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The recently published report of the SELECT evaluation of selenium and vitamin E provided strong evidence that selenium 200mcg/day in the form of selenomethionine does not protect selenium-replete men against prostate or any other cancer. This appears to refute the result of the much smaller Nutritional Prevention of Cancer (NPC) trial of selenium. Since SELECT did not test the NPC agent, is possible that the difference between the two trials stems partly from the use of different agents: selenomethionine in SELECT, selenized yeast in the NPC trial. One of the organic selenium forms suspected of having strong chemopreventive effects, and which may have been present in the NPC agent, is methyl selenocysteine. This study characterizes the single-dose pharmacokinetics of methyl selenocysteine.
Selenium (Se) is an essential nutrient1; inadequate selenium nutrition has been associated with increased cancer vulnerability.2-6 However, the results of chemoprevention trials using different forms of selenium at supra nutritional levels have been largely disappointing.
In the NPC trial, administration of 200 mcg selenium per day in selenized yeast to nonmelanoma skin cancer patients was associated, after 7.4 years, with substantially decreased total cancer incidence, especially of the lung, colon and prostate, and with decreased total cancer mortality.7-9 These endpoints were, to be sure, secondary to the primary endpoint of non melanoma skin cancer recurrence: supplementation actually increased recurrence.10 The association of selenium supplementation with decreased risk was especially marked for prostate cancer.8 In a large trial largely motivated by NPC, Karp and colleagues randomized over 1500 patients with resected non-small cell lung cancer to selenized yeast or to placebo; the trial, designed for a 4-year treatment period, was halted after futility analysis showed that the endpoints of second primary tumors and progression free survival were not likely to be different in selenium and placebo groups 11. In the much larger SELECT study, closed after subjects were followed for an average of 5.5 years, a 200 mcg/day supplement of Se in the form of selenomethionine (SeMet) had no impact on the incidence of prostate or any other cancer. 12 Comparisons between the NPC and SELECT studies bring to light two differences of potential importance to the outcomes of the two studies. First, different forms of Se were used in the two trials:7,12,13 SeMet in SELECT, selenized yeast in NPC. On the other hand, the agent of the randomized trial led by Karp et al was selenized yeast (largely selenomethionine)11, and that showed no evidence of effectiveness. Another possible source of differences among these trials is that substantial numbers of the subjects in the NPC trial were close to being selenium deficient, while few of those in the lung cancer trial or SELECT were; the mean baseline plasma selenium level of NPC participants was approximately 115 ng/ml, that of SELECT participants approximately 136 ng/ml.7,8, 12
The mechanisms by which selenium might inhibit carcinogenesis or otherwise serve as a chemopreventive agent are not known. It has been proposed, but not proven, that a key mechanism may be protection against oxidative stress.14-20 Selenium supplies important proteins that protect against oxidative stress14, 21, so that those with inadequate selenium stores might be at increased risk; whether supranutritional doses of selenium would decrease oxidative stress further and thus protect against carcinogenesis is less clear.
Two major forms of selenium have been most widely used in supplementation: selenomethionine (SeMet) and selenite (S). SeMet and S are metabolized to hydrogen selenide, then to methylselenol: both of these metabolites may exert chemopreventive activity.22-25 Hydrogen selenide is critical to the formation of selenoproteins.25-26 Methyl selenol, with redox activity and possible effects on signaling, may be a key selenium metabolite in cancer prevention.21 SeMet is incorporated into cellular proteins in place of the sulfur-containing amino acid methionine. Given that methionine is common to all proteins, the displacement of a functionally important sulfur atom by selenium has the potential to alter protein structure and function. With continued ingestion, the pool of SeMet accumulates over time within the body; plasma levels increase, virtually without limit, even if toxicity develops.27 S is nonorganic; approximately 35% of a single 200 mcg dose is excreted in urine or feces within 12 days.22 By contrast, only ~ 15% of SeMet will have been similarly excreted within 12 days.23 Plasma selenium from these forms reaches a peak at approximately 8 hours, and persists for up to 24 hours. Approximately equal amounts of selenium in S are recovered from urine and feces,22 while twice as much selenium in SeMet is recovered from urine as from feces. An unidentified selenium isoform present in the NPC yeast, possibly MSC, may have been at least partly responsible for its apparent effects.13
MSC is water-soluble, absorbed in mammals from the gastrointestinal tract, and readily transformed to methylselenol.21, 24, 28 Methylselenol can be demethylated to yield selenide29-30 or methylated to yield dimethyl selenide, then released in the breath; methylated again, it yields trimethyl selenonium, which is excreted in urine. The role of selenium in cancer risk would be clarified by increased understanding of the pharmacokinetics of this methylated selenium compound. The purpose of this study was to characterize the toxicity and the pharmacokinetics of MSC in humans.
