The scientific basis for chelation: animal studies and lead chelation

Abstract

This presentation summarizes several of the rodent and non-human studies that we have conducted to help inform the efficacy and clinical utility of succimer (meso-2,3-dimercaptosuccincinic acid) chelation treatment. We address the following questions: (1) What is the extent of body lead, and in particular brain lead reduction with chelation, and do reductions in blood lead accurately reflect reductions in brain lead? (2) Can succimer treatment alleviate the neurobehavioral impacts of lead poisoning? And (3) does succimer treatment, in the absence of lead poisoning, produce neurobehavioral deficits? Results from our studies in juvenile primates show that succimer treatment is effective at accelerating the elimination of lead from the body, but chelation was only marginally better than the complete cessation of lead exposure alone. Studies in lead-exposed adult primates treated with a single 19-day course of succimer showed that chelation did not measurably reduce brain lead levels compared to vehicle-treated controls. A follow-up study in rodents that underwent one or two 21-day courses of succimer treatment showed that chelation significantly reduced brain lead levels, and that two courses of succimer were significantly more efficacious at reducing brain lead levels than one. In both the primate and rodent studies, reductions in blood lead levels were a relatively poor predictor of reductions in brain lead levels. Our studies in rodents demonstrated that it is possible for succimer chelation therapy to alleviate certain types of lead-induced behavioral/cognitive dysfunction, suggesting that if a succimer treatment protocol that produced a substantial reduction of brain lead levels could be identified for humans, a functional benefit might be derived. Finally, we also found that succimer treatment produced lasting adverse neurobehavioral effects when administered to non-lead-exposed rodents, highlighting the potential risks of administering succimer or other metal-chelating agents to children who do not have elevated tissue lead levels. It is of significant concern that this type of therapy has been advocated for treating autism.

Fig. 1

Study design and timing of testing in the primate lead chelation study (Smith et al. [11]). [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 2

Blood lead levels in the four groups of non-human primates receiving oral lead exposure for either the first 1 or 2 years of life. Each lead exposure group was divided into succimer or vehicle placebo groups, as indicated (n = 10–12/group). Inset panel shows expanded timeline over the first chelation regimen. (Smith et al. [11]). [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 3

Mean ± SE blood total lead levels (as % of day 0 pretreatment values) in succimer and placebo-vehicle-treated 1-year lead-exposed monkeys over the course of the first chelation treatment (days 0–20) and beyond. Placebo and succimer group n = 21–23/group for days 24 to 20, and n = 11/group for days 24 to 60. (Inset) The mean ± SE integrated area under the curve (AUC; days 0 to 20) for the placebo vehicle and succimer groups. ***Statistically different (p < 0.001) from placebo group (comparisons performed only on treatment day 20 and on AUC data). For reference, blood lead levels on day 0 were 43 and 50 mcg/dL for the placebo and succimer groups, respectively (p = 0.08). Data from Smith et al. [11]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 4

a, b Mean ± SE urinary excretion of total lead over the first chelation treatment regimen in 1-year lead-exposed monkeys. Left panel shows daily 24-h total urinary lead elimination, while inset panel shows mean 5-day cumulative total lead in placebo-vehicle- and succimer-treated groups. Placebo and succimer group n = 24/group over days 0 to 5, and n = 8/group for days 10 and 20. ***Statistically different (p < 0.001) from placebo group (comparisons performed only on data in b). Data from Smith et al. [11]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 5

Five-day cumulative urinary total lead excretion by individual succimer- and placebo-treated 1-year lead-exposed monkeys over the first chelation regimen. Each bar represents an individual animal. Symbols above the bar in the succimer group, when present, identify apparent hyper (@) or hypo (#) responders to succimer treatment. Data from Smith et al. [11]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 6

Left panel a—effect of oral succimer on urinary and fecal excretion of total lead (inherent lead + stable lead isotope tracers). Right panel b—total urinary and fecal excretion of orally administered ²⁰⁶Pb tracer over the first 5 days of succimer treatment; right panel c—total urinary and fecal excretion of i.v. administered ²⁰⁴Pb tracer over the first 5 days of treatment. Bars are mean ± SE for the vehicle (n = 7) and succimer (n = 8–9) groups. *Significantly different from placebo-vehicle control (p < 0.05). Data from Cremin et al. [12]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 7

Effects of oral succimer on the retention of orally administered lead absorbed across the gastrointestinal tract (oral ²⁰⁶Pb tracer) and i.v. administered lead (i.v. ²⁰⁴Pb tracer) expressed as a percentage of the administered (²⁰⁴Pb tracer) or calculated (²⁰⁶Pb tracer) internal dose. The internal dose is the amount of the endogenous ²⁰⁴Pb tracer injected i.v. or the amount of the oral ²⁰⁶Pb tracer absorbed from the GI tract. The sum of the lead tracer retained over the first 5 days of succimer treatment is expressed as a percentage of the internal dose, which equals the amount of oral ²⁰⁶Pb tracer absorbed or the amount of ²⁰⁴Pb tracer that was injected. The bars represent mean ± SE for the vehicle (n = 7) and succimer (n = 9) groups. *Succimer group differs from the vehicle group according to an ANOVA (p < 0.05). Data from Cremin et al. [12]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 8

