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Linus PAULING, et al.

Vitamin C Therapy








Sebastian Padayatty, et al. : Intravenously administered vitamin C as cancer therapy: three cases

Ewan Cameron / Linus Pauling : Supplemental ascorbate in the supportive treatment of cancer: Prolongation of survival times in terminal human cancer.
 
Ewan Cameron / Linus Pauling : Supplemental ascorbate in the supportive treatment of cancer -- Reevaluation of prolongation of survival times in terminal human cancer*

Sebastian Padayatty / Mark Levine : New insights into the physiology and pharmacology of vitamin C

Qi Chen, et al : Pharmacologic ascorbic acid concentrations selectively kill cancer cells: Action as a pro-drug to deliver hydrogen peroxide to tissues

L. John Hoffer : Proof versus plausibility -- rules of engagement for the struggle to evaluate alternative cancer therapies

R. Matthias / L. Pauling : US5278189 -- Prevention and treatment of occlusive cardiovascular disease with ascorbate and substances that inhibit the binding of lipoprotein (A)

R. Matthias / L. Pauling : US5230996 -- Use of ascorbate and tranexamic acid solution for organ and blood vessel treatment prior to transplantation

N. Riordan : US5639787 -- Therapeutic method for the treatment of cancer

http://www.paulingtherapy.com

Owen Fonorow : Chronic Scurvy -- The Suppression the Real Nature, Cause, and Cure for Heart Disease, (2005)

Related : KITT, Douglas : DeHydroAscorbic Acid ~ 500% better absorption than Vit. C -- articles, patents, & D-I-Y preparation.



https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1405876/
Riordan et al J Orthomol Med 1998: 13: 7203

Riordan et al PR Health Sci J 2004: 23:115-8
Canada Medical Association Journal (CMAJ) 2006 174:937-42.
doi:  10.1503/cmaj.050346
PMCID: PMC1405876

Intravenously administered vitamin C as cancer therapy: three cases

Sebastian J. Padayatty, Hugh D. Riordan, Stephen M. Hewitt, Arie Katz, L. John Hoffer, and Mark Levine

Abstract

Early clinical studies showed that high-dose vitamin C, given by intravenous and oral routes, may improve symptoms and prolong life in patients with terminal cancer. Double-blind placebo-controlled studies of oral vitamin C therapy showed no benefit. Recent evidence shows that oral administration of the maximum tolerated dose of vitamin C (18 g/d) produces peak plasma concentrations of only 220 μmol/L, whereas intravenous administration of the same dose produces plasma concentrations about 25-fold higher. Larger doses (50–100 g) given intravenously may result in plasma concentrations of about 14 000 μmol/L. At concentrations above 1000 μmol/L, vitamin C is toxic to some cancer cells but not to normal cells in vitro. We found 3 well-documented cases of advanced cancers, confirmed by histopathologic review, where patients had unexpectedly long survival times after receiving high-dose intravenous vitamin C therapy. We examined clinical details of each case in accordance with National Cancer Institute (NCI) Best Case Series guidelines. Tumour pathology was verified by pathologists at the NCI who were unaware of diagnosis or treatment. In light of recent clinical pharmacokinetic findings and in vitro evidence of anti-tumour mechanisms, these case reports indicate that the role of high-dose intravenous vitamin C therapy in cancer treatment should be reassessed.

Thirty years ago Cameron, Campbell and Pauling reported beneficial effects of high-dose vitamin C (ascorbic acid) therapy for patients with terminal cancer.1–4 Subsequent double-blind, randomized clinical trials at the Mayo Clinic failed to show any benefit,5,6 and the role of vitamin C in cancer treatment was discarded by mainstream oncologists.7,8 Vitamin C continues, however, to be used as an alternative cancer therapy.9,10

A key distinction between conventional, science-based medicine and alternative therapy is the presence or absence of scientific plausibility.11 In conventional medicine, the efficacy of treatment is proven by properly conducted clinical trials. Many treatments are still used if there is moderately good, albeit inconclusive evidence of efficacy (“clinical plausibility”), especially when treatment rationale agrees with biologic facts (conferring “biological plausibility”).11 Vitamin C is an alternative cancer therapy because the results obtained in original studies that suggested clinical benefit were not confirmed by controlled clinical trials, and the notion that high-dose vitamin C was selectively toxic to cancer cells was biologically implausible.

New information is available pertaining to biological plausibility. Although similar doses of vitamin C were used in the Cameron–Pauling and Mayo Clinic studies, the Cameron–Pauling studies combined intravenous and oral administration whereas the Mayo Clinic studies used only oral administration.1,2,12–14 Recent pharmacokinetics modeling15 indicates that with oral administration, even very large and frequent doses of vitamin C will increase plasma concentrations only modestly, from 70 μmol/L to a maximum of 220 μmol/L, whereas intravenous administration raises plasma concentrations as high as 14 000 μmol/L. Concentrations of 1000–5000 μmol/L are selectively cytotoxic to tumour cells in vitro,16–20 and emerging evidence indicates that ascorbic acid at concentrations achieved only by the intravenous route may function as a pro-drug for hydrogen peroxide delivery to tissues.20 The in vitro biologic evidence and clinical pharmacokinetics data confer biological plausibility to the notion that vitamin C could affect cancer biology and may explain in part the negative results of the Mayo Clinic trials.13,15,21,22 Thus, sufficient evidence has accumulated, not to use vitamin C as cancer treatment, but to further explore the therapeutic concept. One way to increase the clinical plausibility of alternative cancer therapies is rigorous, well-documented case reporting, as laid out in the US National Cancer Institute (NCI) Best Case Series guidelines (http://www3.cancer.gov/occam/bestcase.html).23,24 Such case series might identify alternative therapies that merit further investigation.23,24

Case reports of apparent responses by malignant disease to intravenous vitamin C therapy have appeared,25–30 including those of 2 of the 3 patients presented below.25,26 However, they were reported without sufficient detail or with incomplete follow-up for evaluation and without conforming to NCI Best Case Series guidelines. They also lacked objective pathologic confirmation, which is a pillar of NCI guidelines. In this article, we use NCI Best Case Series guidelines to report 3 cases of patients with usually progressive malignant disease who received intravenous vitamin C therapy as their only significant cancer therapy and whose clinical courses were unusually favourable. Original diagnostic material obtained before treatment with vitamin C was reviewed by pathologists at the National Institutes of Health (NIH) who were unaware of the diagnoses and treatments.

Patient 1

This case was previously reported but without long-term follow-up, without detail, and with no independent pathologic confirmation.25,26 A 51-year-old woman was found in August 1995 to have a tumour involving her left kidney. At nephrectomy in September 1995 this was shown to be a renal cell carcinoma 9 cm in diameter with thrombus extending into the renal vein. Chest radiography results were normal, and there was no evidence of metastatic disease on CT scan of the chest and abdomen. In March 1996 a CT scan of the chest indicated several new, small, rounded and well-defined soft tissue masses no larger than 5 mm in diameter; they were judged consistent with metastatic cancer. By November 1996 chest radiography revealed multiple cannonball lesions (Fig. 1).

Fig. 1: Chest radiography, November 1996, about 1 month after intravenous vitamin C therapy was started. Cannonball lesions are evident in both lung fields, as indicated by the arrows and lines.

The patient declined conventional cancer treatment and instead chose to receive high-dose vitamin C administered intravenously at a dosage of 65 g twice per week starting in October 1996 and continuing for 10 months. She also used other alternative therapies: thymus protein extract, N-acetylcysteine, niacinamide and whole thyroid extract (Table 1). In June 1997 chest radiography results were normal except for one remaining abnormality in the left lung field, possibly a pulmonary scar (Fig. 2).

Table 1

Fig. 2: Chest radiography, June 1997, showing regression of the lesions; the arrow indicates one residual abnormality.

In October 2001 a new mass 3.5 cm in diameter in the anterior right lung was detected on radiography. A transthoracic biopsy revealed small-cell carcinoma of the lung. The patient opted for intravenous vitamin C injections. The lung mass remained constant in size in radiograpy taken in May and August 2002 but had increased to 4 cm in views taken in October 2002. In early November hyponatremia developed. Two weeks later the patient was admitted to hospital with abdominal distension and constipation. Barium studies revealed slow transit but no intestinal obstruction. Results of a CT scan of the abdomen were normal. She died shortly afterward, and no autopsy was performed. Histopathologic review of the primary renal tumour at the NIH confirmed the diagnosis of clear-cell renal carcinoma, type, nuclear grade III/IV, with the largest diameter measuring 6.5 cm. The tumour involved the renal vein and hilar perinephric fat. Pathologic review of the lung tumour biopsy specimen of October 2001 was not conducted at the NIH. Local pathologists diagnosed this specimen as indicating small-cell lung cancer and not recurrent metastatic renal cell carcinoma.

This case describes the regression of pulmonary metastatic renal cancer in a patient receiving high-dose intravenous vitamin C therapy. According to the NCI Best Case Series guidelines, the credibility of this case would be increased by biopsy proof that the multiple slowly growing bilateral cannonball lung nodules in this patient with known renal cell carcinoma were actually malignant. However, in this case, the clinical characteristics and evolution of the pulmonary lesions, in the absence of bacterial infection or other systemic disease, make any other diagnosis unlikely. The clinician attending the patient deemed a confirmatory biopsy to be unnecessary and inappropriate in this setting. A plausible alternative explanation to the conclusion that this patient's metastatic renal cell cancer responded to intravenous vitamin C therapy is that the tumours spontaneously regressed. Spontaneous regression has been reported in renal cell cancer, but it is rare, occurring in fewer than 1% of cases and typically after nephrectomy, radiation to the primary tumour or primary tumour embolization.31,32 Here, metastatic disease appeared several months after nephrectomy, rather than regressing in response to it. As well, the primary cancer was nuclear grade III/IV and involved the renal vein, factors associated with a highly unfavourable prognosis.31

Of note, more than 4 years after stopping intravenous vitamin C therapy and with the renal cell cancer in complete remission, primary small-cell lung cancer was diagnosed in this patient, who was a long-standing cigarette smoker. The second cancer did not respond to high-dose vitamin C therapy. From the clinical history it appears the tumour remained a constant size for many months and likely slowly progressed until her death about a year after diagnosis despite the resumption of intravenous vitamin C therapy.

