Toxicology: October 2008 Archives

Along with co-applicants Mark and David Geier, TAP Pharmaceuticals* filed an international patent application (PCT/US2007/082866), "Methods of Treating Autism and Autism Spectrum Disorders," in October of last year for the use of Lupron (leuprolide acetate, a GnRH analog), with or without chelation, in children with autism. (A big discovery hat tip for finding this patent application, along with related US patent applications, goes to Kathleen Seidel of the Neurodiversity blog.) The text of this application was published in August May of this year, and treatment descriptions in 7 children can be found if the reader is willing to wade through a lot of repetitive verbiage—including a seemingly endless string of "need in the art," "known in the art," and "skilled in the art."

Essentially, the treatment "invention" of TAP and the Geiers is intended to lower elevated mercury levels in autistic children by giving the chemical castrator Lupron. The applicants base this idea on a 1968 article, which showed that mercuric chloride complexes with testosterone in a hot benzene solution, a condition not possible in living organisms. The patent idea of TAP and the Geiers is to lower testosterone in autistic children by giving them Lupron, which then supposedly frees up toxic mercury. The idea is that the freed-up mercury can then be eliminated with the aid of chelation therapy, if necessary.

However, TAP and the Geiers don't limit their endocrinologic therapy to Lupron, nor their disease targets to autism. They also include the treatment possibilities of antiandrogen hormones (eg, cyproterone [Androcur; Schering-Plough AG]) and birth control pills and propose that mercury toxicity is implicated in Alzheimer's disease, diabetes, heart disease, obesity, amyotrophic lateral sclerosis, asthma, and immune disorders.

What follows is a summary of the pediatric "examples" of TAP's and the Geiers' so-called invention. In all cases of autism, remarkable improvements in gastrointestinal symptoms (if present) and social/cognitive skills are described, sometimes within days of what are described as well-tolerated injections of Lupron.

Subject ages: Four children were of prepubertal age (two 6-year-old boys; a 7-year-old girl; and an 8-year-old boy), and 3 children were within the age range of puberty or beyond (an 11-year-old girl, an 11-year-old boy, and an 18-year-old boy).

Previous treatments: Two of the children (a 6-year-old boy and the 8-year-old boy) had undergone previous chelation therapy with DMSA for approximately 11 and 15 months, respectively. Clinical improvement is described in the case of the 8-year-old boy; the 6-year-old boy did not demonstrate improvement with chelation, according to the patent application. The 11-year-old girl received prescription amphetamines (Adderall; Shire) for the diagnosis of attention deficit hyperactivity disorder, given at the age of 5 years.

Claims of clinical signs of precocious puberty: In no case of the prepubertal or pubertal children is the Tanner stage noted in the patent application. Signs of precocious puberty in 3 of the prepubertal children are vaguely described as increased body, leg, or facial hair; masturbation; "genital development"; or "early sexual behaviors." The 11-year-old girl showed "mild signs of precocious puberty" (whatever those may be) and "fully developed pubic hair" by 8 years of age. These descriptions were presumably obtained in retrospect by history. The girl also began menstruating at the approximate age of 10 years—which is earlier than average, but not precocious.

The performance of GnRH stimulation tests in the patient examples, as recommended by the Lupron Prescribing Information, is not described by the patent applicants. Other diagnostic criteria for central precocious puberty described in the PI, including the documentation of advanced bone age and a number of baseline tests (to exclude congenital adrenal hyperplasia, a chorionic gonadotropin-secreting tumor, a steroid-secreting testicular tumor, and an intracranial [eg, pituitary] tumor), are also not documented in the application. Addendum: Head MR imaging was performed in 2 individuals; however, the timing of the imaging is not provided by the patent applicants.

Mercury assessment: Mercury levels in the absence of chelation therapy are provided only in the case of the 11-year-old boy, whose blood showed "minimal" levels of mercury (1.5 μg/L; reference range, 0.0-14.9 μg/L) and whose urine did not reveal the presence of mercury. The patent applicants claim that urinary porphyrins (specifically urophorphyrin[sic] and hexacarboxylphorphyrins[sic]) were elevated in the 11-year-old girl. Measurements of urinary porphyrins have been proposed by the Geiers to be surrogate markers for mercury toxicity in autism on the basis of a rat study.

