Detoxamin Prostate Studies

Comparison of the Absorption, Brain and Prostate Distribution, and Elimination of CaNa2 EDTA of Rectal Chelation Suppositories to Intravenous Administration

Rita Ellithorpe, MD, Paul Mazur, PhD, Glenwood Gum, PhD, Gerry Button, BS, Julian Le, BS, Ernest H. Pfadenhauer, MS, Robert A. Settineri, MS, Garth Nicolson, PhD

1. Tustin Longevity Center, Tustin, California
2. Biological Test Center, Irvine, California
3. Research Consultant, Sierra Research, Irvine, California
4. The Institute for Molecular Medicine, Laguna Beach, California

ABSTRACT

Rectal suppositories were compared to IV administration of C14-labeled calcium disodium ethylenediaminetetraacetate (CaNa2EDTA) to evaluate the absorption, brain and prostate tissue distribution, and excretion in rats. The absolute bioavailability of CaNa2EDTA in blood following rectal dosing was 36.3% of the IV dose route, which confirmed that rectal dosing is an efficient method for delivering ethylenediaminetetraacetic acid (EDTA) to tissues. The ratio of radioactive residues of EDTA in tissues compared to blood, following IV or rectal dosing of C14 labeled CaNa2EDTA, showed negligible brain localization.
However, prostate tissues were found to have a mean ratio of 3.69 via the IV route and 13.6 rectally. The total recovery of C14 EDTA expressed as percent of administered dosed IV was a mean of 47.3% and 30.3% rectally at eight hours when the test was concluded. The suppository formulation of CaNa2 appears to be well absorbed, delivering high levels of EDTA to prostate tissue.

* Correspondence:
Rita Ellithorpe, MD

Tustin Longevity Center
13422 Newport Avenue # L
Tustin, CA 92780

Phone: 714-544-1521
Fax: 714-544-1904
E-mail: edaly@tlcmd.net

INTRODUCTION

Heavy metal exposures in the twenty-first century are an established global health concern. The FDA has approved EDTA as a chelation agent for the removal of heavy metals. It has been placed on the FDA XGenerally Recognized as Safeg (GRAS) list for the past sixty years. Extensive national and international clinical experiences demonstrate that acute and chronic human exposure to a wide range of heavy metals can be treated with considerable efficacy using EDTA. It is widely administered, with considerable cost to the patient, as an intravenous (IV) solution, which entails 15 to 30 sessions in a physicianhs office,
taking two to five hours per visit. The transrectal delivery of several pharmacological agents is well established. Therefore, using a rat animal model, we set out to determine if the rectal administration of EDTA is absorbed, resulting in significant blood and tissue levels. The pharmacodynamic effects of therapeutic agents differ widely in their route of administration, penetration, absorption, and distribution in body tissues. For medicinal agents to act, they must be absorbed and transported to the appropriate tissue or organ, penetrate to the responding cell surface or sub-cellular and interstitial space, and elicit a
response or alter ongoing processes.1 The parenteral and intramuscular forms of EDTA are well absorbed, but not very practical for routine usage.2 Oral forms of EDTA have been shown to be poorly absorbed (2% to 5%), and topical and subcutaneous forms have been reported as not being absorbed at all.3,4,5,6,7,8,9,10 A relatively new alternative and more convenient route of administration is rectal suppository delivery of a proprietary suppository formula of EDTA (CaNa2 EDTA, Detoxamin,® World Health Products, Draper, Utah), which is the basis of this pharmacokinetic (PK) study. Although IV EDTA dosing is well characterized and has been used for decades, little is known about the absorption of rectal suppositories. In an effort to elucidate the absorption characteristics of CaNa2 EDTA in a suppository form, a rat model was chosen. 14C-labeled EDTA Calcium Disodium salt was administered as a tracer in the suppository and in intravenous forms; blood, urine, and selected tissue levels were evaluated over eight hours.

MATERIALS AND METHODS

14C-labeled EDTA free acid (11.7 mCi/mmol, Lot No. 63151012, purity greater than 98%) was obtained from MP Biomedicals (Irvine, CA). For the IV dosing solution, 14C-labeled EDTA was added to normal saline to achieve concentrations needed to deliver a final dose of 7.53 µCi in approximately 1 gram. The rectal suppository (a proprietary suppository formula of EDTA, CaNa2 EDTA, Detoxamin® Health Products, East Draper, Utah, Lot No. 228-190-0117) was prepared by adding 14C-labeled EDTA solution from Moravek to molten suppository. For the animal dose, approximately 100 µL of the mixture (containing 23.7 µCi per dose) was taken up in a cylindrical glass pipette equipped with a plunger and allowed to cool to
room temperature, where it re-solidified. The radioactive concentration of the IV dosing solution was calculated by Liquid Scintillation Counting (LSC). The prepared dosing solutions were stored and refrigerated. Ten male Sprague Dawley rats were obtained from Taconic, Oxnard, CA. Animals were 6 to 7 weeks old and weighed 157 to 187 grams on Day 1. The animal experiments were performed at the Biological Test Center (BTC), in Irvine, CA. Quarantine and care of animals were performed per BTC Standard Operating Procedures. Prior to dosing, 10 animals were weighed. Cannulated animals (six animals to undergo IV dosing) were randomized for placement into Group A or B. Uncannulated animals (four animals to undergo rectal dosing) were not randomized and were placed into Group C. Treatment groupsare presented below.

