NS and the reference compounds, p < 0.05. (j) Compared to NS, the reference compounds and DL-penicillamine, p < 0.01.

of the important factors, the membrane permeability of N-(Raffects its absorption and transportation as well as its lead detoxification activity. Herein, the Papp value of N-(R-L-arabinose-1-yl)-L-cysteine from the apical side (AP side) to the basolateral side (BL side) is 19.07 × 10-6 cm/s, and the Papp value from the basolateral side to the apical side is 5.99 × 10-6 cm/s. It is 3.18-fold so that the Papp value from the AP side to the BL side is higher than that from the BL side to the AP side. For the positive control, DLpenicillamine, the corresponding Papp values from from the AP side to the BL side and from the BL side to the AP side are 13.91 × 10-6 and 12.56 × 10-6 cm/s, respectively. It is defined that the Papp value from the AP side to the BL side is only 1.11fold higher than the Papp value from the BL side to the AP side. According to the common criterion, an actively absorbed compound generally showed much faster transport from the apical to the basolateral direction, and its Papp value from the apical side to the basolateral side should be more than 10 × 10-6 cm/s (23). On the basis of the Papp value from the apical side to the basolateral side, we consider that N-(R-L-arabinofuranos-1-yl)-L-cysteine is transported actively across the Caco-2 cell monolayer. The comparison of the Papp values of N-(R-Larabinofuranos-1-yl)-L- cysteine and DL-penicillamine suggests that N-(R-L-arabinofuranos-1-yl)-L-cysteine should possess higher lead detoxification activity than DL-penicillamine. Lead Occurred in the Feces and Urine of the Mice Receiving N-(r-L- Arabinofuranos-1-yl)-L-cysteine. To evaluate the effect of N-(R-L-arabinofuranos-1-yl)-L-cysteine on lead excretion, the lead present in the feces and urine of mice receiving N-(R-L- arabinofuranos-1-yl)-L-cysteine, DL-penicillamine (positive control), NS (negative control), L-arabinose, and L-Cys (reference compounds) were measured and the results shown in Figure 2. The data demonstrate that the lead that was present in the feces and urine of the mice receiving N-(R-Larabinofuranos-1-yl)-L-cysteine is significantly higher than that occurring in the feces and urine of mice receiving DLpenicillamine, NS, and the reference compounds. The data also demonstrate that the lead that was present in the feces and urine of mice receiving DL-penicillamine is 1.83-fold and 2.24-fold higher than that occurring in the feces and urine of mice receiving NS, respectively, while the lead that was present in L-arabinofuranos-1-yl)-L-cysteine

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Figure 2. Lead occurred in the fecal and urinary specimen of the treated mice. (a) Data are represented with mg of lead per g of dry fecal matter or per mL of urine (mean ( SD). NS ) vehicle, 6 ) N-(R-Larabinofuranos-1-yl)-L-cysteine, DL-PA ) DL-penicillamine, L-Arab ) L-arabinose, n ) 10, dose ) 0.4 mmol/kg, L-Arabinose, and L-Cys ) reference compounds. (b) Compared to NS and the reference compounds, p < 0.01. (c) Compared to NS and the reference compounds, p < 0.01, to DL-PA p < 0.05. (d) Compared to NS and the reference compounds, p < 0.05. (e) Compared to NS and the reference compounds, p < 0.01.

Figure 3. Essential metals of the tissues of N-(R-L-arabinofuranos-1yl)-L-cysteine treated mice. Data are represented by mg of essential metal per g of tissue (mean ( SD). Dose ) 0.4 mmol/kg, n ) 10, NS ) vehicle, and 6 ) N-(R-L-arabinofuranos-1-yl)-L-cysteine.

the feces and urine of mice receiving N-(R-L-arabinofuranos1-yl)-L-cysteine is 1.25-fold and 1.19-fold higher than that occurring in the feces and urine of mice receiving DLpenicillamine, respectively. These results imply that the increase of lead excretion should be responsible for the higher lead detoxification efficiency of N-(R-L-arabinofuranos-1-yl)-L-cysteine. The fact that the lead in the feces is significantly higher than that occurring in the urine implies that the lead accumulated in the tissues of mice is preferably excreted via feces. Effect of N-(r-L-Arabinofuranos-1-yl)-L-cysteine on the Essential Metals of the Tissues of Treated Mice. To examine the effect of the treatment on essential metals, Fe, Cu, Zn, and Ca in the kidneys, livers, and brains of the mice receiving NS and N-(R-L- arabinofuranos-1-yl)-L-cysteine were measured and shown in Figure 3. The data indicate that these essential metals of the livers, kidneys, and brains of the mice receiving 0.4 mmol/ kg of N-(R-L-arabinofuranos-1-yl)-L-cysteine are at the same levels as those of the mice receiving NS. This implies that the treatment of 0.4 mmol/kg of N-(R-L-arabinofuranos-1-yl)-Lcysteine has no influence on the essential metal levels in treated mice. Dose-Dependent Lead Detoxification of N-(r-L-Arabinofuranos-1-yl)-L-cysteine. Treatments of three doses, i.e., 0.1 mmol/kg, 0.2 mmol/kg, and 0.4 mmol/kg, were used to observe the possible dose-dependent response of the mice receiving

Zhao et al.

