Research On Pharmacokinetics, Toxicity And Effects Of Meso-2, 3-Dimercaptosuccinic Acid On Lead Intoxication

Objective:To develop a quantitative method to analyze DMSA in biological samples, to research distribution of DMSA in tissues in mice and pharmacokinetics in human, and to explore acute toxicity and effects of meso-2, 3-dimercaptosuccinic acid on lead intoxication.Methods:1. The stability of DMSA and mBBr-DMSA derivative was researched, the optimum condition was explored, including pH value of reaction mixture, reaction time and concentration of fluorescent reagent of mBBr in reaction mixture. An assay to determine DMSA in biological samples has been developed.2. Twenty Kunming mice (10 males and 10 females) weighting approximately 21.2±2.3g were acclimatized for 3 days prior to experiment, then were divided into control group and experiment group with 10 mice in each group according to body weight. Fasted for 12 hours, the mice in experiment group received intragastric administration of 160mg DMSA in 24 hours, and the control group received the same volume of normal saline, and then they were observed for 7 days. Blood was collected into heparinized-tubes by removal of eyeball. All mice were sacrificed and brain, heart, liver and kidney were removed, washed with normal saline. The activity or amount of BUN, Scr, AST, ALT, SOD, GSH-PX, MDA and essential trace element were analyzed.3. Sixty Kunming mice (30 males and 30 females) weighting approximately 21.3±2.0g were acclimatized for 3 days prior to experiment, then were divided into control group (10 males and 10 females) and experiment group (20 males and 20 females) according to body weight. The experiment group received intraperitoneal injection of 1 mg lead acetate per gram body weight once daily for 7 days, the control group received the same volume of normal saline. Lead exposure was stopped, and mice were acclimatized for 1 day. The control and experiment group was divided into I and II according to body weight, respectively. Each group had 3 groups, control group, lead group and DMSA group in detail. DMSA group of I received 1.3mg DMSA, orally thrice daily for 3 days, and that of II 1.3mg DMSA, orally twice every other day, and 10 times in all. The other groups received the same volume of normal saline. Blood was collected into heparinized-tubes by removal of eyeball, 24 hours after the last dosing. All mice were sacrificed and brain, heart, lung, liver, kidney, femoral bone and skeletal muscle were removed, washed with normal saline. The activity or amount of BUN, Scr, AST, ALT, SOD, GSH-PX, MDA and essential trace element were analyzed.4. Thirty-six Kunming mice (18 males and 18 females) weighting approximately 18.7±1.3g were acclimatized for 3 days prior to experiment, then were divided into 6 groups with 6 mice (3 males and 3 females) in each group according to body weight. One was control group, and the others experiment groups. The experiment groups orally received 1.3mg DMSA. The control and experiment groups were sacrificed, respectively before and at 0.5, 2, 4, 6 and 12 hours after administration. Blood was collected into heparinized-tubes and brain, heart, lung, intestine, liver, spleen and kidney were removed, washed with normal saline. The amount of DMSA in blood and tissues were determined by HPLC-fluorescence detection and the feature was summarized.5. Twenty normal male volunteers (23.1±1.94years, 65.8±6.47kg) were fasted for 10 hours. Blood and urine samples were collected at zero time. Immediately afterwards 500mg DMSA were given p.o., followed with 200mL deionized water. Blood samples (4mL) were drawn into heparinized-tubes at 0.5,1.0,1.5,2.0,2.5, 3.0, 4.0, 5.0, 6.0, 8.0, 12.0 and 24.0 hours after DMSA administration. Blood samples were divided into 2 tubes, one was immediately stored frozen at -70℃and protected from light, the other was prepared for plasma and stored frozen at -70℃and protected from light. Complete urine collections were made at precisely 2, 4, 6, 8, 12 and 24 hours after DMSA administration, and urine samples were stored frozen at -70℃and protected from light. The amount of unaltered and total DMSA in blood, plasma and urine were determined by HPLC-fluorescence detection. Kinetic parameters were calculated and evaluated using program of DAS.Results:1. DMSA is susceptible to oxidize at ambient temperature and exposed to light, and stable frozen at -20℃and protected from light for at least 3 months without significant loss of DMSA. A method has