Animals were fasted (food withheld) for 16.5 to 19.5 hours before 14C-EDTA administration. Prior to dosing, rats were anesthetized with an intramuscular combination injection of ketamine hydrochloride (40-90 mg/kg) and xylazine (5-10 mg/kg). Water and feed were withheld from animals for four hours after 14C-EDTA administration, and then food and water were given ad libitum.

For Group C, the contents of the colon were removed before dosing by flushing with normal saline heated to 37°C. Rectal doses were administered via a 100 µL glass cylindrical tube, gently heated to allow partial liquefaction of the suppository material. Blood samples of approximately 100 µL were taken. Each sample was placed in combustion cones and stored frozen prior to combustion and LSC analysis. The time of blood collection was recorded.

A terminal blood sample was collected from all animals via heart puncture (1 hour ± 5 minutes after dosing for Group A animals; 8 hours ± 15 minutes after dosing for Group B and C animals). Each animal was anesthetized with an intramuscular combination injection of ketamine hydrochloride (40-90 mg/kg) and xylazine (5-10 mg/kg), and euthanized by exsanguination following heart puncture. As much blood as possible was collected from each rat into heparinized tubes. The time of blood collection was recorded. Four 100-µL aliquots of whole blood were transferred to combustion cones. Two of the aliquots were combusted for determination of radioactivity by LSC, and two were kept frozen as reserve samples.

Absorbent paper was placed in the restrainers to collect urine 0 to 4 hours after dosing. Urine was collected from the individual metabolism cages 4 to 8 hours after dosing. For urine samples collected in absorbent paper, water was added to the paper and urine extracted. For urine samples collected from metabolism cages, the urine was freeze-trapped to avoid atmospheric oxidation, evaporation, and bacterial degradation, and the urine collection pan was rinsed with water.

1. Blood collection times were ± 1 minute for the 5-minute time point; ± 3 minutes for the 15- and 30-minute time points; ± 5 minutes for
the 1-hour time point; and ± 15 minutes for the 2-, 4-, and 8-hour time points.
2. The fourth animal in group C, animal, No. 55905, was dead (attributed to anesthesia) 15 minutes after dosing.

Following euthanasia by exsanguination, the brain and prostate were collected from each animal. Prior to collection, the brain was perfused with approximately 5 mL of saline via the carotid artery. Both organs were stored at -20º C. Following completion of blood kinetics analysis, brains and prostates were combusted for determination of radioactivity by LSC. Brains were homogenized prior to combustion, while prostates were directly combusted.

Duplicate aliquots of each urine sample (0.1 mL) and cage rinse sample (1 mL) were transferred to liquid scintillation counting vials and the amount of radioactivity determined by LSC; Insta-Gel was used as the scintillation fluid. Each of the rectal dosing solution samples, tail vein blood samples, and heart puncture blood samples in combustion cones were combusted. Brain and prostate samples were combusted. Combusted samples were trapped in Carbon-14 Cocktail (R.J. Harvey, Hillsdale, NJ) present in liquid scintillation counting vials, and the amount of radioactivity was determined by LSC.

Sample combustion was performed using a Harvey Sample Oxidizer, Model OX300 (Harvey Instrument, Hillsdale, NJ). All radioactivity measurements were performed using a Beckman Liquid Scintillation Spectrometer. Any radioactivity measurement of less than 100 dpm was considered close to background and was not repeated.

When applicable, summary statistics (mean and standard deviation) were prepared to characterize the data (i.e., radioactivity measurement and percent dose). PK parameters, including Area under the Curve (AUC), half-life, Maximum Concentration in blood (Cmax), Time to Maximum Concentration (Tmax), and bioavailability, were calculated using WinNonlin (Pharsight Corporation, Mountain View, CA).

RESULTS

Individual and mean (± SD) body weights and administered 14C-EDTA doses are presented in Table 1. Radioactivity recovered from blood at different time intervals is presented in Figures 1 and 2. As shown in Figure 2, the absorption phase occurring within the first two hours after dosing for all three rectally-dosed animals was maximal, and the apparent biphasic absorption may have been related to additional material being released from the rectal suppository; the blood levels from the IV doses did not show a biphasic response.

Mean AUC, half-life, Cmax, Tmax, and bioavailability of derived radioactivity in blood are presented in Table 2. The Tmax of EDTA following intravenous dosing occurred at 0.083 hours. The Tmax of EDTA following rectal dosing occurred at 0.417 hours. The half-life of EDTA following intravenous dosing was 1.50 hours, and the half-life of EDTA following rectal dosing could not be calculated since a terminal elimination phase could not be determined. The absolute bioavailability of EDTA in blood following rectal dosing was 36.3 compared to the IV bolus of 100%. Radioactivity recovered from urine at different time intervals is presented in Table 3. Following intravenous dosing, the amount of radioactivity excreted in urine decreased over the 8-hour study period (46.3% of dosed radioactivity excreted at the 0 to 4 hour interval, and 0.935% of dosed radioactivity excreted at the 4 to 8 hour interval). Following rectal dosing, the amount of radioactivity excreted in urine remained relatively constant over the 8-hour study period (15.8% of dosed radioactivity excreted at the 0 to 4 hour interval, and 14.4% of dosed radioactivity excreted at the 4 to 8 hour interval).

Figure 1. EDTA levels in blood over time following intravenous administration of 14C-EDTA.