Patient 2

A 49-year-old man presented to his physician in 1996 with hematuria and was found at cystoscopy to have a primary bladder tumour with multiple satellite tumours extending 2–3 cm around it. Transurethral resection of the primary tumour and its surrounding tumour satellites was carried out until apparently normal muscle was reached and the tumour base was fulgurated. The patient declined systemic or intravesical chemotherapy or radiotherapy and instead chose intravenous vitamin C treatment. He received 30 g of vitamin C twice per week for 3 months, followed by 30 g once every 1–2 months for 4 years, interspersed with periods of 1–2 months during which he had more frequent infusions. Histopathologic review at the NIH revealed a grade 3/3 papillary transitional cell carcinoma invading the muscularis propria. Now, 9 years after diagnosis, the patient is in good health with no symptoms of recurrence or metastasis. The patient used the following supplements: botanical extract, chondroitin sulfate, chromium picolinate, flax oil, glucosamine sulfate, α-lipoic acid, Lactobacillus acidophilus and L. rhamnosus and selenium (Table 2).

Table 2

Complete or partial bladder removal is the standard treatment for stage T2 (muscle invasive) bladder cancer, since the presence of muscle invasion appears to be the best predictor of aggressive behaviour. When treated only locally, as in this case, invasive transitional cell bladder cancer almost invariably develops into clinically apparent local or metastatic disease within a short period.33–35 There are reports of transurethral tumour resection being offered as the sole initial therapy in carefully selected patients with T2 disease. In one report 20% of patients with muscle invasive bladder cancer treated only with transurethral resection remained free of recurrent disease after 3–7 years of follow-up.36 However, such minimal therapy is considered an option only when the cancer is solitary, well defined and completely excised as documented by pathologic evaluation,37 whereas this patient presented with multiple tumours and associated muscle invasion.

Patient 3

This case was previously reported but without detail and without independent pathologic confirmation.26,30 A 66-year-old woman was found in January 1995 to have a large left paraspinal mass medial to the iliopsoas muscle at the L4–5 level. On imaging the mass measured 3.5–7 cm transversely and 11 cm in the craniocaudal direction, with evidence of extension into the posterior paraspinal muscle and bone invasion. Chest radiography results were normal. An open biopsy specimen was diagnostic of a diffuse large B-cell lymphoma. The patient's oncologist recommended local radiation therapy and chemotherapy. Although she agreed to a 5-week course of local radiation therapy, the patient refused chemotherapy, electing instead to receive vitamin C intravenously. She received 15 g of vitamin C twice per week for about 2 months, 15 g once to twice per week for about 7 months, and then 15 g once every 2–3 months for about 1 year. This began in mid-January 1995 concurrently with the radiation therapy, which was given as AP/PA parallel opposed 18 MEV x-rays and between 1–18 and 2–28–95, 5040 Centi Gray in 28 fractions delivered to the mid-plane of the body with 3:2 loading from the back. At this time a left axillary lymph node 1 cm in diameter and a right axillary lymph node 1.5 cm in diameter were palpable.

Two weeks later, in early February 1995, the right and left axillary lymph nodes remained palpable and a new left cervical lymph node 1 cm in diameter and a new left supraclavicular lymph node larger than 1 cm were apparent on physical examination. Intravenous vitamin C therapy continued. Three weeks later the supraclavicular and cervical lymph nodes were no longer palpable, the left axillary node had disappeared, and the right axillary node had decreased in size to less than 1 cm. After a further 3 weeks, in mid-March 1995, there was no lymphadenopathy in the neck and no palpable axillary lymphadenopathy. In late April 1995 a new left cervical lymph node was detected, and histopathologic review identified a biopsy specimen as identical to the original tumour. The patient once again refused chemotherapy and continued her program of intravenous vitamin C injections. Two months later, in June 1995, there was marked left supraclavicular lymphadenopthy 3 cm in size, with shotty right axillary nodes but no adenopathy in the left axilla. Four months later, in October 1995, a single right submandibular node was palpable, but the supraclavicular and all other areas, including the axillas, had no palpable lymph nodes. In May 1996 a left anterior cervical node 1.5 cm in size was present, but there was no other adenopathy. Intravenous vitamin C therapy continued through late December 1996, at which time the patient was in normal health and had no clinical sign of lymphoma. The patient remains in normal health 10 years after the diagnosis of diffuse large B-cell lymphoma, never having received chemotherapy. The patient used additional products: β-carotene, bioflavonoids, chondroitin sulfate, coenzyme Q10, dehydroepiandrosterone, a multiple vitamin supplement, N-acetylcysteine, a botanical supplement and bismuth tablets (Table 3). Histopathologic examination of the original paraspinal mass at the NIH confirmed a diffuse large B-cell lymphoma at stage III, with a brisk mitotic rate.

Table 3

Patients with untreated stage III diffuse B-cell lymphoma have a dismal prognosis. This case, like the preceding one, is unusual in that the patient refused chemotherapy, which might have produced a long-term remission. It appears, nonetheless, that a cure occurred in connection with intravenous vitamin C infusions.

Discussion

These cases were analyzed in accordance with the NCI's Best Case Series process, which reports and interprets apparent responses to alternative therapies.23,24 Apart from its implications regarding vitamin C, this article illustrates the use of the Best Case Series approach in assessing the clinical plausibility of novel therapies. None of these case histories provides definitive proof that intravenous vitamin C therapy was responsible for the patient's unusually favourable clinical course. It is often difficult to prove definitively that a given treatment is responsible for a specific clinical outcome. When the treatment is unorthodox, alternative explanations, even if highly unlikely, tend to be preferred.38,39 However, although they do not provide grounds for advocating intravenous vitamin C therapy as a cancer treatment, these cases increase the clinical plausibility of the notion that vitamin C administered intravenously might have effects on cancer under certain circumstances.

The overall plausibility of ascorbic acid administered intravenously as a cancer therapy is enhanced by recent insights into clinical pharmacokinetics and in vitro cancer-specific cytotoxicity of vitamin C.15–20 Pharmacokinetics data show that orally administered vitamin C results in tightly controlled plasma and cell concentrations. Subjects consuming 200–300 mg per day of vitamin C in 5 or more daily servings of fruits and vegetables have fasting steady state plasma concentrations of about 70–80 μmol/L.40,41 Even with maximally tolerated oral doses of 3 g every 4 hours, peak plasma concentrations are estimated to not exceed 220 μmol/L.15 Intravenous administration of vitamin C bypasses tight control for several hours, until homeostasis is restored by renal excretion. Depending on the dose and infusion rate, peak plasma concentrations obtained intravenously are estimated to reach 14 000 μmol/L, and concentrations above 2000 μmol/L may persist for several hours. Emerging in vitro data show that extracellular ascorbic acid selectively kills some cancer but no normal cells by generating hydrogen peroxide.20 Death is mediated exclusively by extracellular ascorbate, at pharmacologic concentrations that can be achieved only by intravenous administration. Vitamin C may serve as a pro-drug for hydrogen peroxide delivery to extravascular tissues, but without the presence of hydrogen peroxide in blood. These data are consistent with clinical pharmacokinetics of vitamin C administered intravenously.15 Of note, only a minority of cancer patients reported by Cameron and colleagues responded to intravenous and oral vitamin C therapy,1–4 and not all cancer cells were killed by ascorbic acid in vitro.20 Further basic investigation of pharmacologic vitamin C concentrations in mediating cell death will facilitate discovery of the mechanisms responsible for sensitivity and resistance in vitro and in vivo. On the basis of emerging clinical and in vitro data, early-phase clinical trials of intravenous vitamin C therapy alone and in combination with conventional chemotherapy are currently in the planning and execution phase, including a formal phase I trial in progress at McGill University.42–44

The cases reported here do not prove that vitamin C induced the favourable outcomes observed. These patients received other alternative medicine therapies. Spontaneous remission of some tumours may occur rarely, although the 3 cancers reported here are dissimilar. Accretion of more cases meeting NCI Best Case Series guidelines may indicate whether vitamin C or other factors contribute to such remissions.

It is likely that high vitamin C intakes have low toxicity, except under certain conditions.45,46 Intravascular hemolysis was reported after massive vitamin C administration in people with glucose-6-phosphate dehydrogenase deficiency.46 Administration of high-dose vitamin C to patients with systemic iron overload may increase iron absorption and represents a contraindication.46,47 Ascorbic acid is metabolized to oxalate, and 2 cases of acute oxalate nephropathy were reported in patients with pre-existing renal insufficiency given massive intravenous doses of vitamin C.48,49 Therefore, patients with renal insufficiency or renal failure, or who are undergoing dialysis, should not receive high doses of vitamin C.46 It is controversial whether high-dose vitamin C use is associated with oxalate kidney stones, and patients with hyperoxaluria or a prior history of oxalate kidney stones have a relative contraindication to high-dose vitamin C.46 Rare cases of acute tumour hemorrhage and necrosis were reported in patients with advanced cancer within a few days of starting high-dose intravenous vitamin C therapy, although this was not independently verified by pathologic review.1,50 Although tumour hemorrhage suggests an anticancer potential for ascorbate, there is the potential for risk to some patients.

The cases reported here are of tumours confirmed by histopathologic examination to have poor prognosis but that instead had long clinical remissions. Most previous case reports lacked independent pathologic confirmation of the tumour and did not follow the NCI Best Case Series guidelines, which makes their interpretation difficult. Recent findings show that only high-dose intravenous, but not oral, vitamin C therapy results in very high plasma vitamin C concentrations (e.g., 14 000 μmol/L). At these concentrations, the vitamin is toxic to some cancer cells, possibly because at these concentrations the vitamin is a pro-drug for hydrogen peroxide formation in extracellular fluid. Accumulated data confer some degree of biological and clinical plausibility to the notion that high-dose intravenous vitamin C therapy may have anti-tumour effects in certain cancers. When all available data are considered, further clinical study as to safety and efficacy of intravenous vitamin C is warranted.