Thiol levels: Given that the Geiers have proposed a "decreased detoxification capacity" in children with autism, defined by certain thiol levels, these metabolites were measured in 2 children. Levels of plasma cysteine and reduced glutathione (GSH) were measured during and after the 6-year-old boy's initial chelation therapy, and selected thiol levels were measured in the 8-year-old boy after his Lupron/chelation therapy. The plasma cysteine levels, although below the reference range contained in the patent application, are within the control ranges of those in the literature. In the case of plasma GSH, the levels (when converted to units of μmol/L) are orders of magnitude greater than those found in relevant medical/science articles.

Plasma Metabolite, μmol/L	8-Year-Old Boy	6-Year-Old Boy	Patent Reference	Literature Reference
Homocysteine	5	—	Not given	6.0 ± 1.3a
Cysteineb	226	212	255-320	207 ± 22a
Sulfateb	302	—	302	369-451c
Reduced GSHb	651	651	>1041	2.2 ± 0.9a

^a From James et al, 2006.
^b Presented by the patent applicants in units of mg/dL.
^c From Chattaraj and Das, 1992. 

Testosterone levels: According to the patent applicants, baseline serum testosterone levels were elevated in 2 of their subjects: a 6-year-old boy (23 ng/dL; reference range, 0-20 ng/dL) and the 7-year-old girl (18 ng/dL; reference range, 0-10 ng/dL). They also emphasize high-normal levels of serum testosterone in the 8-year-old boy (25 ng/dL; reference range, 0-25 ng/dL) and the other 6-year-old boy (20 ng/dL). Follow-up testosterone levels predictably rose and then dropped during the Lupron therapy. (According to the Lupron PI, "During the early phase of therapy, gonadotropins and sex steroids rise above baseline because of the natural stimulatory effect of the drug. Therefore, an increase in clinical signs and symptoms may be observed.")

Lupron therapy: The Lupron therapy for the 7 pediatric subjects is tabulated. Therapy was not uniform and, in some cases, involved supplementation with the non-depot (subcutaneous) formulation of Lupron. The recommended starting dosage for Lupron Depot-Ped, according to the PI, is 0.3 mg/kg every 4 weeks: 7.5 mg if ≤25 kg (≤55 lb); 11.25 mg if 25-37.5 kg (55-82.5 lb); and 15 mg if >37.5 kg (>82.5 lb). How the doses of 22.5 mg IM in the cases of the 8-year-old boy and a 6-year-old boy were derived is not stated by the patent applicants. The Lupron PI also indicates that, if downregulation is not achieved (via GnRH stimulation testing and Tanner staging), the dose should be titrated upward in increments of 3.75 mg every 4 weeks.

Subject	Lupron Therapy
8-year-old boy	Depot, 22.5 mg IM on 11/24/04, 1/20/05, 3/25/05, 5/25/05, and 7/14/05
6-year-old boy	Depot 22.5 mg IM on 4/2/05, 5/21/05, and 7/9/05
6-year-old boy	Depot 15 mg IM followed immediately by 0.2 mL (55 μg/kg) sq à gradually increased in 0.1-mL increments to final dose of 0.4 mL (83 μg/kg) sq qd
7-year-old girl	0.3 mL (55 μg/kg) sq qd à increased by using Depot 15 mg IM to a final dose of 2.0 mg/d (74 μg/kg)
18-year-old boy	Depot 15 mg IM; augmented with 0.2 mL sq qd à gradually increased in 0.1-mL increments to 0.5 mL (45 μg/kg) sq qd
11-year-old boy	Depot 15 mg IM; augmented with 0.4 mL sq qd à gradually increased in 0.1-mL increments to 0.7 mL (32 μg/kg) sq qd
11-year-old girl	Depot 15 mg IM q 28 d plus 3.5 mg sq qd

The Lupron PI states that therapy should be monitored with a GnRH stimulation test, measurements of sex steroids, and Tanner staging. Bone age for advancement should be assessed every 6-12 months. Confirming GnRH stimulation tests and Tanner staging are not described by the patent applicants, nor is there a discussion of the monitoring of bone age during therapy.