Figure 2. EDTA levels in blood over time following rectal administration of 14C-EDTA

Table 1. Body Weights and Administered 14C-EDTA Doses

Table 2. Mean AUC, half-life, Cmax, Tmax, and bioavailability of EDTA in blood following intravenous or rectal administration of 14C-EDTA

Where Xtestg data is the rectal data, and Xref g (reference) data is the intravenous data.

1. The terminal elimination phase was not observed, therefore, the half-life could not be calculated.
References for the above formula are as follows:

• Kwon Y. Handbook of Essential Pharmacokinetics, Pharmacodynamics, and Drug Metabolism for Industrial Scientists.
  New York: Kluwer Academic/Plenum Publishers, 2001.
•  Shargel L, Yu A. Applied Biopharmaceutics and Pharmacokinetics, 4th ed. Norwalk, Connecticut: Appleton & Lange, 1999

Radioactivity recovered from tissues (brain and prostate) expressed as a ratio of the radioactivity in blood is  presented in Table 4 and Figure 3. The prostate retained higher levels of radioactivity than the brain following both intravenous and rectal dosing, with the highest level of radioactivity found in the prostate following rectal dosing. The total recovery of radioactivity from urine and tis- sues expressed as percent of dose is presented in Tables 5 and 6. Total recovery represents the combined total percent of dose in urine and tissues. Following intravenous dosing, 41.4% and 47.3% of the radioactive dose was recovered 1 hour and 8 hours after dosing, respectively; of which virtually all was in urine. Following rectal dosing, 30.3% of the radioactive dose was recovered 8 hours after dosing, of which virtually all was also in urine.

DISCUSSION

This study has shown that the proprietary formula of Ca Na2 EDTA has been effectively absorbed from the lower enteral route in rats, through the anal portal into the rectum or lower intestine to reach blood and tissue levels via rectal suppositories. Bioavailability has now been established for this mode of administration in an animal model and is strong evidence that EDTA suppositories are an adequate and medically acceptable approach to providing the benefits of chelation. Intravenous dosing resulted in greater elimination of radioactivity in urine at the 0 to 4 hour time point, but the percent of dose recovered drastically decreased by the 4 to 8 hour time point, while the level of recovery was relatively steady at both time points following rectal dosing. The slow and consistent movement of CaNa2 EDTA via rectal administration may have lesser toxicity since there are significant blood and tissue levels to chelate metals without a high dose EDTA IV drip over many hours. These data point to the ability of rectal suppositories to deliver a continuous lower dose concentration of EDTA for longer periods of time compared with IV administration, allowing EDTA to bind metals efficiently and effectively.

In tissues, significant amounts of radioactivity were recovered from the prostate following intravenous or rectal dosing, with the highest level of dosed radioactivity (179.6 ppm) recovered 8 hours following rectal dosing. This observation of rectal administration, revealing higher amounts of EDTA in prostate tissue as compared to IV, can have far-reaching implications of a more complete distribution of EDTA into interstitial and intracellular spaces, further leading to more efficient chelation of compartmentalized heavy metal content with CaNa2 suppositories.

EDTA is not bio-transformed in the body. It is excreted in hair, urine, feces, saliva, and perspiration. This study shows that animals excreted 47.3% and 30.3% of dosed radioactivity in urine during the 8 hours following intravenous and rectal dosing, respectively. The 30.3% excretion of EDTA in the urine corresponds closely to the rectal dose bioavailability calculated from the blood levels (36.3%).

Blood samples were taken over an 8-hour period, and during this time, the rectal administration showed high levels of absorbed ETDA with no apparent elimination phase observed. If further blood samples had been taken, the bioavailability calculated for rectally administered EDTA would have undoubtedly been much higher, since the bioavailability calculation presented here only used up to 8- hour blood level data. No extrapolation of the AUC could be done since the levels at 8 hours were actually increasing in two out of three animals. Further research is indicated over a longer time span to quantify the actual half life of the suppository form of administration.

Figure 3. Ratio of tissue and blood radioactive residues (ppm) following intravenous or rectal administration of 14C-EDTA.

Table 3. 14C-EDTA-derived radioactivity excreted in urine expressed as percent of administered dose following intravenous or rectal administration of 14C EDTA

CONCLUSIONS

This proprietary suppository formulation appears to be a viable dosing mechanism for delivery of CaNa2 EDTA to the bloodstream in this rat model, showing substantial circulating levels of EDTA for least 8 hours after administration. EDTA appears to be favorably distributed to the prostate, but not the brain, following both IV and rectal dosing. The excretion of rectal CaNa2 EDTA administration in urine corresponds well with the rectal dose bioavailability of blood levels. The absolute bioavailability of EDTA in blood following rectal dosing was 36.3% within the 8-hour period. Additional testing is required to confirm and duplicate these results in humans.

Table 4. Ratio of radioactive residues of EDTA in tissues (µg/g) to blood (µg/g) following intravenous or rectal administration of 14C-EDTA

Table 5. Total recovery of radioactivity expressed as percent of administered dose following intravenous or rectal administration of 14C-EDTA.

ACKNOWLEDGMENT

Funding for this study was provided by World Health Products, Draper, Utah. One of the authors, Robert Settineri, MS, is also a consultant to World Health Products. The other authors at The Biological Test Center are independent contract researchers paid for their research services by World Health Products.

REFERENCES:

1. University of the SC/Remington. The science and practice of pharmacy. Columbia, S.C.: Lippincott Williams & Wilkins/Wolters Kluwer, 21:2006.