Acknowledgments

The writing of this paper was supported in part by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Z01 DK 54506).

Footnotes

Editor's take

• Intravenous administration of the maximum tolerated dose of vitamin C produces plasma levels 25 times that achieved when the same dose is administered orally. At high plasma concentrations vitamin C is toxic to some cancer cells but not to normal cells in vitro.

• Using the National Cancer Institute Best Case Series guidelines, the authors reviewed 3 cases of advanced cancer where patients had unexpectedly long survival times after receiving high-dose intravenous vitamin C therapy.

Implications for practice: In a setting of biological plausibility and clinical plausibility, further research into vitamin C as a treatment for cancer is warranted.

REFERENCES

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48. McAllister CJ, Scowden EB, Dewberry FL, et al. Renal failure secondary to massive infusion of vitamin C. JAMA 1984;252:1684.
49. Wong K, Thomson C, Bailey RR, et al. Acute oxalate nephropathy after a massive intravenous dose of vitamin C. Aust N Z J Med 1994;24:410-1.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431183/
Proc Natl Acad Sci U S A. 1976 Oct; 73(10): 3685–3689.
PMCID: PMC431183

Supplemental ascorbate in the supportive treatment of cancer: Prolongation of survival times in terminal human cancer.

E Cameron and L Pauling

Abstract

Ascorbic acid metabolism is associated with a number of mechanisms known to be involved in host resistance to malignant disease. Cancer patients are significantly depleted of ascorbic acid, and in our opinion this demonstrable biochemical characteristic indicates a substantially increased requirement and utilization of this substance to potentiate these various host resistance factors. The results of a clinical trial are presented in which 100 terminal cancer patients were given supplemental ascorbate as part of their routine management. Their progress is compared to that of 1000 similar patients treated identically, but who received no supplemental ascorbate. The mean survival time is more than 4.2 times as great for the ascorbate subjects (more than 210 days) as for the controls (50 days). Analysis of the survival-time curves indicates that deaths occur for about 90% of the ascorbate-treated patients at one-third the rate for the controls and that the other 10% have a much greater survival time, averaging more than 20 times that for the controls. The results clearly indicate that this simple and safe form of medication is of definite value in the treatment of patients with advanced cancer.




https://www.ncbi.nlm.nih.gov/pmc/articles/PMC336151/
Proc Natl Acad Sci U S A. 1978 Sep; 75(9): 4538–4542.
PMCID: PMC336151

Supplemental ascorbate in the supportive treatment of cancer: Reevaluation of prolongation of survival times in terminal human cancer

Ewan Cameron and Linus Pauling

Abstract


A study has been made of the survival times of 100 terminal cancer patients who were given supplemental ascorbate, usually 10 g/day, as part of their routine management and 1000 matched controls, similar patients who had received the same treatment except for the ascorbate. The two sets of patients were in part the same as those used in our earlier study [Cameron, E. & Pauling, L. (1976) Proc. Natl. Acad. Sci. USA 73, 3685-3689]. Tests confirm that the ascorbate-treated patients and the matched controls are representative subpopulations of the same population of “untreatable” patients. Survival times were measured not only from the date of “untreatability” but also from the precisely known date of first hospital attendance for the cancer that eventually reached the terminal stage. The ascorbate-treated patients were found to have a mean survival time about 300 days greater than that of the controls. Survival times greater than 1 yr after the date of untreatability were observed for 22% of the ascorbate-treated patients and for 0.4% of the controls. The mean survival time of these 22 ascorbate-treated patients is 2.4 yr after reaching the apparently terminal stage; 8 of the ascorbate-treated patients are still alive, with a mean survival time after untreatability of 3.5 yr.



https://www.ncbi.nlm.nih.gov/pmc/articles/PMC80729/
CMAJ. 2001 Feb 6; 164(3): 353–355.
PMCID: PMC80729

New insights into the physiology and pharmacology of vitamin C

Sebastian J. Padayatty and Mark Levine

Pharmacokinetic data are now available for the absorption, plasma concentrations and renal excretion of vitamin C. With these new insights, it is worth reconsidering the clinical role of vitamin C in scurvy and subclinical deficiency, the amount of vitamin C required for good health and, in particular, the speculative use of high doses of vitamin C to treat cancer.

Scurvy, the dread of sailors past, conjures up images of perilous sea voyages and rough tattooed men laid low by ignorance of a vital amine, later designated vitamin C (which, as it happens, is not an amine). While scurvy devastated seafarers, it was endemic in the landbound, occurring widely wherever fruit and vegetables were in short supply. Military campaigns from the Crusades to the Napoleonic Wars, the American Civil War, and even World War I were stymied by widespread and often fatal scurvy among the troops. The antiscorbutic principle, identified and named ascorbic acid (vitamin C) in 1932, is a simple water-soluble sugar-like molecule. Early experiments, though perhaps flawed, showed that the consumption of as little as 10 mg of vitamin C a day would prevent signs of clinical scurvy.1 Although minute amounts will forestall death, the optimum requirements for good health are not known. Vitamin C is concentrated in many tissues, but these tissue stores are easily depleted. James Lind's Treatise on Scurvy, first published in 1753, reported the onset of the disease in sailors after a month and a half at sea and described lassitude as its early and invariable symptom.2 Depletion–repletion studies in volunteers using a diet free of vitamin C have shown that plasma vitamin C falls below 10 μmol/L in less than a month. At these concentrations, fatigue is invariably present,3 and physical signs appear soon after.1 A person who has been consuming 100 mg of vitamin C daily will not develop scurvy for a month, even if the intake of vitamin C is stopped altogether.3 Recently, on the advice of the Food and Nutrition Board of the US National Academy of Sciences, US and Canadian recommended dietary allowances were increased from 60 mg per day to 75 mg per day for women and 90 mg per day for men. Vitamin C intake is less than 60 mg in 20%–30% of US adults. It is even lower among many population subgroups, including children. Subclinical vitamin C deficiency is much more common than is generally recognized,4 especially because the first symptom of deficiency is fatigue, a nonspecific and common complaint.

As an electron donor, vitamin C acts as a cofactor for 8 enzymes involved in collagen hydroxylation, biosynthesis of carnitine and norepinephrine, tyrosine metabolism and amidation of peptide hormones.5 Vitamin C also has many nonenzymatic actions. It is a powerful water-soluble antioxidant and, at physiological concentrations, probably does not produce reactive intermediaries. It protects low-density lipoproteins from oxidation, reduces harmful oxidants in the stomach and promotes iron absorption. Its antioxidant role in vivo is, however, unclear. Plasma ascorbic acid concentrations may be low in chronic or acute oxidant states such as in diabetes, in smokers, or following acute pancreatitis or myocardial infarction. Ascorbic acid is easily oxidized to the unstable dehydroascorbic acid. Dehydroascorbic acid is not normally detectable in plasma but may occur transiently during oxidant stress. Ascorbic acid is transported into the cell by sodium-dependent vitamin C transporters SVCT1 and SVCT2, one or both of which are found in most tissues.6 Dehydroascorbic acid is transported by glucose transporters GLUT1 and GLUT3, and, in insulin-sensitive tissues, also by GLUT4. When exposed to bacteria, neutrophils oxidize extracellular ascorbic acid to form dehydroascorbic acid, which is transported into the neutrophil and rapidly reduced to ascorbic acid by the protein glutaredoxin (Fig. 1). As a result of this recycling of extracellular ascorbic acid, the neutrophil internal concentration of ascorbic acid increases 10-fold.7 Ascorbic acid may quench oxidants generated during phagocytosis and, thus, protect the neutrophil and surrounding tissues from oxidative damage. Brain, adrenal cortex, liver, spleen, pancreas and kidney tissues concentrate vitamin C for unknown reasons.

Fig. 1: Ascorbic acid (AA) transport, dehydroascorbic acid (DHA) transport and recycling in human neutrophils. AA, transported by sodium-dependent vitamin C transporters (SVCT), maintains mmol/L concentrations of AA inside neutrophils. Activated neutrophils ...

When given orally, ascorbic acid is well absorbed at lower doses, but absorption decreases as the dose increases. Thus, median bioavailability following an oral dose is 87% for 30 mg, 80% for 100 mg, 72% for 200 mg and 63% for 500 mg. Less than 50% of a 1250-mg dose is absorbed, and most of the absorbed dose is excreted in the urine.3,8 Ascorbic acid is not protein bound, so it is filtered and reabsorbed by the kidneys in healthy subjects but is lost in patients who have been hemodialyzed. Ascorbic acid begins to appear in urine at doses above 100 mg/day, corresponding to a plasma concentration of about 60 μmol/L, at which point plasma is 70% saturated and circulating white blood cells are fully saturated. Decreased bioavailability and renal excretion keep plasma vitamin C at less than 100 μmol/L, even with an oral dose of 1000 mg. In men at steady state, a 30-mg daily intake results in a mean plasma concentration of 9 μmol/L, 60 mg results in25 μmol/L, 100 mg in 56 μmol/L and 200 mg in 75 μmol/L. Thus, the dose–concentration relationship is sigmoidal, with the steep portion of the curve lying between 30 mg and 100 mg of oral vitamin C daily.3,8 Doses greater than 500 mg daily contribute little to plasma or tissue stores. Circulating white blood cells contain 10–30 times the plasma concentrations of vitamin C.