In 5 cases, the total duration of Lupron therapy is not specified in the patent application. In the case of the 18-year-old boy, his serum testosterone level dropped from a baseline of 559 ng/dL (reference range, 241-827 ng/dL) to a follow-up level of 28 ng/dL. The serum testosterone level of the 11-year-old boy dropped from a baseline of 153 ng/dL to 35 ng/dL after "several months of treatment." Essentially both boys were subjected by the applicants to chemical castration with Lupron at the end or beginning of puberty, respectively.

The 11-year-old girl, who began menstruating at the age of 10 years, most likely underwent chemically induced menopause with her Lupron therapy. This girl was also treated with "low dose birth control pills," presumably in conjunction with her Lupron therapy. The rationale for prescribing OCPs with Lupron in the 11-year-old girl is not stated by the applicants. (I'm out of my medical territory here, but I've only heard of prescribing Lupron with OCPs in the setting of infertility therapy.)

The Lupron PI states that discontinuation of the drug, when used for central precocious puberty "should be considered before age 11 for females and age 12 for males." This recommendation is presumably to allow timely puberty to begin. In adults, Lupron is FDA indicated for the treatment of prostate cancer, endometriosis, and uterine fibroids.

Chelation therapy: Only 2 subjects, the 8-year-old boy and a 6-year-old boy underwent chelation therapy. In the case of the 6-year-old boy, chelation preceded Lupron therapy. The 8-year-old boy underwent chelation after the initiation of Lupron therapy. The rationale for starting chelation therapy before Lupron injections is not stated by the applicants.

Other hormonal therapies: In addition to the OCPs prescribed for the 11-year-old girl, the 8-year-old boy began therapy with the antiandrogen cyproterone acetate (Androcur; Schering-Plough AG) 50 mg tid between his 2nd and 3rd doses of Lupron. Cyproterone acetate was prescribed for an unspecified period of time.

TAP's name* on this international patent application, along with the Geiers, is more than just a little troubling, given that pediatric subjects were treated with the company's proprietary drug in a maverick, off-label fashion on the basis of dubious theories about autism. Moreover, this off-label treatment, with TAP's evident involvement or knowledge, was performed without clinical-trial protocols (specifically those for the protection of human subjects) being noted in the patent application. In addition, adherence to on-label treatment guidelines, as recommended by the company's prescribing information, is not described. Clearly 3 autistic pediatric patients, who were at the beginning or end of puberty, received a drug that is known to disrupt reproductive function.

DMSA = dimercaptosuccinic acid; GnRH = gonadotropin-releasing hormone.

* Given the dissolution of TAP, it's unclear whether Abbott or Takeda is now the primary applicant for this international patent. Abbott has apparently taken over the Lupron franchise.

In their recently published study in the Journal of the Neurological Sciences, Geier et al not only presented values for transsulfuration metabolites in children with autism spectrum disorder (ASD); they also measured urinary porphyrins. Their rationale for doing so was to test a pet theory that autism is due to a reduced ability to excrete environmental mercury, as a result of an innate "decreased detoxification capacity"—which is proposed to be characterized by altered levels of transsulfuration metabolites. (The questionable values for some transsulfuration metabolites presented by Geier et al in the JNS article and elsewhere have been discussed here, here, here, and here.) In addition, because investigators have not been able to consistently find elevated mercury levels in children with ASD by direct measurement, Geier et al have turned to the use of "mercury intoxication-associated urinary porphyrins" as markers of mercury toxicity (most notably from thimerosal-containing vaccines) in children with ASD. In the JNS article, they write,

[I]t was previously demonstrated that the transsulfuration pathyway products of glutathione [15] and sulfate [16] were related to mercury excretion rates, and that the heme synthesis pathway products of urinary porphyrins can provide specific profiles that reflect mercury toxicity [17].

The references cited by Geier et al in this statement are worth examining.

First, reference "15" is a 1985 article from Ballatori and Clarkson, with the relatively straightforward (if not bone-dry) title, "Biliary secretion of glutathione and of glutathione-metal complexes." This study is an examination of the excretion of methylmercury in the bile in rats, which was found by the authors to closely parallel the biliary secretion of reduced glutathione (GSH). Important pharmacokinetic differences between methylmercury and ethylmercury (which is in the vaccine preservative thimerosal) have been discussed at this blog and by many others. Also the biochemical/biophysiologic leap from rodents to humans should have been acknowledged by Geier et al.