2. Foreman H. Metal Binding Med, Proc Symposium, Philadelphia, 1959. 1960;82-94.

3. Sbrova J Teisinger J, Arc. Gewerbepathol. 1957;15:572.

4. Foreman H. Metal Binding Med, Proc Symposium, Philadelphia, 1959. 1960;82-94.

5. Foreman H, Vier M, Magee M. The metabolism of C14 labeled ethylenediamine tetraacetic acid in the rat, J Biol Chem. 1953;203:1045.

6. Yang, SS. Ethylenediaminetetraacetate, Disodium and Calcium Disodiu Salts. Seventeenth Report of the Joint FAO/WHO Expert Committee on Food Additives. Wld Hlth Org.techn. rep. ser., 1974, No. 539; FAO Nutrition Meetings Report Series, 1974, No. 53. http://www.inchem.org/documents/jecfa/jecmon /v05je25.htm

7. Cleton F, Turnbull A, Finch, CA. Synthetic chelating agents in iron metabolism. J Clin Invest. 1963 March; 42(3): 327-337.

8. Foreman H, Trujillo TT. Synthetic chelating agents in iron metabolism. J Lab Clin Med. 1954 Apr;43(4):566-571.

9. MacPhail AP, Bothwell TH, Torrance JD, et al. Factors affecting the absorption of iron from Fe(III)EDTA. Br J Nutr. 1981 Mar;45(2):215-227.

10.Bjarnason I, OhMorain C, Levi AJ, Peters TJ. Absorption of 51 chromium-labeled ethylenediaminetetraacetate in inflammatory bowel disease. Gastroenterol. 1983 Aug;85(2):318-322.

Draft Submittal for Publication in the World Journal of Urology

Anti-Microbial plus CaNa2EDTA Chelation Suppository Therapy for Chronic Prostatitis/Pelvic Pain Syndrome with or without Prostatic Hyperplasia: Preliminary Study

Rita Ellithorpe1, MD, Larry Clapp2, JD, Tony Jimenez3, MD, Brett Jacques4, ND, Robert Settineri5, MS, PhD, and Garth L. Nicolson6, PhD

Tustin Longevity Center, Tustin, CA, Prostate Health Resources, Santa Monica, CA,  Hope Cancer Institute, Baja California, Mexico, Tustin Longevity Center,Tustin, CA, Sierra Research, Irvine, CA, and 6 Institute for Molecular Medicine, Laguna Beach, CA

Abstract

Patients with chronic prostatitis/pelvic pain syndrome are characterized by treatment failures, a high incidence of prostate calcium deposits and poor quality of life. We used proprietary suppositories containing calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA) to remove metal and calcium cations. Participants (N=31) with chronic prostatitis, with or without prostate hyperplasia, and prostate calculi (mean age=61) were treated with tetracycline (500 mg/day) and CaNa2EDTA suppositories (750 mg 4X/week) for 90 days. Using the NIH Chronic Prostatitis Symptom Index significant post-treatment mean reductions in symptoms (p<0.0106) and pain (p<0.0122) were found along with a significant improvement in mean quality of life (p<0.0022). Overall, the mean total scores showed significant post-treatment reduction (p<0.0006). In addition, use of the International Prostate Symptom Score indicated significant reductions in 5/7 symptom categories and significant reduction of mean overall scores (p<0.0001). Analysis of blood and stool post-treatment indicated significant changes in mobilized, secreted cations (cadmium, copper, boron, lead, molybdenum, magnesium and calcium). In addition, the blood cholesterol/high density lipoprotein ratio was significantly decreased (p<0.0005). However, using the Erectile Function Index Questionnaire there was significant mean improvement in only 7/15 questions, resulting in non-significant overall mean improvement, and there were nonsignificant reductions in prostate calcifications at this suppository dose level. The data suggest that combining CaNa2EDTA suppositories with tetracycline can significantly reduce symptoms and pain in refractory chronic prostatitis with or without prostate hyperplasia.

Keywords: Prostatitis symptoms, antibiotic, EDTA suppository, bacterial biofilm, prostate calcification.

Corresponding Author: Prof. Garth Nicolson, Dept. of Molecular Pathology, The Institute for Molecular Medicine, P.O. Box 9355, Laguna Beach, CA 92652.

Email: gnicolson@immed.org

Introduction

Prostatitis is a common urological condition, and it has been estimated that up to one-half of men will suffer from symptoms of this disease at some time during their lives [1]. At any one time 2-10% of men suffer from prostatitis [2]. Prostatitis is classified within a complex series of syndromes (NIH category I-IV prostatitis) that vary widely in clinical presentation and response to treatment. Acute bacterial prostatitis (category I) and chronic bacterial prostatitis (category II) are characterized by uropathogenic infections of the prostate gland that respond well to antimicrobial treatment, whereas chronic prostatitis/chronic pelvic pain syndrome (category III, accounting for 90%-95% of prostatitis cases) is marked by a mixture of pain, urinary and ejaculatory symptoms and presence of prostate calculi with no uniformlyeffective therapy [3]. Several different classes of bacterial infections have been found in acute and chronic
prostatitis, including Gram-positive and znegative bacteria [1, 2, 4, 5], cell wall-deficient forms (Mycoplasma, Ureaplasma, Chlamydia) [4, 6, 7] and biofilm-forming bacteria [8-10]. The latter forms are likely to be important in chronic prostatitis and have proven to be particularly difficult to effectively treat [10, 11]. Biofilms contain bacterial glycosaminoglycans, salts (especially calcium and other cations) and other molecules [12, 13]. According to Parsek and Singh [14] biofilms support bacterial: (a) adhesion, (b) clustering, (c) localized infection, and (d) increased resistance to antibiotic treatment in the
host. They do this by providing a protective structure for bacterial colonies so that they can evade mechanical stresses, host responses and antimicrobial agents.
Calcium and other cations are thought to play a structural role in biofilms as well as in cellular and tissue deposits, such as calculi, a calcium apatite-containing mineral deposit that is often associated with category III chronic prostatitis/chronic pelvic pain syndrome [15]. In some studies the presence of calculi is a feature that is associated with antimicrobial resistance and treatment failure [16, 17]. Although the presence of calculi in chronic prostatitis/chronic pelvic pain syndrome and its association with disease symptoms are controversial [17], Geramoutsos et al. [18] found that larger deposits of calculi were associated with chronic prostatitis symptoms.