In addition to the physiological role of ascorbic acid, it may have unrelated pharmacological effects. When ascorbic acid is administered intravenously, the limiting absorptive mechanism is bypassed and very high plasma levels are attained. Following the administration of 1.25 g intravenously, a peak plasma level of 1000 μmol/L is reached, even though 100 μmol/L is not exceeded by oral dosing.8 When 5–10 g is given intravenously, the resulting plasma levels may be as high as 5000 μmol/L.9

This difference between the oral and intravenous administration of high doses was not adequately appreciated in studies of the treatment of cancer with vitamin C. In vitro, ascorbic acid is cytotoxic to many malignant cell lines10 at concentrations that can be achieved in plasma by intravenous, but not oral, administration. Whether similar effects would occur in vivo is not known. The unconventional studies of Cameron and Campbell,11 later joined by Linus Pauling, used high-dose intravenous vitamin C to treat terminal cancer. They reported clinical benefits and improved survival.12,13 Because these studies were not randomized or placebo controlled, Moertel and colleagues carried out 2 randomized placebo-controlled clinical trials at the Mayo Clinic to check these findings.14,15 Using high-dose oral vitamin C, they found no benefit. Given the recent appreciation of the differing plasma levels resulting from oral versus intravenous administration, it is difficult to compare the studies carried out by Moertel and coworkers with those of Cameron and his colleagues. The cause of cancer patients will be better served if advocates and sceptics concerning the efficacy of vitamin C re-examine these issues with both open minds and scientific rigour.

We now know that plasma vitamin C concentrations are tightly controlled and that the vitamin is concentrated by many tissues. The optimum intake of vitamin C, its function in various tissues and its antioxidant actions in vivo remain to be elucidated. In the meantime, we should rigorously explore the potential anticancer effects of vitamin C, when administered intravenously at high doses, in patients with well-documented cancer16 in whom other options have been exhausted. If these studies show promise, then randomized clinical trials should follow.

Footnotes

Clinicians who wish to submit cases for review can contact Drs. Padayatty and Levine for instructions at the address below.

This article has been peer reviewed.

Competing interests: None declared.

Correspondence to: Dr. Mark Levine, Molecular and Clinical Nutrition Section, Bldg. 10, Rm. 4D52–MSC 1372, National Institutes of Health, Bethesda MD 20892–1372; fax 301 402-6436; vog.hin.kddin.artni@LkraM

References

1. Hodges RE, Baker EM, Hood J, Sauberlich HE, March SC. Experimental scurvy in man. Am J Clin Nutr 1969;22:535-48.
2. Lind J. Lind's Treatise on Scurvy. In: Stewart CP, Guthrie D, editors. Bicentenary Volume. Edinburgh: Edinburgh University Press; 1953. p. 69-112.
3. Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, et al. Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci U S A 1996;93:3704-9.
4. Johnston CS, Thompson LL. Vitamin C status of an outpatient population. J Am Coll Nutr 1998;17:366-70.
5. Levine M, Rumsey SC, Daruwala R, Park JB, Wang Y. Criteria and recommendations for vitamin C intake. JAMA 1999;281:1415-23.
6. Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen XZ, Wang Y, et al. A family of mammalian Na+-dependent L-ascorbic acid transporters. Nature 1999;399:70-5.
7. Rumsey SC, Levine M. Absorption, transport, and disposition of ascorbic acid in humans. J Nutr Biochem 1998;9:116-30.
8. Graumlich JF, Ludden TM, Conry-Cantilena C, Cantilena LR Jr, Wang Y, Levine M. Pharmacokinetic model of ascorbic acid in healthy male volunteers during depletion and repletion. Pharm Res 1997;14:1133-9.
9. Riordan NH, Riordan HD, Meng X, Li Y, Jackson JA. Intravenous ascorbate as a tumor cytotoxic chemotherapeutic agent. Med Hypotheses 1995;44:207-13.
10. Koh WS, Lee SJ, Lee H, Park C, Park MH, Kim WS, et al. Differential effects and transport kinetics of ascorbate derivatives in leukemic cell lines. Anticancer Res 1998;18:2487-93.
11. Cameron E, Campbell A. The orthomolecular treatment of cancer. II. Clinical trial of high-dose ascorbic acid supplements in advanced human cancer. Chem Biol Interact 1974;9:285-315.
12. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal human cancer. Proc Natl Acad Sci U S A 1978;75:4538-42.
13. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer. Proc Natl Acad Sci U S A 1976;73:3685-9.
14. Moertel CG, Fleming TR, Creagan ET, Rubin J, O'Connell MJ, Ames MM. High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy. A randomized double-blind comparison. N Engl J Med 1985;312:137-41.
15. Creagan ET, Moertel CG, O'Fallon JR, Schutt AJ, O'Connell MJ, Rubin J, et al. Failure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer. A controlled trial. N Engl J Med 1979;301:687-90.
16. Hawkins MJ, Friedman MA. National Cancer Institute's evaluation of unconventional cancer treatments. J Natl Cancer Inst 1992;84:1699-702. Current version of Best Case Series Program is available: http://occam.nci.nih.gov (accessed 2000 Dec 21).



https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1224653/
Proc Natl Acad Sci U S A. 2005 Sep 20; 102(38): 13604–13609.
Published online 2005 Sep 12. doi:  10.1073/pnas.0506390102
PMCID: PMC1224653

Pharmacologic ascorbic acid concentrations selectively kill cancer cells: Action as a pro-drug to deliver hydrogen peroxide to tissues

Qi Chen, Michael Graham Espey, Murali C. Krishna, James B. Mitchell, Christopher P. Corpe, Garry R. Buettner, Emily Shacter, and Mark Levine


Abstract

Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test whether ascorbate killed cancer cells selectively, and if so, to determine mechanisms, using clinically relevant conditions. Cell death in 10 cancer and 4 normal cell types was measured by using 1-h exposures. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC50 values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC50 of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H2O2 formation. Cell death from H2O2 added to cells was identical to that found when H2O2 was generated by ascorbate treatment. H2O2 generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H2O2, ascorbate addition to blood generated no detectable H2O2 and only trace detectable ascorbate radical. Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H2O2, and that blood can be a delivery system of the pro-drug to tissues. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for treatment of infections where H2O2 may be beneficial.


Ascorbic acid (vitamin C, ascorbate) has a controversial history in cancer treatment (1). Observational reports described ascorbate, given in pharmacologic doses of 10 g daily, as effective in treating some cancers and in improving patient well-being (2-4). Subsequently, the same dose had no effect on patient well-being and survival in two double-blind placebo-controlled trials, and ascorbate was discarded as a treatment modality (5, 6). Recent clinical evidence, however, indicates that the role of ascorbate in cancer treatment should be examined anew (7). The originally reported observational studies used i.v. and oral ascorbate, but the subsequent double-blind placebo-controlled studies used only oral ascorbate. It was not recognized that the route of ascorbate administration might produce large differences in plasma concentrations. Recent pharmacokinetics studies in men and women show that 10 g of ascorbate given i.v. is expected to produce plasma concentrations of nearly 6 mM, which are >25-fold higher than those concentrations from the same oral dose (7-9). As much as a 70-fold difference in plasma concentrations is expected between oral and i.v. administration, depending on dose. Despite inconsistencies, some in vitro studies showed that ascorbate killed cancer cells, although mechanisms and physiologic relevance were unclear (10-12). Complementary and alternative medicine practitioners worldwide currently use ascorbate i.v. in some patients, in part because there is no apparent harm (13-15).

Given its potential safety and benefit, there is merit in investigating i.v. ascorbate as a possible novel cancer treatment modality. It is essential first to learn whether ascorbate acts as an anticancer agent in vitro, and if so, by what mechanisms. Our goals were to address the following: Does ascorbate in pharmacologic concentrations kill cancer cells, but not normal cells, using conditions that mimic i.v. use and a clinically relevant time course? Is action dependent on extracellular ascorbate, intracellular ascorbate, or both? If effective, what are the mechanisms? Can ascorbate be delivered to tissues without harm? Are there implications for other diseases?

We studied ascorbate at physiologic (0.1 mM) and pharmacologic (0.3-20 mM) concentrations using 1-h incubations to mimic clinical i.v. use (7-9). The data showed that pharmacologic concentrations of ascorbate killed cancer but not normal cells, that cell death was dependent only on extracellular but not intracellular ascorbate, and that killing was dependent on extracellular hydrogen peroxide (H2O2) formation with ascorbate radical as an intermediate. Ascorbate generated detectable levels of H2O2 in extracellular medium in the presence of trace serum protein but not in whole blood. The findings indicate that ascorbate at pharmacologic concentrations in blood may be a pro-drug for H2O2 delivery to tissues, with major therapeutic implications.

Materials and Methods

Cells and Reagents. Human Burkitt's lymphoma cells (JLP-119) were obtained and studied as described in ref. 16. Other cell lines were purchased from American Type Culture Collection and were grown at 37°C in 5% CO2/95% air in recommended media containing 10% FBS (GIBCO). Human lymphocytes and monocytes were isolated by apheresis (17) from at least six healthy subjects and used immediately. Ascorbic acid was always buffered to pH 7.0 with sodium hydroxide and prepared immediately before use. Dehydroascorbic acid was freshly prepared (18). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Molecular Probes and bacto-agar was from Difco. Other reagents, enzymes, and media were from general commercial sources.

Cell Death. Nuclear staining with Hoechst 33342 (Hoechst Pharmaceuticals) and propidium iodide (PI) was used for morphological assessment of apoptosis, necrosis, and pyknosis/necrosis by fluorescence microscopy as described in ref. 19. Briefly, 2.5 × 105 cells per ml were incubated with ascorbate or H2O2 for 1 h, washed with PBS, and suspended in fresh media. After 18-22 h, at least 200 cells were stained with Hoechst/PI and visualized under fluorescence microscopy.

MTT was used as a screening assay and performed as described in ref. 20. Cells in 96-well plates were treated with ascorbate (0.1-20 mM) for 1 h, washed, and incubated for an additional 24 h. The EC50 value was the concentration that reduced survival by 50%.

For colony formation on soft agar plates, cells were treated with 5 mM ascorbate for 1 h, washed, and plated. A two-layer agar system was used, and colonies were visualized after 10-14 days (21).