An IRB approved, phase I single-dose, dose-escalation pharmacokinetic/toxicity study of MSC was conducted at Roswell Park Cancer Institute (RPCI). Healthy male volunteers were recruited as subjects by public announcement in Buffalo, NY. After granting informed consent verbally and in writing, subjects were randomized, double-blinded, to receive either a single dose of MSC at one of three different concentrations or placebo. In the first wave, 5 patients received 400 mcg of selenium, and one received placebo; in the second wave, 5 patients received 800 mcg of selenium, and one received placebo; in the third wave, 5 patients received 1200 mcg of selenium, and one received placebo. The intent of placebo-group inclusion was to decrease the likelihood of participant reporting of inconsequential, subjective symptoms. The placebo arm experience is included in the results, although the statistical precision of estimates, with only 3 subjects, is very limited. Subjects were required to have normal hepatic, renal and bone marrow function as assessed by history, physical, and clinical chemistry analysis. They could not have given blood within 30 days of MSC administration, had to be 18 or older, had to have an Eastern Cooperative Oncology Group (ECOG) performance status31 of 0 or 1, and to weigh between 50 and 115 Kg. Eligibility was restricted to males, because the most powerful effect of selenium was believed to be against prostate cancer. Subjects could not be taking prescription or nonprescription drugs, vitamins or herbal supplements known to affect gastric acidity within three days of drug administration. Subjects arrived at RPCI at 7:00 am on the day of their pharmacokinetic analysis after a fast beginning at 10:00 pm the previous night. After a brief review of concurrent medications, vital signs and symptoms, subjects had an intravenous catheter placed in one arm. A baseline pre-dose blood sample was drawn through the catheter, after which subjects ingested the assigned agent along with 8 ounces of water, under direct supervision. Subjects remained in hospital for 12 hours, returning at 24 and 48 hours. In each cohort of 6 men, five were randomized to MSC, one to placebo. Blood was drawn at baseline and at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24 and 48 hours after dosing. Urine was collected within time spans of 0-4, 4-8 and 812, and at 12 and 24 hours. Subjects in the first, second and third cohorts received 400, 800, and 1200 mcg of Se in the form of MSC or placebo, respectively. All subjects in each cohort were treated and evaluated for toxicity prior to treating subjects on the next cohort. The occurrence of grade 2 or greater toxicity thought at least possibly due to drug was to preclude escalation to the next higher dose.
Quantitative descriptors of subjects—age, race, baseline plasma selenium, toenail selenium, height, weight and ECOG31 performance status--were compared by means and standard deviations. Statistical significance of between-group differences was evaluated by one-way analysis of variance (ANOVA). The statistical significance of category variation in race (white or non-white) among the treatment groups was considered by chi square. Baseline comparisons among the four different treatment groups showed no statistically or substantively significant difference in age, race, plasma or toe nail selenium, height, weight or ECOG performance status (Table 1). The ECOG score ranges from 0 to 5, with zero indicating that the subject is fully active and able to carry on all activities of daily living without restriction, five indicating death. That the mean ECOG score was zero means that subjects were in general quite healthy, experiencing no noteworthy limitations.
The pharmacokinetic parameters of maximum concentration (Cmax) and area under the curve (AUC), were compared using one-way ANOVA, with alpha = .05. Paired comparisons were used to evaluate the significance of mean differences of each treatment group from the placebo group. The pharmacokinetic program WinNonlin was used to estimate half life for each dose-specific treatment group. Box-plot graphics were also used to describe in more detail differences in Cmax and AUC. As the statistical power of the comparison of each group to the placebo patients is limited by the small number of placebo patients, interpretation of the findings is necessarily conservative.
Toxicity was evaluated in all subjects. History was reviewed at baseline; physical examinations were conducted at baseline, at 12 hours and at one week post-dose. Vital signs were checked at baseline, and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 and 48 hours, and at one week post-dose. Clinical laboratory studies, which included a hemogram, SGOT/SGPT, total bilirubin, serum electrolytes with BUN and creatinine, and urinalysis, were performed at baseline, 24 hours and one week post-dose. At 30 days post-dose, subjects were contacted by telephone for toxicity assessments. All toxicities for all consented subjects were recorded and graded according to the National Cancer Institute's Common Toxicity Criteria (CTC) version 3.0.