Patients with NIH Category III chronic prostatitis/chronic pelvic pain syndrome have a high incidence of calculi, antibiotic treatment failures and poor quality of life [16, 17]. They also show evidence of biofilm-forming bacterial infections [17]. Therefore, Shoskes et al. [17] initiated a preliminary complex treatment study with 16 patients using an antibiotic (500 mg tetracycline per day) plus a calcium chelator (1500 mg ethylenediaminetetraacetic acid,
EDTA) and a proprietary vitamin-mineral nutraceutical and found significant decreases in the NIH Chronic Prostatitis Symptom Index (NIH-CPSI) in most patients and decreases in prostatic calcium-containing stones (calculi) in one-half of the patients examined. Toxic heavy metals, such as mercury, lead, cadmium, nickel and others [19,20], can also be chelated with EDTA, and in some cases these heavy metals are carcinogenic to prostate tissue [21, 22]. Their removal from prostate tissue along with excess calcium can be accomplished with long-term EDTA administration. Thus we initiated a study based on the
work of Shoskes et al. [17] to test the hypothesis that combinations of oral tetracycline and a patented, proprietary suppository (Detoxamin®) containing CaNa2EDTA that is known to produce a long tissue half-life of EDTA in rats [23] would decrease chronic prostatitis symptoms along with cation deposits and increase quality of life.

Patients and methods

Patients

Criteria for inclusion in the study included: men aged 40 years or older (N=31, mean age=61, range 41-73) with a diagnosis of chronic prostatitis and the presence of prostate calcifications on ultrasound, absence of painful pelvic side wall spasm on rectal palpation, and the absence of allergy to tetracycline. Exclusionary criteria included: chronic debilitating condition other than prostatitis, such as chronic inflammatory or irritable bowel disease, chronic diarrhea or constipation, tetracycline allergy, cognitive decline (unable to comprehend instructions and answer questions), renal or hepatic dysfunction, inability to
tolerate anal suppositories and absence of prostate calcifications. Of the participants, all but 7 showed evidence of prostate hyperplasia, varying from mild (48%) to moderate or severe (29%) as assessed by prostate sonogram. Participants were entered into an open label treatment trial where laboratory and clinical data were collected before and after treatment. Prior to entry all patients had a complete medical history and urological examination.

Laboratory/Clinical Tests

The following laboratory/clinical tests were performed pre- and post-treatment: non-fasting comprehensive blood chemistry and lipid panel (Southern California Reference Laboratory), Tustin, CA), blood analysis for heavy metals and essential minerals, fecal metals analysis (Doctors Data, Inc., St. Charles, IL), digital rectal exam and prostate sonogram using a Fukuda Denshi Model FF sonic UF-750XT Power Color Doppler (PCD) with 3D imaging
capability. Analysis of elements was performed by ICP-Mass Spectroscopy following digestion of the specimen in a closed microwave system. For a given mineral element, these procedures measure the sum of the amounts of surface-adhering and intracellular content, regardless of chemical form. Participants also took the NIH-CPSI [24], International Prostate Symptom Score (IPSS) [25] and Erectile Function Index (EFI) [26] surveys.

Study Design

The study received Institutional Review Board (IRB) approval, and before admission each patient signed an informed consent document. Participants in the study were examined and tested (as above) before and after three months administration of tetracycline (HPN Pharmaceuticals, Torrance, CA; 500 mg PO daily) and use of Detoxamin® (World Health Products, Draper, Utah) time-release suppositories (4-times per week) each containing 750
mg disodium calcium EDTA (CaNa2EDTA). Each day during the trial participants took two tabs of a multi-vitamin, mineral and trace mineral supplement (Health Genesis, World Health Products) to replace essential minerals and one capsule of a probiotic mixture in an oil matrix capsule (stored refrigerated; Healthy Trinity, Natren, Westlake Village, CA) to replace depleted gut flora [27,28]. At the end of therapy laboratory and clinical tests and clinical
surveys were completed as described above and compared to pre-treatment tests and surveys.

Statistical analyses

Post-pre measurements were analyzed by paired t-test comparisons with Bonferroni corrections. Reliability was evaluated by Cronbachhs alpha coefficient. In some cases parametric statistical results were checked by nonparametric Wilcoxon tests.

Results

Blood and fecal elements

A panel of mineral element tests were performed on packed red blood cells pre/post treatment and compared among participants. Post-treatment analyses indicated that were significant increases in mean red blood cell copper, boron, molybdenum and magnesium levels and a borderline significant increase in calcium and decreases in arsenic, cadmium and lead, suggesting that the CaNa2EDTA was mobilizing some tissue elements during the trial
(Table 1). Analysis of fecal metals indicated that mean copper, tungsten and to a lesser degree beryllium levels were significantly increased post-treatment, whereas cadmium was significantly decreased (Table 2).