To determine the effects of red blood cells on ascorbate-induced cell death, red blood cells were prepared by centrifugation of heparinized human blood at 500 × g for 30 min. Human Burkitt's lymphoma cells at 2.5 × 105 cells per ml were mixed with red blood cells, 25% or 50% hematocrit. Cell mixtures were treated with 2 mM ascorbate for 1 h. Lymphoma cells were recovered by using Vacutainer CPT tubes (Becton Dickinson) according to the manufacturer's instructions. After washing, lymphoma cells were returned to fresh medium and assessed after 18 h by nuclear staining as above.

Quantitative Procedures. Catalase activity was determined by using Amplex Red (Molecular Probes) (22). Glutathione was detected by using 5,5′-dithio bis-2-nitrobenzoic acid, and glutathione peroxidase activity was measured by a coupled reaction with glutathione reductase (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's instructions.

Ascorbate radical in culture media and blood was detected by using electron paramagnetic resonance (23, 24). Spectrometer (E9 series, Varian) settings were as follows: microwave power, 20 mW; modulation amplitude, 1.0 G; time constant, 0.25 s; scan range, 4 × 10 G; and scan time, 4 min. Radical quantitation was performed by using 3-carboxyproxyl as a standard (23).

Because ascorbate interferes with most peroxidase-based detection methods, H2O2 was measured by using a Clark-type oxygen electrode (5/6 Oxygraph, Gilson Medical Electronics, Middleton, WI). Oxygen evolution was measured upon introduction of catalase: 2H2O2 → 2H2O + O2. Calibration was performed with freshly prepared solutions of H2O2 (10-200 μM) (25).

Ascorbate was measured by HPLC with coulometric electrochemical detection (26). Protein was determined by using bicinchoninic acid (27). Cell volumes were determined by using a Coulter Multisizer II cell counter. Intracellular ascorbate concentrations were calculated by converting cell protein to a measured intracellular volume (18).

Results

Effects of Ascorbic Acid in Pharmacologic Concentrations on Survival of Tumor and Normal Cells. We first investigated whether ascorbate in pharmacologic concentrations selectively affected the survival of cancer cells by studying nine cancer cell lines, four normal cell types, and clinically relevant conditions. Clinical pharmacokinetics analyses show that pharmacologic concentrations of plasma ascorbate, from 0.3 to 15 mM, are achievable only from i.v. administration (7). These concentrations are cleared within hours by renal filtration and excretion. In contrast, plasma ascorbate concentrations from maximum possible oral doses cannot exceed 0.22 mM because of limited intestinal absorption, which is bypassed with i.v. administration (7-9). To mimic potential clinical i.v. use, tested cells were incubated for 1 h with either pharmacologic ascorbate concentrations (0.3-20 mM) or a high physiologic concentration (0.1 mM) as control. Once ascorbate was removed, cell survival was determined by nuclear staining or MTT after 24 h (Fig. 1A). For five of the nine cancer cell lines, ascorbate concentrations causing a 50% decrease in cell survival (EC50 values) were less than 5 mM, a concentration easily achievable from i.v. infusion (7). All tested normal cells were insensitive to 20 mM ascorbate.

Fig. 1.
Effects of pharmacologic ascorbic acid concentrations on cancer and normal cells. Concentrations in this and all figures indicate final concentrations. (A) EC50 values of ascorbate in human and mouse cancer cells and normal human cells. All cells were ...

Colony formation assays were used as an additional means to determine cell survival (21). Four cancer cell lines were incubated with 5 mM ascorbate or untreated media for 1 h. Cells were diluted and plated and growth assessed after 14 days (Fig. 1B). All four untreated cell lines grew in soft agar, whereas three of four exposed to ascorbate displayed at least 99% growth inhibition.

Effects of Ascorbic Acid on Death of Human Lymphoma Cells. Human lymphoma cells (JLP-119) were studied in detail to determine the effects of ascorbate on cell death. Lymphoma cells were selected because of their sensitivity to ascorbate (Fig. 1A), the suitability of these cells for nuclear staining to characterize the mode of cell death (16, 19, 28), and the report of a positive clinical response of lymphoma to i.v. ascorbate (14) (unpublished work). Cells were incubated for 1 h with 0.1-5 mM ascorbate and washed, and Hoechst/PI nuclear staining was performed 18 h later to determine the amount and type of cell death (Fig. 2A). Ascorbate induced concentration-dependent cell death, which was nearly 100% at 2 mM. As ascorbate concentration increased, the pattern of death changed from apoptosis to pyknosis/necrosis, a pattern suggestive of H2O2-mediated cell death (19). We determined the time necessary for cell death after exposure to 2 mM ascorbate for 1 h (Fig. 2B). Apoptosis occurred by 6 h after exposure, and cell death by pyknosis was ≈90% at 14 h after exposure. In contrast to lymphoma cells, there was little or no killing of normal lymphocytes and monocytes by ascorbate (Fig. 2C).

Fig. 2.
Effects of ascorbic acid on human Burkitt's lymphoma cells. Cells were treated for 1 h, washed, and recultured without ascorbate. Amounts and types of cell death were determined 18-22 h later by nuclear staining with Hoechst/PI. Types of cell death: necrosis ...

The roles of intracellular versus extracellular ascorbate in causing cell death were examined, using ascorbate and its oxidized product dehydroascorbic acid. Ascorbate is transported into cells as such by sodium-dependent transporters, whereas dehydroascorbic acid is transported into cells by glucose transporters and then immediately reduced internally to ascorbate (29). By using either external ascorbate or external dehydroascorbic acid, lymphoma cells were loaded to equal internal concentrations of ascorbate over 1 h (data not shown). Despite similar intracellular ascorbate concentrations under both conditions, cells died only when ascorbate was present externally (Fig. 2D).

Similar to most cultured cells, lymphoma cells contain no ascorbate unless the vitamin is added to the extracellular medium (data not shown) (17). In contrast, excepting red blood cells, all cells in vivo or acutely isolated contain ascorbate, usually in millimolar concentrations. We investigated whether the prior presence of intracellular ascorbate affected death mediated by extracellular ascorbate. Lymphoma cells were preloaded with physiologic concentrations of ascorbate to produce millimolar intracellular concentrations, similar to normal lymphocytes (8, 9). Their response to external ascorbate was compared with unloaded cells (Fig. 2E). Whether or not intracellular ascorbate was preloaded, extracellular ascorbate induced the same amount and type of death. Taken together, the data in Fig. 2 A-E indicate that extracellular ascorbate in pharmacologic concentrations mediates death of lymphoma cells by apoptosis and pyknosis/necrosis, independently of intracellular ascorbate.

Mechanism of Ascorbate-Mediated Cell Killing. To determine the mechanism of ascorbate-mediated lymphoma cell death, we tested the effects of the membrane-impermeant H2O2-scavenger catalase, the membrane-permeant H2O2-scavenger tetrakis (4-benzoic acid) meso-substituted manganoporphyrin (MnTBAP) (30), and the thiol-reducing agent Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (31). We also tested whether adventitious transition metals were responsible, by using the membrane impermeant chelator diethylenetriamine-pentaacetic acid (DTPA) (32) and the membrane permeant chelator N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED) (33-35) (Fig. 3A). The H2O2 scavengers were completely protective, identifying H2O2 as the effector species mediating pharmacologic ascorbate-induced cell death. The effect of ascorbate was not due to chelatable, trace redox-active metals, because the two chelators had no effect on preventing death. Superoxide dismutase was not protective (data not shown), consistent with its action in producing but not degrading H2O2 (36).

Fig. 3.
Extracellular ascorbate kills human Burkitt's lymphoma cells by generating H2O2. Cell death determined and symbolized as in Fig. 2; H2O2 measured by oxygen electrode (see Materials and Methods). (A) Effects of reactive oxygen species quenchers/scavengers, ...

Because these data implicated H2O2 in cell killing, we added H2O2 to lymphoma cells and studied death patterns using nuclear staining (19, 28). The death patterns found with exogenous H2O2 exposure were similar to those found with ascorbate. For both ascorbate and H2O2, death changed from apoptosis to pyknosis/necrosis as concentrations increased (Fig. 3B).

As a specific test of ascorbate action, the amount of H2O2 formed in the presence of ascorbate was measured by using an oxygen electrode. We compared the effects on cell death of H2O2 amounts formed in the presence of ascorbate to effects from exogenously added H2O2. H2O2 generated by ascorbate oxidation and exogenously added H2O2 produced cell death curves that were indistinguishable (Fig. 3C).

Sensitivity to direct exposure to H2O2 was greater in lymphoma cells compared with normal lymphocytes and normal monocytes (Fig. 3D), consistent with the cytotoxicity pattern found above with pharmacologic ascorbate exposure. Taken together, these data are consistent with the conclusion that extracellular ascorbate induced cell death by formation of H2O2.

We investigated whether activities of intracellular H2O2-removal systems correlated with ascorbate-mediated cell death, for all cells studied. There was no association between the EC50 for ascorbate-mediated cell death and intracellular glutathione concentrations, catalase activity, or glutathione peroxidase activity (data not shown).

Mediators and Inhibitors of H2O2 Generation. H2O2 concentrations generated by ascorbate were similar with tumor cells, normal cells, or in medium without cells (data not shown), as measured by using an oxygen electrode as above. H2O2 generation was dependent on time, ascorbate concentration, and the presence of trace amounts of serum in media (Fig. 4 A and B).

Fig. 4.
Enhancing factors for ascorbate-mediated H2O2 generation in cell culture medium. H2O2 was measured by oxygen electrode, and ascorbate radical was measured by electron paramagnetic resonance. (A) H2O2 formation as function of time and ascorbate concentration: ...

Based on these data, the most cogent explanation of ascorbate action in forming H2O2 is that the first step is ascorbate oxidation to its radical. We measured H2O2 concentration as a function of ascorbate radical concentration and found a linear relationship (Fig. 4C). These data imply that ascorbate radical is a surrogate marker for H2O2 formation.