The primary endpoint of this study was toxicity. A total of 25 adverse events were reported in 18 subjects (Table 2); all were grade 1. We observed no association between assignment to MSC and the occurrence of adverse events; nor was any association between the dose of MSC and the occurrence of adverse events apparent.
Figure 1 describes the courses of mean plasma Se concentration among subjects. Each subject's plasma selenium is expressed as a deviation from its baseline level. The most distinct concentration curve is for the 1200 mcg dose, although the curve of the 800 mcg dose slightly exceeds that of the 400 mcg dose, and that of the 400 mcg dose exceeds that of placebo. For those receiving MSC, maximum concentration times are similar, ranging between 3 and 5 hours for the 400 through 1200 mcg cohorts.
The pharmacokinetic parameter estimates of those curves are in Table 3. Mean Cmax for the placebo group reflects values of of 9, 10 and 11 ng/ml; two of those maximum values are seen near 24 hours, the other at around 4 hours. The mean Cmax increases in dose-response fashion from 10 for placebo to 22.8, 30.75 and 63.2 ng/ml for 400, 800 and 1200 mcg subjects, respectively. Mean Cmax for the 1200 mcg subjects is significantly and substantially greater than that of placebo subjects: approximately twice that of subjects who received the 800 mcg dose. For those receiving MSC, mean Cmax times are similar, ranging between 3 and 5 hours for the 400-1200 mcg cohorts. The time of Cmax for the 1200 mcg cohort is in fact the shortest.
Figure 2 describes in greater detail the pattern of Cmax among the four treatment groups. In this box-plot graphic, the longest horizontal bar refers to the median, and the shorter bars to the 25th and 75 percentile values. The individual subject values are displayed with distinct symbols for the different doses. It can be seen that Cmax increases substantially with increased selenium dose. The increase is not monotonic, however, as median Cmax of the 1200 mcg dose is roughly three times that of the 400 mcg dose, twice that of the 800 mcg dose.
Area under the curve (AUC) values are also in Table 3. The mean and median values for the 400 and 800 mcg cohorts are greater than those of the placebo cohort, and their excesses over those of placebo statistically significant. However, the mean and median AUC of the 1200 mcg cohort are nearly twice those of the 800 mcg cohort. It is not possible to derive convergent estimates of half life for subjects receiving the 400 or 800 mcg doses; for those who received 1200 mcg, however, half life is estimated to be 29 hours (not shown). Figure 3 graphically describes this analysis: the AUCs of both the 400 and 800 mcg doses are elevated, although the difference is slight; the elevation of the 800 mcg dose is significantly greater than that of the placebo. On the other hand, the AUC for the 1200 mcg dose is significantly greater than that of placebo, nearly twice that of the 800 mcg dose.
Figure 4 describes urinary concentration of Se for subjects receiving each dose by interval of excretion. Since the baseline levels of subjects were in general below the level of detection, those concentrations are not expressed as deviations from baseline levels. Most of the urine values for subjects receiving placebo remain below the limits of detection, so those are not presented. The mean values of subjects who received 400 or 800 mcg of Se exhibit discernible peaks 4 to 8 hours post dose. Excretion among subjects who received the 800 mcg dose is slightly higher than that of subjects who received 400 mcg. For subjects receiving 1200 mcg Se, the mean peak concentration is approximately twice that of subjects who received 400 or 800 mcg doses; the maximum point of this excretion is seen 8-12 hours post dose.
These data represent a first examination of methyl selenocysteine (MSC) as administered to humans. There was no evidence of toxicity.
A substantial body of preclinical data indicates that selenium is important to protection against oxidative stress.14-21, 29, 32-34 Selenium deficiency is also associated with increased cancer risk;22-23, 35-36 whether supplementation of those who are selenium deficient might decrease vulnerability to oxidative stress is not well known; whether an agent that protects against oxidative stress would protect against carcinogenesis is not known. SELECT suggests that, among men who are selenium replete, selenium offers no such protection 12.