Blood Serum Chemistries

The non-fasting comprehensive blood chemistry panel showed the cholesterol/HDL ratio was significantly decreased (p<0.0005) post-treatment, but no significant changes in creatinine, BUN/creatinine ratio or calcium levels were observed (Table 3)

Chronic prostatitis symptom index (NIH-CPSI)

Examination of the NIH-CPSI domains indicated that there were significant decreases in mean Urinary Symptom Score, mean Pain Symptom Score and mean Quality of Life Score post-treatment (Table 4). The mean Urinary Symptom Score post-treatment decreased from a pre-treatment mean of 3.709±2.08 to a mean of 2.84±2.25, a mean decrease of 0.87±1.99 post-treatment (p<0.0106), whereas the pain symptom mean score decreased from 4.35±4.54
to 3.10±3.67, a mean decrease of 1.25±2.95 (p<0.0122). The mean quality of life index score decreased from 4.68±2.81 to a mean of 3.45±2.53, a mean decrease of 1.23±2.22 posttreatment (p<0.0022). Overall the NIH-CPSI mean total scores decreased from 12.74±8.31 to 9.39±6.16, a mean decrease of 3.35±0.93 (p<0.0006).

International prostate symptom score (IPSS)

Using the International Prostate Symptom Score (IPSS) survey form indicated significant reductions in 5 out of 7 categories and significant reduction of overall symptoms postcompared to pre-treatment (Table 5). Significant reductions were found in incomplete 17 emptying of bladder (p<0.0082), frequency of urination (P<0.0044), urgency of urination (p<0.00001), and intermittency of urination (P<0.0741), weak stream (p<0.0003), quality of life score (p<0.0191) and overall score (p<0.0001) (Table 5). Non-significant reductions were found in urination straining and nocturia (Table 5).

Erectile function index (EFI)

Using the EFI survey scores on 7 of 15 questions showed positive changes, indicating improvement in self-evaluated conditions, whereas 8 of 15 showed negative changes (Table 6). Thus overall there was not a significant overall change in EFI scores. The lack of significant improvement was confirmed by use of nonparametric Wilcoxon tests.

PCD sonography

Transrectal Power Color Doppler (PCD) sonography showed that there were no significant decreases in prostatic calcifications at the treatment levels used in this study (data not shown).

Discussion

Similar to Shoskes et al. [17] we found that patients with NIH Category III chronic prostatitis could be successfully treated in an open label trial with tetracycline (500 mg/day) and Detoxamin® suppositories containing 750 mg of CaNa2EDTA used 4-times per week for 90 days. In the present study the dose of CaNa2EDTA was half of the amount used by Shoskes et al. [17]. Using the NIH-CPSI and IPSS survey forms significant post-treatment reductions in mean index of prostatitis symptoms and pain were found along with significant improvements in mean quality of life and mean overall scores. The IPSS is also used as a
validated instrument for benign prostatic hyperplasia [29], suggesting the positive influence of symptom reduction for this condition as well as prostatitis. Approximately 77% of the patients in our study had benign prostatic hyperplasia or cancerous prostate lesions.

We did not see a significant increase in mean overall Erectile Function Index post-treatment; however, in 7 of 15 questions there were significant improvements. Since this patient group is generally unresponsive to multiple courses of antibiotics alone and other therapies, we agree with Shoskes et al. [17] that this approach represents a significant improvement in therapy of Category III chronic prostatitis. As in Shoskes et al. [17], this open label study is 18 limited in terms of the number of patients entered and lack of blinded control arms, and thus no conclusions can be drawn as to mechanism of action and durability of the effect.

In our study, Detoxamin® CaNa2EDTA suppositories were used to loosen and remove calcium and heavy metals from patients with established prostatic calcifications and chronic prostatitis while on tetracycline. That some metals and calcium were being mobilized by the CaNa2EDTA suppositories was shown in blood and fecal tests. In particular, the mean levels of copper, boron, molybdenum, magnesium and calcium were significantly increased in packed red blood cells as well as significant decreases in arsenic, cadmium and lead in fecal matter in the post-treatment tests, suggesting that tissue stores of these elements were being affected. The presence of heavy metals and cations like calcium varies widely in different patients, and we found differences among patients in the amounts of these elements present in blood and stool when CaNa2EDTA suppositories were used. Since approximately 77% of the patients in our study had a diagnosis of benign or cancerous prostate lesions, the removal of potentially carcinogenic heavy metals was a desirable effect of the therapy. Certain heavy metals, such as arsenic, cadmium, chromium and lead, among others, are known have carcinogenic potential, and their long-term exposure is associated with an increased incidence of prostate and other cancers [30,31]. However, the potential role of heavy metals in the pathogenesis and progression of already established cancers and in affecting the symptoms of chronic prostatitis remain uncertain.

We previously studied the pharmacokinetics and tissue concentrations (bioavailability) of Detoxamin® CaNa2EDTA and compared it to IV administration of CaNa2EDTA [23]. We found that there were substantially higher tissue-to-blood concentration ratios of EDTA via the suppository route compared to IV administration. Prostate tissues were found to have 8 hr mean tissue-to-blood ratios of 13.6 (rectal suppository) and 3.69 (IV), respectively [23]. This finding supports the mechanism of action of CaNa2EDTA chelation suppositories on the target tissue in question (prostate) and was an initial justification for this clinical trail.