been developed in which DMSA was converted to a highly fluorescent and stable derivative by reaction with mBBr in aqueous solution at pH 8.2. The reaction was rapid and complete within 5 min. At least 20-fold excess of mBBr was recommended for quantitative analysis of DMSA in reaction mixture and biological samples.2. Given 160mg DMSA in 24 hours, gastrointestinal symptoms are main side effects. During the observation, experiment group lost weight due to the decrease of foodintake. And some mice had slight hydroabdomen. The amount of BUN and Scr significantly rose due to the damage of kidney (P < 0.001). The aminopherase rosed significantly in blood (P < 0.001), but not in liver and kidney (P > 0.05), which was related to acute toxicity on liver. The effect of DMSA on antioxidant systems of mice was examined. High dose of DMSA caused a significant inhibition of GSH-PX without significant loss of Se, possibly due to formation of disulfide between DMSA and cysteine. The activity of SOD was inhibited in accordance with the change of Mn, Cu and Zn, but no significance. The hepatic cell was damaged according to the raise of MDA in liver.3. The BUN and Scr significantly rose after lead exposure (P < 0.05), and rose further without significance after administration of DMSA (P > 0.05), which was possibly due to the renal excretion of DMSA, lead and chelate compounds. DMSA could ameliorate lead-induced change of aminopherase in blood and liver and inhibition of antioxidant systems. The essential trace element lost balance due to the competition of lead, especially Mn, Cu and Zn, and DMSA could increase excretion of beneficial element.4. After oral administration, DMSA rapidly distributed into blood and other tissues. The concentration of DMSA reached the maximum at 0.5 hours after administration of DMSA in blood, intestine, liver and heart, and 2 hours in kidney, lung, spleen and brain. DMSA mainly distributed in kidney, then blood, intestine, lung, liver, spleen, heart and brain. DMSA was metabolized in liver and kidney, and excreted in kidney, which could explain the damage to liver and kidney. The amount was lowest in brain, however, lead in brain was effectively excreted.5. DMSA pharmacokinetic parameters of single dosage in volunteers were as followed: t_1/2, 7.94±2.10h; C_max, 3.77±1.26μg·mL^-1; t_max, 2.45±0.22h; AUC, 24.35±7.37μg·mL^-1·h; nAUC, 27.63±8.42μg·mL^-1·h; aumc, 188.24±51.89μg·mL^-1; MRT, 7.81±0.50h; Ke, 0.99±0.70h^-1; Vd, 1.10±0.70L·kg^-1; CL, 0.33±0.11L·kg^-1·h^-1. Approximately 50% of total DMSA was free, unaltered in blood, and no free, unaltered DMSA was detected in plasma. The contribution of the plasma total DMSA concentration to the blood total DMSA concentration was determined by adjusting the plasma data using the experimentally determined hematocrits. This was done by multiplying the plasma data by (1-hematocrit). The mean adjusted plasma curve was the same as the mean blood curve (P > 0.05) indicating that DMSA does not enter blood cells. The excreted total DMSA in 24 hours represented 30.18%～47.74% of the dosage, however, only 0.26%～1.54% of total DMSA was found free and unaltered in urine.Conclusions:An assay has been developed to determine DMSA in blood, plasma, urine and tissue homogenate. It is sensitive, selective, valid, reproducible, simple and timesaving.After oral administration, DMSA rapidly distributed into blood and other tissues, and mainly in kidney, then blood, intestine, lung, liver, spleen, heart and brain. DMSA was metabolized in liver and kidney, and excreted in kidney, which could explain the damage to liver and kidney. The amount was lowest in brain, however, lead in brain was effectively excreted.High dosage could do damage to liver and kidney, inhibit the antioxidant systems, and increase excretion of beneficial element. Therapeutic dose could excrete lead effectively, protect liver and kidney from lead-caused damage, and ameliorate lead-induced disorder of essential trace element, meanwhile, excrete beneficial element. So periodic inspection of hepatic and renal function should be taken during clinical application of DMSA, and it is better to combine DMSA, vitamin C and trace element in therapy.Approximately 50% of total DMSA was free, unaltered in blood, and no free, unaltered DMSA was detected in plasma. The majority of DMSA excreted in kidney. The excreted total DMSA in 24 hours represented 30.18%～47,74% of the dosage, however, only 0.26%～1.54% of total DMSA was found free and unaltered in urine.