Preclinical toxicology studies of MSC—long and short term—have been performed by the National Cancer Institute, through the Division of Cancer Prevention (DCP) Rapid Access to Preventive Intervention Development (RAPID) Program.37-38 The studies, conducted on rats and dogs, showed dogs to be the most sensitive species. The no adverse effect level after 28 and after 90 days of dosing in dogs based on histopathologic and hematologic findings was 0.3 mg MSC/kg-bw/day (0.13 mg Se/kg-bw/day); an equivalent value extrapolated to humans for a 70 kg person is 21,000 mcg MSC/day, or 9,100 mcg Se/day. Single, bolus doses of MSC were largely converted to excretory metabolites in breath and urine; even chronic, high doses of MSC may lead to very modest tissue accumulations of Se.34,36 NCI, DCP-sponsored genotoxicity studies with MSC were negative.38
Selenium is a natural dietary constituent, so that varying baseline concentrations were present in the plasma of subjects. In order to accurately gauge the pharmacokinetic parameters, we took these baseline values into consideration. It will be important to take a similar approach in future investigations. This factor, along with the single-dose nature of the study design and the relatively low doses investigated, limits the conclusions that can be drawn. However, these are inherent to the study of a first-time-in-human chemopreventive agent, and represent safeguards purposefully built into the design of the study. These factors notwithstanding, this study provides some important initial findings.
There was little difference in the pharmacokinetic parameters of the 400 and 800 mcg doses. The values observed were clearly above those observed in the placebo cohort. There are important differences when the 1200 mcg cohort is compared to the other two MSC cohorts. Together, they are consistent with the possibility of saturation in metabolism and/or excretion. This is supported by the finding of a shorter time of maximum concentration in the 1200 mcg cohort; that time trends downward as dose increases, and it is coupled with a delay in the peak urinary excretion time. The time of maximum concentration for the 1200 mcg dose of MSC appears to be shorter than for SeMet,23 although it needs to be evaluated in a direct pharmacokinetic comparison.
The Cmax and AUC for the 1200 mcg cohort are proportionally greater than the respective values for the 800 mcg cohort. There are several potential implications to this for design of multi-dose studies. Specifically, given the possibility for accumulation, multi-dose studies should not exceed the 1200 mcg dose until additional information becomes available. This value is approximately one eighth the value extrapolated from tests on the most sensitive non-human mammal. Second, this finding is not consistent with MSC moving into a large storage reservoir. This contrasts sharply with SeMet; as SeMet accumulates within essentially all proteins, the body provides a vast storage reservoir. This may explain why selenium concentration increases so directly with multiple doses of SeMet.
Preclinical research indicates MSC provides a more efficient route than SeMet to the formation of methyl selenol, a metabolite that may impart a chemopreventive effect.24-25, 35, 39 It will be important in the future to accurately speciate methyl selenol and related plasma selenium metabolites. This represents an evolving field, and work is actively progressing.30, 36 Once the technology is in place, it can be applied to stored samples. The major downstream protein products of selenium supplementation, selenoprotein P and glutathione peroxidase, which are the key and most abundant selenoproteins in plasma, can be readily evaluated. Indeed, in that the function of selenium resides primarily in the proteins to which it gives rise, a focus on these key selenoproteins may be highly informative.27 Analysis of these is under way.
Work by Ip and others suggests that methyl selenocysteine (MSC) might prove more physiologically relevant than SeMet or S. It has efficacy in preclinical prevention models, and it may therefore represent an important potential chemopreventive agent, if only for those who are selenium deficient.14, 21, 24-26, 28, 39 MSC is a close derivative of selenocysteine (SeCys). SeCys is referred to in the literature as the physiologic form of selenium30; it is necessary for the synthesis of selenoproteins, and these proteins appear to be responsible for selenium's physiological effects in humans.
The SELECT results leave little room for hope that selenium as SeMet will prove of chemopreventive efficacy for selenium-replete subjects 12. Whether supplementation will be of benefit to those who are not selenium replete is less clear. Moreover, the form of selenium administered in SELECT, SeMet, may have important limitations. It is important not to extrapolate without adequate evidence the findings of SELECT (i.e. SeMet) to all Se compounds and all populations. Preclinical findings, as well as findings in humans now available through the current study, demonstrate in a corroborative fashion that there may be important differences between MSC and SeMet.21, 24-27 Taken together, prior studies and findings from the current study support continued investigation of the role of MSC in cancer risk among humans.
Manuscript supported by NCI contract N01-CN-35157, NWU 04-4-02: “Phase I study of single oral dose of semethylseleno-L-cysteine (MSC) in adult men”.
Special thanks to Linda Schmieder, NP at Roswell Park Cancer Institute for her oversight of the protocol implementation and follow up.