Figure 4. Effect of the dose of N-(R-L-arabinofuranos-1-yl)-L-cysteine on the lead accumulated in the tissues of the treated mice. (a) Data are represented with mg of lead per g of tissue (mean ( SD). NS ) vehicle, and n ) 10. (b) Compared to NS, p < 0.05. (c) Compared to 0.1 mmol/ kg of N-(R-L-arabinofuranos-1-yl)-L-cysteine, p < 0.001; (d) Compared to 0.2 mmol/kg of N-(R-L-arabinofuranos-1-yl)-L-cysteine, p < 0.001. (e) Compared to NS, p < 0.05; f) Compared to 0.1 mmol/kg of N-(RL-arabinofuranos-1-yl)-L-cysteine p < 0.01. (g) Compared to 0.2 mmol/ kg of N-(R-L-arabinofuranos-1-yl)-L-cysteine, p < 0.001. (h) Compared to NS, p < 0.05. (i) Compared to 0.1 mmol/kg of N-(R-L-arabinofuranos1-yl)-L-cysteine, p < 0.01. (j) Compared to 0.2 mmol/kg of N-(R-Larabinofuranos-1-yl)-L-cysteine, p < 0.01. (k) Compared to NS, p < 0.05. (l) Compared to 0.1 mmol/kg of N-(R-L-arabinofuranos-1-yl)-Lcysteine, p < 0.05. (m) Compared to 0.2 mmol/kg of N-(R-Larabinofuranos-1-yl)-L-cysteine, p < 0.05.

N-(R-L-arabinofuranos-1-yl)-L-cysteine. Lead accumulated in the livers, kidneys, left femurs, and brains of three-dose treated mice was measured and shown in Figure 4. The data indicate that lead accumulation progressively decreased with increasing dose. Therefore, N-(R-L-arabinofuranos-1-yl)-L-cysteine dose-dependently moves the lead accumulated in the tissues. ADMET Hepatotoxicity of N-(r-L-Arabinofuranos-1-yl)-Lcysteine. To estimate the prospect of N-(R-L-arabinofuranos-1yl)-L-cysteine as a lead detoxification agent, its hepatotoxicity was calculated according to the absorption, distribution, metabolism, elimination, and toxicity (ADMET) program (24, 25), which was developed from available literature data of 382 compounds known to exhibit liver toxicity (i.e., positive dosedependent hepatocellular, cholestatic, neoplastic, etc.) or trigger dose-related elevated aminotransferase levels in more than 10% of the human population. In general, two scores are defined to predict hepatotoxicity of the compounds from all individual trees that comprised the ensemble recursive partitioning model, namely, 0 and 1 as nonhepatotoxin and hepatotoxin, respectively. The prediction gave N-(R-L-arabinofuranos-1-yl)-Lcysteine a hepatotoxicity score of 0.059. The predicted score of hepatotoxicity is close to 0, which suggests that N-(R-Larabinofuranos-1-yl)-L-cysteine possesses no hepatotoxicity. Comparison of Lead Detoxification of the r- and β-Anomers. In the lead detoxification assay, the activities of R- and β-anomers of N-(L-arabinofuranos-1-yl)-L-cysteine were compared and shown in Figure 5. The data indicate that after the treatment of 0.4 mmol/kg of N-(L-arabinofuranos-1-yl)-Lcysteine the lead that accumulated in the livers, kidneys, femurs, and brains of the mice receiving the R-anomer is significantly lower than that accumulated in the livers, kidneys, femurs, and brains of mice receiving the β-anomer. This observation implies that the lead detoxification activity of N-(L-arabinofuranos-1yl)-L-cysteine significantly depends on the anomeric configuration. Comparison of the Membrane Permeability of the rand β-Anomers. In the membrane permeability assay of N-(Larabinofuranos-1-yl)-L-cysteine, the R-anomer had a 19.07 × 10-6 cm/s Papp value from the apical side to the basolateral side and a 5.99 × 10-6 cm/s Papp value from the basolateral side to

Lead Detoxification ActiVity

Figure 5. Lead levels of the tissue of mice after the treatment of the R- and β-anomers of N-(L-arabinofuranos-1-yl)-L-cysteine. (a) Data are represented with mg of lead per g of tissue (mean ( SD); n ) 10 and dose ) 0.4 mmol/kg. (b) Compared to the β-anomer, p < 0.01. (c) Compared to the β-anomer, p < 0.05.

the apical side, while the β-anomer had a 15.04 × 10-6 cm/s Papp value from the apical side to the basolateral side and a 8.00 × 10-6 cm/s Papp value from the basolateral side to the apical side. The active transport of the R-anomer is significantly higher than that of the β-anomer. These observations imply that the absorption of N-(L-arabinofuranos-1-yl)-L-cysteine significantly depends on the anomeric configuration.

Conclusions The lead accumulated in mice tissues could be efficiently removed by 0.1 mmol/kg of N-(R-L-arabinofuranos-1-yl)-Lcysteine, perhaps due to the Pb2+-N-(R-L- arabinofuranos-1-yl)L-cysteine complex having a high stability constant (15). The essential metals of mice could not be removed by 0.4 mmol/kg of N-(R-L-arabinofuranos-1-yl)-L-cysteine. Silico modeling predicted N-(R-L-arabinofuranos-1-yl)-L-cysteine having no hepatotoxicity. In Caco-2 cell monolayer tests, the Papp value of 19.07 × 10-6 cm/s from the apical side to the basolateral side identified N-(R-L-arabinofuranos-1-yl)-L-cysteine to be an actively absorbed agent. Therefore, N-(R-L-Arabino-furanos-1-yl)-L-cysteine should be a promising agent for lead detoxification therapy. Acknowledgment. This work was finished in the Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, supported by PHR (IHLB, KZ200810025010, and KM200910025009), the National Natural Scientific Foundation of China (30801426), and Special Project of China (2008ZX09401-002).

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