Another aspect of the use of CaNa2EDTA suppositories is that bacterial biofilms contain high amounts of divalent cations like calcium that are important in maintaining their structures [32]. The presence of biofilm-producing bacteria has been associated with chronic prostatitis  [14,17,32], and the use of calcium supplements is associated with increased risk of urinary 19 tract infections [33]. Calcium is also important in bacterial adhesion element in the urinary
tract either as an adhesion promoting cation or inhibitor of secreted host glycoproteins that prevent bacterial adhesion to the urinary tract [34]. Thus removal of excess calcium ion and calcium complexed to extracellular bacterial proteoglycans and glycosylaminoglycans could be important in reducing bacterial adherence and chronic prostatitis symptoms.

There is a rather long history of the use of EDTA in clinical studies [35-39]. Animal experiments have shown that EDTA is extremely safe and relatively high doses are well tolerated without noticeable side effects [40,41]. However, in a few reports a few subjects receiving IV EDTA had reversible renal damage [42,43], and temporary numbness and tingling in IV administered EDTA are relatively common [44]. None of these adverse effects were found in the current study using Detoxamin® CaNa2EDTA suppositories. The Detoxamin® CaNa2EDTA suppositories were found to be well tolerated and safe. Interestingly, although not the focus of this study, we found that mean HDL and LDL lipid blood panel determinations showed significant improvements post-treatment. We and others [17] have concluded that the addition of EDTA suppositories to antibiotic therapy of chronic prostatitis significantly reduced symptoms and improved outcome.

Table 1. Red Blood Cell Elements Pre-Post Treatment Comparisons*

Table 2. Fecal Elements Pre-Post Treatment Comparisons*

Table 3. Comprehensive Blood Chemistry Panels Pre-Post Treatment Comparisons

Table 4. NIH-CPSI (Chronic Pain Symptom Index) Scores Pre-Post Treatment Comparison

Table 5. International Prostate Symptom Scores (IPSS)Matched pair t-tests for each question, quality of life and total score

Table 6. Erectile Function Index (EFI) Scores Matched pair t-tests for each question and EFI total score