The same criticism can be applied to the use of reference "16," a 2004 review of organ systems in the American lobster (!) that regulate and detoxify environmental heavy metals.

Reference "17" is a 1996 study by James S. Woods from the University of Washington. In rats, Woods demonstrated changes in urinary porphyrins after prolonged exposure to—again—methylmercury. Specifically levels of 4- and 5-carboxyl porphyrins and the expression of precoproporphyrin were demonstrated in the exposed animals. Woods also conducted a possibly controversial pilot study in Mexican dental workers who were exposed to mercury-containing dental amalgam. Woods showed that the excretion of mercury increased and that levels of these urinary porphyrins decreased with chelation.*

In the JNS study, Geier et al measured urinary porphyrins in their 28 subjects with ASD (age range, 2-16 years), but not in their control children—as they did when assessing transsulfuration metabolites. (The reason for not measuring urinary porphyrins in the control group is not explained by the authors.) So without a true control group, Geier et al compared urinary porphyrin values** in their 14 children with "mild" ASD (CARS score ≤38.5) with those in 14 children with "severe" ASD (CARS score ≥38.5). (It's not clear what the authors did with the kids who hit the 38.5 mark.)

Not too surprising, Geier et al claimed significant differences in urinary levels of pentacarboxyporphyin (ie, 5-carboxyl porphyrins) and precoproporphyrin (Table 2) between the mild and severe ASD groups, which would be consistent with the methylmercury rat data of Woods. The authors also ostensibly monkeyed around with the various ratios of urinary porphyrins and found other significant differences between the 2 ASD groups. Additional fiddling demonstrated relationships between the CARS score and some porphyrin ratios. These data are intended to show that the authors' surrogate markers for mercury intoxication—urinary pentacarboxyporphyin and precoproporphyrin—are associated with the severity of autism.

Last, Geier et al assessed the plasma oxidized glutathione (GSSG) levels—as a "strong indicator of cellular oxidative stress"—among ASD children with low urinary porphyrins or high urinary porphyrins and claimed significantly increased levels of plasma GSSG in subjects with high urinary pentacarboxyporphyin or precoproporphyrin levels. Again, Geier et al intended to demonstrate that their surrogate markers for mercury intoxication are associated with a reduced capacity to excrete mercury, per the GSSG level, in ASD children. The main problem with this particular finding is that the plasma GSSG values presented by Geier et al are considerably different (eg, by 3 orders of magnitude) from those published elsewhere, including references cited by the authors.

Also published data suggest that Geier et al should have controlled for age-related differences in urinary porphyrin excretion—especially given that their subjects ranged in age from 2-16 years. A 1996 study by Minder and Schneider-Yin (Age-dependent reference values of urinary porphyrins in children) found distinctive age-related changes in the urinary excretion of 3 porphyrins, which may be explained—depending on the porphyrin—by age-related changes in the physiologic development of the excretion system and heme synthesis. For instance, their data showed that the urinary excretion of coproporphyrin III decreases steadily from the age of approximately 2 years to late adolescence. In an e-mail response, lead author Elisabeth Minder stated, in reference to the JNS study by Geier et al, that "one should control the data for age."

CARS = Childhood Autism Rating Scale. 

* Curiously enough, Woods is a coauthor of a 2006 JAMA article, which reported no significant differences in urinary mercury levels or neurologic function between Portuguese children who received dental amalgam and those who received a resin-based composite for routine dental work. The study's ethics were criticized in the Petition to Order Mercury Amalgam Withdrawn From Interstate Commerce. According to the JAMA article, "Urinary...porphyrins were monitored as indicators of renal responses to mercury..and will be reported separately."

** Urinary porphyrin levels from the subjects, who were recruited in Dallas, Texas, were shipped to and measured at, for some inexplicable reason, the Laboratoire Philipe Auguste in Paris. A coauthor of the JNS article is Robert Nataf from the same Paris lab. Nataf is the lead author of a retrospective 2006 study, which reported relatively elevated coproporphyrin and precoproporphyrin levels in children with autism.

For some completely unknown reason, today's WSJ gives free publicity to Pamlab, LLC, a Louisiana company that sells an oral, "high-dose" vitamin B supplement for peripheral neuropathy. 