For ascorbate to be useful clinically, it should increase the steady-state concentration of H2O2 in the extracellular milieu but not in blood. We predicted that steady-state concentrations of H2O2 generated by ascorbate oxidation would be undetectable in blood for several reasons. First, if any ascorbate radical is generated in blood, only very low concentrations are expected, and such concentrations should be lower than that needed to form detectable steady-state concentrations of H2O2 (37). Second, whatever H2O2 is generated should be removed by glutathione peroxidase and catalase within red blood cells, because H2O2 is membrane permeable (38-41). These predictions were explored in the following experiments. First, ascorbate (0-10 mM) was added to whole blood and to medium, and ascorbate radical was measured by electron paramagnetic resonance. Ascorbate radical in whole blood was not detectable when ascorbate concentrations were <3 mM and was present at minimal concentrations thereafter. In contrast, there was robust ascorbate radical generation in medium, a surrogate for extracellular fluid (Fig. 5A). Second, as direct tests, H2O2 concentrations were measured under the following conditions: In whole blood in the presence of varying concentrations of ascorbate, in whole blood after exogenous H2O2 addition, and in medium with varying concentrations of ascorbate (Fig. 5B). H2O2 was not detected in whole blood under either condition, even in the presence of far higher added concentrations than could be generated by ascorbate oxidation. Control formation of H2O2 as a function of ascorbate concentration in medium proceeded as expected. These data indicate that even if ascorbate radical was formed in blood and H2O2 was generated, it would be immediately scavenged to concentrations below detection limits. Based on these data, an additional functional experiment was conducted, based on the prediction that blood would protect tumor cells from ascorbate-mediated cell death. Lymphoma cells were incubated in the presence or absence of red blood cells, with and without added ascorbate. Red blood cells completely protected lymphoma cells from ascorbate-mediated cell death (Fig. 5C). Taken together, these data indicate that ascorbate cannot generate sustainable H2O2 concentrations in whole blood. The data are consistent with the hypothesis that ascorbate in pharmacologic concentrations is a pro-drug for H2O2 generation in the extracellular milieu but not in blood.

Fig. 5.
Human blood inhibits H2O2 and ascorbate radical generation from ascorbate. Ascorbate radical was measured by electron paramagnetic resonance, H2O2 was measured by oxygen electrode, and cell death was measured and displayed as in Fig. 2. (A) Ascorbate ...

Discussion

Our data show that ascorbic acid selectively killed cancer but not normal cells, using concentrations that could only be achieved by i.v. administration and conditions that reflect potential clinical use. The effect was due only to extracellular and not intracellular ascorbate, consistent with clinical i.v. dosing. Ascorbate-mediated cell death was due to protein-dependent extracellular H2O2 generation, via ascorbate radical formation from ascorbate as the electron donor. Like glucose, when ascorbate is infused i.v., the resulting pharmacologic concentrations should distribute rapidly in the extracellular water space (42). We showed that such pharmacologic ascorbate concentrations in media, as a surrogate for extracellular fluid, generated ascorbate radical and H2O2. In contrast, the same pharmacologic ascorbate concentrations in whole blood generated little detectable ascorbate radical and no detectable H2O2. These findings can be accounted for by efficient and redundant H2O2 catabolic pathways in whole blood (e.g., catalase and glutathione peroxidase) relative to those in media or extracellular fluid (38-41). The totality of the data are consistent with the interpretation that ascorbic acid administered i.v. in pharmacologic concentrations may serve as a pro-drug for H2O2 delivery to the extracellular milieu, but without H2O2 accumulation in blood.

Although it is possible that H2O2 might accumulate in blood, this would occur only under specific conditions that reflect on the general safety of i.v. ascorbate. Ascorbate administered i.v. is likely to be safe in most patients, with virtually no toxicity compared to most currently available cancer chemotherapeutic agents. The occurrence of one predicted complication, oxalate kidney stones, is controversial (13). In patients with glucose-6-phosphate dehydrogenase deficiency, i.v. ascorbate is contraindicated because it causes intravascular hemolysis (13). The mechanism of this previously unexplained observation is now straightforward, based on the results here. H2O2 generated in blood is normally removed by catalase and glutathione peroxidase within red blood cells, with internal glutathione providing reducing equivalents. The electron source for glutathione is NADPH from the pentose shunt, via glucose-6-phosphate dehydrogenase. If activity of this enzyme is diminished, the predicted outcome is impaired H2O2 removal causing intravascular hemolysis, the observed clinical finding.

Ascorbate as a potential cancer therapeutic agent has a controversial and emotionally charged past (1, 3-6). Clinical observational studies reported possible benefit in selected patients, but double-blind placebo-controlled studies reported no benefit, and ascorbate was discarded as a potential therapy by conventional practitioners. Only recently has it been understood that the discordant clinical findings can be explained by previously unrecognized fundamental pharmacokinetics properties of ascorbate (7). In vitro effects of ascorbate on death and survival of cell lines have been reported, but there are multiple experimental concerns. For example, reports compared an experimental condition to that with no ascorbate at all (43, 44), but such a condition has had unclear physiologic relevance, because ascorbate outside and inside cells is always present unless there is severe scurvy. It was unclear whether observed effects were due to extracellular or intracellular ascorbate, or both (12, 43-46). Some experiments have used widely varying incubation times and ascorbate concentrations that have had no corresponding clinical context, making interpretation difficult. H2O2 generation by ascorbate oxidation in culture media was variously interpreted as artifact (47, 48), even though chelators had no effect (49), or reported to mediate damage internally due to diminished intracellular ascorbate, but using an H2O2 assay in which ascorbate could interfere (43, 44).

The experiments presented here provide a clear clinical context for ascorbate action. Conditions were selected to reflect peak ranges of i.v. ascorbate concentrations, which clinically might last a few hours at most, depending on the infusion rate (7). Intracellular transport of ascorbate is tightly controlled in relation to extracellular concentration (8, 9, 29). Intravenous ascorbate infusion is expected to drastically change extracellular but not intracellular concentrations (8, 9). For i.v. ascorbate to be clinically useful in killing cancer cells, pharmacologic but not physiologic extracellular concentrations should be effective, independent of intracellular ascorbate concentrations. This was what was observed here. The experiments here provide a cohesive explanation for ascorbate action in generating H2O2 outside cells, without H2O2 accumulation in blood, leading to the conclusion that ascorbate at pharmacologic concentrations in blood is a pro-drug for H2O2 delivery to tissues.

We observed that H2O2 generation was independent of metal chelators and dependent on at least 0.5% extracellular protein. The responsible proteins were between 10 and 30 kDa (data not shown). It is reasonable that extracellular milieu contains these proteins, given that extracellular milieu protein is as much as 20% of serum protein, and favors lower-molecular-weight proteins (50). Although identities of the proteins responsible are unknown, we postulate that they may have redox-active metal centers. While chelators may marginally affect these metals, they could participate in the oxidation of ascorbate when it is at pharmacologic concentrations, with subsequent formation of superoxide and H2O2 (34). It is also possible that in vivo, cell membranes and their associated proteins could harbor metals accessible to extracellular fluid and could react similarly. In either case, ascorbate, an electron-donor in such reactions, ironically initiates pro-oxidant chemistry and H2O2 formation (34, 51).

It is unknown why ascorbate, via H2O2, killed some cancer cells but not normal cells. There was no correlation with ascorbate-induced cell death and glutathione, catalase activity, or glutathione peroxidase activity. The data here showed that ascorbate initiated H2O2 formation extracellularly, but H2O2 targets could be either intracellular or extracellular, because H2O2 is membrane permeant (38, 52). For example, extracellular H2O2 might target membrane lipids, forming hydroperoxides or reactive intermediates that are quenched or repaired in normal cells but not in sensitive cancer cells. In sensitive but not resistant cancer cells, intracellular H2O2 could target DNA, DNA repair proteins, or mitochondria because of diminished superoxide dismutase activity (53). New insights may follow from future studies of a very broad range of tumor cells or from microarray analysis of resistant and sensitive cells derived from the same genetic lineage.

H2O2, as the product of pharmacologic ascorbate concentrations, has potential therapeutic uses in addition to cancer treatment, especially in infections. H2O2 is a potent mammalian antimicrobial defense mechanism (54). Neutrophils generate H2O2 from superoxide, in turn formed by NADPH oxidase-catalyzed reduction of molecular oxygen. There may be particular therapeutic application in patients with chronic granulomatous disease who have diminished superoxide production (55). Old observational animal experiments, although uncontrolled, suggest that i.v. ascorbate is effective in some viral infections (56, 57). This finding is also consistent with in vitro experiments, in which H2O2 is toxic to hepatitis C (58). Use of ascorbate as an H2O2-delivery system against sensitive pathogens, viral or bacterial, has substantial clinical implications that deserve rapid exploration.

To proceed clinically in potential treatment of infectious diseases and cancer, clear safety documentation of i.v. ascorbate administration is necessary. More than 100 patients have been described, presumably without glucose-6-phosphate dehydrogenase deficiency, who received 10 g or more of i.v. ascorbate with no reported adverse effects other than tumor lysis (3, 4, 15, 59). However, these descriptions lack formal safety documentation. Complementary and alternative medicine practitioners worldwide currently use ascorbate i.v. in doses as high as 70 g over several hours (14, 15, 59). Because i.v. ascorbate is easily available to people who seek it, a phase I safety trial in patients with advanced cancer is justified and underway.

Acknowledgments

This work was supported in part by the Intramural Research Programs of the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute (National Institutes of Health Grant Z01 DK 54506).

Notes

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC80728/
CMAJ. 2001 Feb 6; 164(3): 351–353.
PMCID: PMC80728

Proof versus plausibility: rules of engagement for the struggle to evaluate alternative cancer therapies

L. John Hoffer

The debate that has been taking place in CMAJ about alternative cancer therapies is extremely valuable, especially when considered against the backdrop of intense public interest in this subject.1,2,3,4,5,6 Like it or not, government agencies and medical research centres are willing to evaluate alternative therapies, and money from public and private sources is available to pay the costs. The paradox, of course, is that by definition unconventional, unorthodox or complementary therapies — whatever one's favourite term may be — lack scientific credibility. How, then, does one select the most promising of them for evaluation, and what assessment procedures will be regarded as sufficiently thorough and definitive by mainstream medical scientists, government agencies and the proponents of alternative therapy?