References

1. Domingue GR, Hellstrom WJG. Prostatitis. Clin Microbiol Rev 1998; 11:604-613.
2. Habermacher GM, Chason JT, Schaeffer AJ. Prostatitis/chronic pelvic pain syndrome. Annu Rev Med 2006; 57;195-206.
3. Krieger JN, Ross SO, Deutsch L, Riley DE. The NIH Consensus concept of chronic prostatitis/chronic pelvic pain syndrome compared with traditional concepts of nonbacterial prostatitis and prostatodynia.Curr Urol Rep 2002; 3:301-6.
4. Domingue GJ. Cryptic bacterial infection in chronic prostatitis: diagnostic and therapeutic implications. Curr Opin Urol 1998; 8:45-49.
5. Beckman TJ, Edson RS. Methicillin-resistant Staphylococcus aureus prostatitis. Urology 2007; 69:779-783.
6. Skerk V, Krhen I, Schonwald S, Cajic V, Markovinovic L, Roglic S, Zekan S, Andracevic AT, Kruzic V. The role of unusual pathogens in prostatitis syndrome. Int J Antimicrob Agents 2004; 24 (Suppl 1): S53-S56.
7. Mandar R, Raukas E, Turk A, Korrovitis P, Punab M. Mycoplasmas in semen of chronic prostatitis patients. Scand J Urol Nephrol 2005; 39:479-482.
8. Wood HM, Shoskes DA. The role of nanobacteria in urologic disease. World J Urol 2006; 24:51-54.
9. Kanamaru S, Kurazono H, Terai A, Monden K, Kumon K, Kumon H, Mizunoe Y, Ogawa O, Yamamoto S. Increased biofilm formation in Escherichia coli isolated from acute prostatitis. Int J Antimicrob Agents 2006; 28(Suppl 1):S21-S25.
10. Soto SM, Smithson A, Martinez JA, Horcajada JP, Mensa J, Vila J. Biofilm formation in uropathogenic Escherichia coli strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance. J Urol 2007; 177:365-368.
11. Tunuguntla HSGR, Evans CP. Management of prostatitis. Pros Cancer Pros Dis 2002; 5:172-179.
12. van Houdt R, Michiels CW. Role of bacterial cell surface structures in Escherichia coli biofilm formation. Res Microbiol 2005; 156:626-633.
13. Reisner A, Krogfelt KA, Klein BM, Zechner EL, Molin S. In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors. J Bacteriol 2006; 188:3572-3581.
14. 14. Parsek MR, Siingh PK. Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol
    2003; 57:677-701.
15. Shoskes DA, Hakin L, Ghoniem G, Jackson CL. Long-term results of multimodal therapy for chronic prostatitis/ chronic pelvic pain syndrome. J Urol 2003; 169:1406-1410.
16. Rugendorff EW, Weidner W, Ebeling L, Buck AC. Results of treatment with pollen extract (Cernilton N) in chronic prostatitis and prostatodynia. Br J Urol 1993; 71:433-438.
17. Shoskes DA, Thomas KD, Gomez E. Anti-nonobacterial therapy for men with chronic chronic prostatitis/chronic pelvic pain syndrome and prostatic stones: preliminary experience. J Urol 2005; 173:474-477.
18. Geramoutsos I, Gyftopoulos K, Perimenis P, Thanou V, Liagka D, Stamblis D, Barbalias G. Clinical correlation of prostatic lithiasis with chronic pelvic pain syndromes in young adults. Eur Urol 2004; 45:333-337.
19. Lippmann M, Thurston GD. Exposure assessment: input into risk assessment. Arch Environ Health 1988; 43:113-123.
20. Goyer RA. Nutrition and metal toxicity. Am J Clin Nutr 1995; 61(Suppl 3):646S-650S.
21. Magos L. Epidemiological and experimental aspects of metal carcinogenesis: physiochemical properties, kinetics and active species. Environ Health Persp 1991; 95:157-189.
22. Piscator M. Role of Cadmium in carcinogenesis with special reference to cancer of the prostate. Environ Health Persp 1981; 40:107-120.
23. Ellithorpe R, Mazur P, Gum G, Button G, Pfadenhauer EH, Settineri RA, Nicolson GL. Comparison of the absorption, brain and prostate distribution and elimination of CaNa2EDTA of rectal chelation suppositories to intravenous administration. J Am Nutricutical Assoc 2007; 10: in press.
24. Propert KJ, Litwin MS, Wang Y, Alexander RB, Calhoun E, Nickel JC, O’Leary MP, Pontari M, McNaughton-Collins M. Responsiveness of the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI). Qual Life Res. 2006; 15:299-305.
25. Rosen RC, Riley A, Wagner G, Osterloh IH, Kirkpatrick J, Mishra A. The international index of erectile function (IIEF): a multidimensional scale for assessment of erectile dysfunction. Urology. 1997; 49:822- 830.
26. Barry MJ, Fowler FJ Jr, O’Leary MP, Bruskewitz RC, Holtgrewe HL, Mebust WK, Cockett AT. The American Urological Association symptom index for benign prostatic hyperplasia. The Measurement Committee of the American Urological Association. J Urol 1992; 148:1549-1557.
27. Madden JA, Plummer SF, Tang J, Garaiova I, Plummer NT, Herbison M, Hunter JO, Shimada T, Cheng L, Shirakawa T. Effect of probiotics on preventing disruption of the intestinal microflora following antibiotic therapy: a double-blind, placebo-controlled pilot study. Int Immunopharmacol. 2005; 5:1091-1097.
28. Bondarenko VM, Lykova EA, Matsulevich TV. Microecological aspects of small intestinal bacterial overgrowth syndrome. Zh Mikrobiol Epidemiol Immunobiol. 2006; 6:57-63.
29. Madsen FA, Bruskewitz RC. Clinical manifestations of benign prostatic hyperplasia. Urol Clin North Am 1995; 22:291-298.
30. Doll R, Fishbein L, Infante P, Landrigan P, Lloyd JW, Mason TJ, Mastromatteo E, Norseth T et al. Problems of epidemiological evidence. Environ Health Perspect 1981; 40:11-20.
31. Kazantzis G. Role of cobalt, iron, lead, manganese, mercury, platinum, selenium and titanium in carcinogenesis. Environ Health Perspect 1981 40:143-161.
32. Jones BV, Mahenthiralingam E, Sabbuba NA, Stickler DJ. Role of swarming in the formation of crystalline Proteus micrbilis biofilms on urinary catheters. J Med Microbiol 2005; 54:807-813.
33. Apicella LL, Sobota AE. Increased urinary tract infection associated with the use of calcium supplements. Urol Res 1990; 18:213-217.
34. Sobota AE, Apicella LL. Reduction in the anti-adherence activity of Tamm-Horsfall protein with increasing concentration of calcium. Urol Res 1991; 19:177-180.
35. Belknap EL. EDTA in the treatment of lead poisoning. Indust Med Surg 1952; 21:305-306.
36. Mosher LE, McCann DS. Some in vivo effects of chelation-I: Rheumatoid arthritis. J Chronic Dis 1963; 16:325-328.
37. Chappell LT, Wilson J. Chelation therapy for vascular disease. Circulation. 1999; 99:164-165.
38. Clarke NE Sr. Atherosclerosis, occlusive vascular disease and EDTA. Am J Cardiol 1960; 6:233-236.
39. Chisolm JJ Jr: The use of chelating agents in the treatment of acute and chronic lead intoxication in childhood. J Pediat 1968; 73:1-38.
40. Castellino N, Aloj S. Effects of calcium sodium ethylenediaminetetra-acetate on the kinetics of the distribution and excretion of lead in the rat. Brit J Indust Med 1965; 22:172-178.
41. Aronson AL, Hammond PB, Strafuss AC. Studies with calcium ethylenediaminetetraacetate in calves; toxicity and use in bovine lead poisoning. Toxicol Appl Pharmacol 1968; 12:337-349.
42. Betieman V, Landy E, Moseka JK and Wedsen RP. Contribution of lead hypertension with renal impairment. N Engl J. Med. 1983; 309:17-21.
43. Mc Dragh, EW, Rudolph DJ, Cherskin E. The effect of EDTA chelation therapy plus supportive multivitamin trace mineral supplementation upon renal function: as study on serum creatinine. J. Holistic Med 1982; 4:146-151.
44. Chen IW, Park HM, King LR, Bahr GK, Goldsmith RE. Radioimmunoassay of parathyroid hormone: peripheral plasma immunoreactive parathyroid hormone response to ethylenediaminetetraacetate. J Nucl Med 1974; 15:763-769.