Treatment of neuropathy with various forms of vitamin B has been around for at least decades, and the promotion of B supplementation rears up periodically in the lay press (as in the case of today's WSJ) and mostly bottom-feeding medical journals. The popularity of vitamin B6 (pyridoxine), specifically, as a neuropathy treatment probably has its genesis in the use of the vitamin to reduce the risk of isoniazid-induced neuropathy when treating tuberculosis. We're talking Eisenhower era.

The problem is that clinicians forget (or never knew) that high-dose B6, itself, can cause a toxic sensory neuropathy, which was documented in 1983 in the NEJM. Reversible B6 neuropathies were also described in 172 women in 1987 who used lower vitamin dosages (mean, 117 mg/d ± 92). These clinical neuropathies were more likely to occur with longer durations of treatment.

Pamlab's supplement contains 25 mg of the active form of pyridoxine, with a recommended dosage of 1-2 tablets per day. This dosage is within the standard deviation of the B6 range reported in the 1987 case series. Pamlab says that its supplement (which is only available by prescription and costs a stunningly high $40-$70 per month) has been shown in unpublished clinical trials to improve foot sensation and pain, writes the WSJ. (The supplement also contains 2.8 mg of L-methylfolate [the active form of folate, or B9] and 2 mg of methylcobalamin [a form of B12].)

The WSJ also cites a Cochrane review from July, which concluded that there is currently insufficient evidence to use vitamin B supplementation for neuropathy. The Cochrane authors found only 13 randomized or "quasi"-randomized studies (N = 741) from the mid-1960s to 2005, which compared vitamin B supplementation with placebo in general peripheral neuropathy. A considerably fewer number of trials compared vitamin B complex with an active control. Placebo-controlled trials provided mixed efficacy results, and active-comparator trials suggested that vitamin B supplementation is less efficacious in the short term than other agents. (Specific forms of vitamin B, with the exception of thiamine, are not highlighted in the Cochrane abstract.)

Pamlab informed the WSJ of an unblinded study of its vitamin B supplement, which was presented last month by a podiatrist at an unidentified scientific conference. The NIH Clinical Trials database includes a randomized, double-blind, placebo-controlled phase 4 study of the proprietary product in patients with type 2 diabetic neuropathy. The study is currently recruiting patients at selected US locations.

Why the WSJ chose to showcase Pamlab's vitamin B product, without 1) having more definitive scientific information to support its use in neuropathy and 2) citing the risk of neuropathy with B6 supplementation, is a minor mystery. 

D'oh! Just when you thought you dodged an IOC bullet, the overseer of the Olympic Games announced yesterday that it will further analyze blood and urine samples collected from athletes at this summer's Beijing games. First on the retroactive testing list is Roche's long-acting red-cell booster Mircera.* But the IOC also warns that it will store the Beijing samples for 8 years to enable additional analyses when new drug tests become available.

In Beijing, 4770 doping tests were conducted on blood or urine, covering the period from July 27th to August 24th. But only 6 out of approximately 11,000 athletes suffered sanctions as a result of positive tests, an unexpected, low number (Table). Therefore the IOC will retest samples with a recently validated assay for Mircera. The announcement comes on the heels of news this week that 3 more Tour de France racers tested positively for the substance.

Disqualified Athlete		Country	Sport	Highest Placement	Detected Substance
1	Lyudmila Blonska	Ukraine	Heptathlon	2nd	methyltestosterone
2	Igor Razoronov	Ukraine	Weightlifting	6th	nandrolone
3	Fani Halkia	Greece	Hurdles	—	methyltrienolone
4	Jong Su Kim	North Korea	Shooting	2nd	propanolol
5	Isabel Moreno	Spain	Cycling	—	Epo
6	Thi Ngan Thuong Do	Vietnam	Gymnastics	15th	furosemide

(IOC decisions regarding 3 other alleged Olympic doping cases, Belarussian hammer throwers Vadim Devyatovskiy and Ivan Tiskhan [testosterone] and Polish kayaker Adam Seroczynski [clenbuterol], are pending.)

* A pegylated version of recombinant erythropoietin.

Image of a freakishly bulked-up Lyudmila Blonska from Wikipedia.