Practitioners of alternative medicine and community physicians can, no doubt, form successful partnerships with the aim of providing high-quality, holistic care. Many mainstream Canadian physicians already integrate elements of alternative therapy into their practices. Alternative and Western science–based medicine are either partners or amicable neighbours in most parts of the world.

But it would be a triumph of hope over experience to assume that alternative therapists and clinical researchers will easily develop partnerships to test alternative cancer therapies. This situation has to change because each side needs to learn from the other. The alternative therapists need expertise in rigorous documentation and the principles of clinical investigation, as well as the physical and intellectual resources necessary to design and conduct informative clinical trials. Clinical researchers need information about the specific aims of a given alternative therapy, insight into how it is used and the perspective necessary to design pragmatic trials that assess it fairly. The effort should be made to identify genuinely promising new approaches and to explore, rather than refute, them.7,8,9 Young scientific investigators may be disinclined to travel this rocky road. My own dean — now retired — encouraged my interest in alternative medicine, but added, “Keep your day job.”

It was with such considerations in mind that I, with Dr. Carmen Tamayo and Dr. Mary Ann Richardson of the University of Texas Center for Alternative Medicine Research, organized a 2-day research workshop in Montreal last spring to consider the background and current status of the most luminously controversial of all biological, alternative cancer therapies, high-dose vitamin C. Our workshop brought together mainstream physicians, research oncologists, alternative practitioners and a representative of the US Food and Drug Administration. Funding was provided by the Lotte and John Hecht Memorial Foundation, a charitable organization endowed in British Columbia in 1962 with a major objective of supporting the investigation of alternative and complementary therapies, especially for cancer. A selection of some of the workshop presentations has appeared elsewhere,10,11,12,13 and a full summary report is in preparation, but it is appropriate to relate here a powerful personal lesson from this experience.

The vitamin C and cancer controversy began in 1974 with the publication, by Cameron and Campbell, of a careful description of the responses of 50 consecutive patients with advanced, untreatable cancer to high-dose intravenous and oral vitamin C.14 Most patients did not respond, but extraordinary things happened to a significant minority of them. These included several tumour regressions and 4 cases (2 of them fully documented at autopsy) of catastrophic tumour hemorrhage and necrosis occurring within 3–6 days after starting vitamin C therapy. Cameron had never seen anything like this in his long and distinguished career as an oncologic surgeon, and he concluded that an important phenomenon was occurring that merited further investigation. Two-time Nobel laureate Linus Pauling championed the cause. As a result of the ensuing controversy, a double-blind controlled clinical trial of 10 g/day oral vitamin C was carried out at the Mayo Clinic in patients with a variety of untreatable, terminal cancers.15 The results were negative. Pauling and Cameron objected that, unlike in Scotland, all the Minnesota patients had received prior cytotoxic therapy and that this could have mitigated the restorative biological effects of vitamin C.

A second clinical trial was carried out in patients with colorectal cancer at an earlier stage than in the first trial, but for which the only cytotoxic therapy then available, fluorouracil, was known to be ineffective and hence could ethically be withheld.16 The results of this trial were also negative, but Pauling and Cameron objected to the way that it was designed and carried out. A patient's course of vitamin C (or placebo) was terminated as soon as there was clear evidence that his or her tumour was continuing to progress, whereas Cameron and Pauling's key claim was that vitamin C prolongs the life of cancer patients when given continuously. Indeed, they had previously emphasized the danger of withdrawing vitamin C abruptly, having found this to be associated with a rebound acceleration of the disease. They also objected to the lack of effort to ensure compliance with the study medication or to screen the control group for illicit use of vitamin C.17,18 Another obvious problem was the lack of statistical power. Cameron had found that no more than a minority of patients responded in the prompt and dramatic fashion typical of cytotoxic drugs, yet the latter was the type of response the Mayo Clinic trial was designed to detect. It is interesting to note that the year in which the second Mayo Clinic trial was published, the New England Journal of Medicine also published, to wide acclaim, the clinical responses of an uncontrolled series of cancer patients treated with the biological agent, interleukin-2, at the US National Cancer Institute (NCI).19 As with Cameron's Scottish patients, only a minority of the patients responded. Had interleukin-2 been assessed as vitamin C was in the Mayo Clinic study, it, too, might well have been found to be ineffective.

In 1989 Pauling visited the head of the NCI, Samuel Broder, and described cases of what he claimed were complete cancer remissions in response to vitamin C. Broder was sufficiently interested to convene an NCI panel to review 25 case histories of patients to be selected by Cameron as providing plausible evidence that high-dose vitamin C could have important biological effects in human cancer. The cases selected included 2 complete remissions experienced by a patient with stage IV non-Hodgkin's lymphoma following courses of vitamin C therapy,20,21 the disappearance of multiple brain lesions diagnosed as metastatic on clinical grounds and CT scanning in a patient with bronchogenic carcinoma, tumour regression in a patient with metastatic renal cell carcinoma, and autopsy-confirmed tumour hemorrhage in a patient within 3 days of initiating vitamin C therapy.18 In 1991 Pauling received a letter informing him of the panel's conclusion that vitamin C had not been shown to be responsible for improved outcome in any cancer case, either because the cancer diagnosis was not sufficiently proven or because an explanation other than vitamin C therapy might have accounted for the patient's clinical course. In some cases, extraordinarily long survivals were not credited to vitamin C because of lack of information about such long survivals in the natural history of the disease.

When submitting their case histories, Cameron and Pauling understood that they would be evaluated with regard to the plausibility of the hypothesis that vitamin C could have important biological effects in human cancer. Instead, considering each case separately from all the others, the NCI panel looked for proof that vitamin C must have been responsible for the clinical effects reported and exact confirmation, not plausibility, of the tissue diagnosis. Proof is necessary to change medical practice, plausibility to justify testing a clinical hypothesis. Neither side in this exchange was wrong, but it would have been helpful if they had understood each other's position.

The lesson to be learned from this is that the parameters of the debate about alternative therapies — the “rules of engagement” — between mainstream cancer researchers and proponents of alternative therapy need to be clearly defined and the goals must be explicit and common to both parties. To do otherwise leads to the risk of unintended confusion and heightening of the barrier of mistrust that already stands between many individuals involved in this debate. Proponents of alternative therapy have an obligation to provide grounds for biological plausibility, such as sound theoretical or preclinical data, or for clinical plausibility, in the form of authentic, well-prepared case reports, in order to justify the investment of time and energy in exploring the merits of a novel anticancer therapy. But plausibility, not proof, should be sufficient to initiate the process.

Since the Mayo Clinic trials were published, rational guidelines for testing biological agents like vitamin C have been developed,22 and new information has emerged since the NCI review took place about the biological effects and clinical pharmacokinetics of vitamin C.18,23,24 In this issue (page 353),25 Sebastian Padayatty and Mark Levine (Levine was also a member of the NCI panel that reviewed the cases submitted by Pauling and Cameron) describe these new developments in our understanding of vitamin C biology and their relevance to the question of a role for vitamin C in cancer therapy. Perhaps it is time to revisit the issue of the clinical and biological plausibility of a role for vitamin C in cancer therapy.

Footnotes

Competing interests: None declared.

Correspondence to: Dr. L. John Hoffer, Lady Davis Institute for Medical Research, 3755 Chemin de la Côte-Sainte-Catherine, Montreal QC H3T 1E2

References

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20. Cameron E, Campbell A, Jack T. The orthomolecular treatment of cancer. III. Reticulum cell sarcoma: double complete regression induced by high-dose ascorbic acid therapy. Chem Biol Interact 1975;11:387-93.
21. Campbell A, Jack T, Cameron E. Reticulum cell sarcoma: two complete ‘spontaneous’ regressions, in response to high-dose ascorbic acid therapy. A report on subsequent progress. Oncology 1991;48:495-7.
22. Hoffer LJ. Nutrients as biologic response modifiers. In: Quillan P, Williams RM, editors. Adjuvant nutrition in cancer treatment. Arlington Heights (IL): Cancer Treatment Research Foundation; 1993. p. 55-79.
23. Block G, Henson DE, Levine M. Vitamin C: a new look. Ann Intern Med 1991;114:909-10.
24. Levine M, Rumsey SC, Daruwala R, Park JB, Wang Y. Criteria and recommendations for vitamin C intake. JAMA 1999;281:1415-23.
25. Padayatty SJ, Levine M. New insights into the physiology and pharmacology of vitamin C. CMAJ 2001;164(3):355-7. Available: www.cma.ca/cmaj/vol-164/issue-3/355.htm



Prevention and treatment of occlusive cardiovascular disease with ascorbate and substances that inhibit the binding of lipoprotein (A)
US5278189


RATH MATTHIAS; PAULING LINUS

A method is provided for prevention and treatment of cardiovascular disease, such as atherosclerosis, by administering therapeutically effective dosages of a drug comprised of ascorbate, lipoprotein(a) binding inhibitors, and antioxidants

Description

TECHNICAL FIELD

The present invention relates generally to the prevention and treatment of cardiovascular disease and more particularly to methods and compounds that inhibit the binding of lipoprotein (a) to components of the arterial wall.