Example of the oral lead doses (triangles, mg Pb/kg body wt.) and resultant blood lead levels over the lead exposure (filled circles) and chelation treatment (open diamonds) periods in a representative monkey from the succimer treatment group. The treatment schedule is indicated below the x-axis. Data from Cremin et al. [12]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 9

Lead concentrations in blood of the vehicle- (filled circle, n = 4–5) and succimer- (open diamond, n = 5–6) treated groups over the succimer treatment period. The values presented are the means ± SE of blood lead concentrations. Statistical analyses were conducted on data adjusted to starting (day 0) blood lead levels for each animal, in order to control for variation in the initial blood lead concentrations among animals. Comparisons of mean blood lead values: means with different superscript letters (e.g., a, b, and c) are statistically significantly different from one another. Data from Cremin et al. [13]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 10

Lead levels in brain prefrontal cortex (PFC, pre- and post-treatment), frontal lobe, hippocampus, and striatum of the vehicle- (solid bars, n = 5) and succimer- (hatched bars, n = 6) treated groups (lead levels expressed as a percentage of the pretreatment PFC level within each animal). Values are means (±SE). Mean values of lead concentrations in the pretreatment PFC biopsy for the vehicle- and succimer-treated groups were 1,980 and 1,410 ng/g dry wt., respectively. Symbols below the x-axis indicate: * = pre- and post-treatment mean values were significantly different; a, b, and c comparisons of post-treatment brain regions. Means with different letters were significantly different from one another. Data from Cremin et al. [13]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 11

Relative enrichment of ²⁰⁴Pb tracer levels in brain prefrontal cortex (PFC, pre- and post-treatment), frontal lobe (FL), hippocampus (H), and striatum (S) of the vehicle- (solid bars, n = 4) and succimer-(hatched bars, n = 6) treated groups (²⁰⁴Pb tracer relative enrichment levels expressed as a percentage of the pretreatment PFC level within each animal). Values are means (±SE). Mean (relative enrichment levels are expressed as a percentage of the pretreatment PFC level within each animal. Mean relative enrichment values of the pretreatment PFC were 0.291 and 0.317 % for the vehicle- and succimer-treated groups, respectively. Symbols below the x-axis indicate: NS pre- and post-treatment mean values were not significantly different; a, b comparisons of post-treatment brain regions; means with different letters were significantly different from one another. # trend (p < 0.10) towards a significant difference between the vehicle and succimer groups. Data from Cremin et al. [13]. [Reprinted from Toxicology and Applied Pharmacology with permission from Elsevier]

Fig. 12

Relative benefit of none, one, or two succimer chelation regimens for reducing blood and brain lead levels in rats. Data highlight the additional benefit of a second regimen of succimer treatment (vs. one regimen and vehicle treatments) in reducing blood and brain lead levels in rats, as a function of time since the second regimen ended. Data from Stangle et al. [14]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 13

Study design and timing of testing in the rodent succimer chelation study (Stangle et al. [20]; Beaudin et al. [21]) [Stangle reproduced with permission from Environmental Health Perspectives; Beaudin reprinted from Neurotoxicology and Teratology with permission from Elsevier]

Fig. 14

Succimer treatment significantly improved learning ability of the Mod-Pb rats. a Visual discrimination task (Mod-Pb–succimer vs. Mod-Pb; main effect contrast, p = 0.03). b Attention task 1. Data points are means ± SEs. *p = 0.056; **p ≤ 0.03; #p < 0.01, Mod-Pb vs. control. ##p = 0.03; †p = 0.006, Mod-Pb–succimer vs. Mod-Pb. Data from Stangle et al. [14]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 15

The impaired learning ability of the high-Pb rats was only somewhat alleviated by succimer chelation. Data show percent premature responses in attention task 1 as a function of the stage of testing (stages divided into blocks of sessions). Superscripts above symbols indicate level of significance for the contrasts between the high-Pb group and controls, #p < 0.07, *p < 0.05. Data from Stangle et al. [14]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 16

Heightened reactivity to errors of the high-Pb rats was completely normalized by succimer treatment. Left panel shows percent omission errors on the attention task 2 across session blocks for trials that followed a correct response on the prior trial (left panel), and for trials that followed an error on the prior trial (right panel). Note that no significant lead or succimer effects were evident for trials follow a correct response (left panel), but there was a significant lead effect on trials following an error on the prior trial, and succimer alleviated this effect (right panel). **p < 0.01, high-Pb vs. control. #p < 0.01, high-Pb + succimer vs. high-Pb. Data from Stangle et al. [20]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 17

Succimer treatment of the non-lead-exposed rats impaired performance in a visual discrimination task (main effect contrast, p = 0.04), b attention task 1, and c the sustained attention task (treatment × cue duration, p = 0.004). Data points are means ± SEs. *p = 0.07; **p < 0.05, succimer-only vs. controls. Data from Stangle et al. [20]. [Reproduced with permission from Environmental Health Perspectives]

Fig. 18

This figure depicts the percent inaccurate responses in the selective attention task, as a function of whether or not a distractor was presented on the current trial and whether the prior trial was correct (a) or incorrect (b). The rats treated with succimer in the absence of lead exposure performed significantly worse than controls, with the largest impairment seen for trials that both included a distractor and followed an error. Data are means ± SEs. *p = 0.02; **p < 0.01, succimer-only vs. controls. Data from Stangle et al. [20]. [Reproduced with permission from Environmental Health Perspectives]