BACKGROUND OF THE INVENTION

Lipoprotein(a) ("Lp(a)") was first identified by Blumberg, B. S., et al. (1962) J. Clin. Invest. 41: 1936-1944 , and Berg, K. (1963) Acta Pathol. 59: 369-382. The structure of Lp(a) resembles that of low-density lipoprotein ("LDL") in that both share a lipid apoprotein composition, mainly apolipoprotein B-100 ("apo B"), the ligand by which LDL binds to the LDL receptors present on the interior surfaces of arterial walls. The unique feature of Lp(a) is an additional glycoprotein, designated apoprotein(a), apo(a), which is linked to apo B by disulfide groups. The cDNA sequence of apo(a) shows a striking homology to plasminogen, with multiple repeats of kringle 4, one kringle 5, and a protease domain. The isoforms of apo(a) vary in the range of 300 to 800 kDa and differ mainly in their genetically determined number of kringle 4 structures. McLean, J. W., et al. (1987) Nature 300: 132-137. Apo(a) has no plasmin-like protease activity. Eaton, D. L., et al., (1987) Proc. Natl Acad. Sci. USA, 84: 3224-3228. Serine protease activity, however, has been demonstrated. Salonen, E., et al. (1989) EMBO J. 8: 4035-4040. Like plasminogen, Lp(a) has been shown to bind to lysine-sepharose, immobilized fibrin and fibrinogen, and the plasminogen receptor on endothelial cells. Harpel, P.C., et al. (1989) Proc. Natl. Acad. Sci. USA 86:3847-3851; Gonzalez-Gronow, M., et al. (1989) Biochemistry 28: 2374-2377; Miles, L. et al. (1989) Nature 339: 301-302; Hajjar, K. A., et al. (1989) Nature 339: 303-305. Furthermore, Lp(a) has been demonstrated to bind to other components of the arterial wall like fibronectin and glycosaminoglycans. The nature of these bindings, however, is poorly understood.

Essentially all human blood contains lipoprotein(a); however, there can a thousand-fold range in its plasma concentration between individuals. High levels of Lp(a) are associated with a high incidence of cardiovascular disease. Armstrong, V. W., et al. (1986) Atherosclerosis 62: 249-257; Dahlen, G., et al. (1986) Circulation 74: 758-765; Miles, L. A., et al. (1989) Nature 339: 301-302; Zenker, G., et al. (1986). Stroke 17: 942-945 (The term occlusive cardiovascular disease will be used hereafter as including all pathological states leading to a narrowing and/or occlusion of blood vessels throughout the body, but particularly atherosclerosis, thrombosis and other related pathological states, especially as occurs in the arteries of the heart muscle and the brain.)

For some time, general medical practice has focused on the role of LDL, the so called "bad cholesterol," in occlusive cardiovascular disease. A great many studies have been published ostensibly linking occlusive cardiovascular disease with elevated levels of LDL. As a result, most therapies for the treatment and prevention of arteriosclerosis rely on drugs and methods for the reduction of serum levels of LDL's. Such therapies have had mixed results. The efficacy of such approaches to the problem of occlusive cardiovascular disease continues to be major source of debate.

There exists therefore a need for a drug therapy for reducing the binding of Lp(a) to vessel walls, for reducing the overall level of Lp(a) in the circulatory system and for promoting the release of existing deposits of Lp(a) on vessel walls.

SUMMARY OF THE INVENTION

The foregoing needs in the treatment and prevention of cardiovascular disease are met by the methods and compositions of the present invention.

A method is provided for the treatment of occlusive cardiovascular disease, comprising the step of administering to a subject an effective amount of ascorbate and one or more binding inhibitors, as a mixture or as a compound comprising ascorbate covalently linked with binding inhibitors, which inhibit the binding of Lp(a) to blood vessel walls, such as arterial walls. This effect may also be obtained by administering an effective amount of one or more inhibitors, without ascorbate. The term binding inhibitor throughout the specification and claims is intended to include all substances that have an affinity for the lysine binding site present on the interior walls of blood vessels, particularly arteries, the site of Lp(a) binding. Most of these substances compete with plasmin for the lysine binding site and some of these compounds, in high doses, are in clinical use for the treatment of hyperfibrinolytic states.

A method is further provided for the prevention of atherosclerosis comprising the step of administering to a subject an effective amount of ascorbate and one or more binding inhibitors as previously discussed but further comprising one or more antioxidants. The term antioxidant throughout the specification and the claims is intended to exclude ascorbate which has as one of its chemical properties a potent antioxidant effect.

It is thus an object of the invention to provide a method for treatment of occlusive cardiovascular disease by administering to a subject an effective amount of ascorbate and one or more binding inhibitors, or an effective amount of one or a mixture of binding inhibitors.

It is another object of the invention to provide a method for preventing of occlusive cardiovascular disease, by administering to a subject an amount of ascorbate effective to lower the amount of Lp(a) in the plasma of the subject.

Yet another object of the present invention is to provide a method for prevention of cardiovascular disease by administering to a subject an effective amount of ascorbate and one or more binding inhibitors, or an effective amount of one or more binding inhibitors.

A further object of the present invention is to provide a pharmaceutically acceptable agent for the treatment of occlusive cardiovascular disease.

Still another object of the present invention is to provide a pharmaceutically acceptable agent for the prevention of cardiovascular disease.

These and other objects will be more readily understood upon consideration of the following detailed descriptions of embodiments of the invention and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immunoblot of the plasma of guinea pigs form from the test described in Example 1.

FIG. 2A a photograph of the aorta of a guinea pig receiving an adequate amount of ascorbate from the test diet in Example 1.

FIG. 2B is a photograph of an aorta of a guinea pig receiving a hypoascorbic diet after three weeks from the test diet in Example 1.

FIG. 3 is an immunoblot of plasma and tissue of guinea pigs from the test shown in Example 2.

FIG. 4 shows the potential mechanism of binding inhibitors in the therapy for atherosclerosis.

FIG. 5 show the potential mechanism of ascorbate in the binding of Lp(a) to the arterial wall.



DETAILED DESCRIPTION OF THE INVENTION

Our invention is based in part on our discovery that animals which have lost the ability to produce ascorbate, such as higher primates and guinea pigs, uniformly produce Lp(a). Most animals which possess the ability to synthesize ascorbate generally do not produce Lp(a). Further, we have found that ascorbate deficiency in humans and guinea pigs tends to raise Lp(a) levels and causes atherosclerosis by the deposition of Lp(a) in the arterial wall, from which we conclude that ascorbate administration lowers plasma Lp(a) levels.

We have also discovered that substances that inhibit the binding of Lp(a) to components of the arterial wall, particularly to fibrinogen, fibrin and fibrin degradation products herein identified as binding inhibitors, such as lysine or .epsilon.-aminocaproic acid used alone or in combination with ascorbate, cause release of Lp(a) from the arterial wall. Thus, ascorbate and such binding inhibitors are not only useful for the prevention of occlusive cardiovascular disease, but also for the treatment of such disease. The present invention, then, provides methods and pharmaceutical agents for the both the treatment and prevention of occlusive cardiovascular disease in vivo.

GENERAL APPLICATIONS

The present invention provides a method and pharmaceutical agent for the treatment and prevention of occlusive cardiovascular disease generally, by administering to a subject an effective amount of ascorbate and one or more binding inhibitors. Throughout the specification and claims, the term binding inhibitor is intended to cover any substance which has as at least one of its chemical properties the ability to inhibit the binding of Lp(a) to blood vessel wall components, particularly to fibrin or fibrinogen. As used herein, the term "ascorbate" includes any pharmaceutically acceptable salt of ascorbate, including sodium ascorbate, as well as ascorbic acid itself. Binding inhibitors include, but are not limited to .epsilon.-aminocaproic acid, lysine, tranexamic acid (4-aminomethylcyclohexane carboxylic acid), p-aminomethylbenzoic acid, p-benzylamine sulfuric acid, o-N-acetyl-lysine-methyl ester, PROBUCOL (a compound comprised of 2 butyl hydroxy tocopherol groups linked together by a disulphide group), Aprotinin, trans-4-aminomethylcyclohexanecarboxylic acid (AMCA), and benzamidine derivatives such as amidinophenylpyruvic acid (APPA) and 1-naphthyl-(1)-3-(6-amidinonaphthyl-(2))-propanone-1 HCl (NANP). An effective amount of a binding inhibitor or a mixture of binding inhibitors may also be used, without ascorbate. Other substances used in the treatment of occlusive cardiovascular disease may be co-administered, including antioxidants, such as tocopherol, carotene and related substances; vitamins; provitamins; trace elements; lipid-lowering drugs, such as hydroxy-methyl-glutaryl coenzyme A reductase inhibitors, nicotinic acid, fibrates, bile acid sequestrants; and mixtures of any two or more of these substances.

Although ascorbate can be used alone or in varying combinations with one or more representative constituents of the above classes of compounds, we prefer when treating a pre-existing cardiovascular condition to combine ascorbate with at least one each of the binding inhibitors, antioxidants and lipid lowering drugs elements in the dosages (per kilogram of body weight per day (Kg BW/d)) provided in Table 1. It should be noted that Table 1 provides differing concentration ranges of each constituent, depending upon whether the agent is to be administered orally or parenterally. The variance in dosages is reflective of variation in disease severity. It will be realized therefore that if the subject has been diagnosed for advanced stages of atherosclerosis, dosages at the higher end of this range can be utilized. However, if only prevention of an atherosclerosis condition is the object, dosages at the lower end of this range can be utilized.

As an alternative, a pharmaceutical agent identical to the one just described, but omitting ascorbate, may be employed.

Where ascorbate and binding inhibitors are utilized in the same agent, they may simply be mixed or may be chemically combined using synthesis methods well known in the art, such as compounds in which ascorbate and the inhibitor are covalently linked, or form ionically bound salts. For example, ascorbate may be bound covalently to lysine, other amino acids, or .epsilon.-aminocaproic acid by ester linkages. Ascorbyl .epsilon.-aminocaproate is such an example. In this form the ascorbate moiety may be particularly effective in preventing undesirable lipid peroxidation.

In the case of oral administration, a pharmaceutically acceptable and otherwise inert carrier may be employed. Thus, when administered orally, the active ingredients may be administered in tablet form. The tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid, and/or a lubricant such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring agents may be used. If administration is by parenteral injection, in isotonic saline, a phosphate buffered solution or the like, may be used as a pharmaceutically acceptable carrier.

The advisability of using binding inhibitors in treating occlusive cardiovascular disease will depend to some extent on the subject's general health, particularly with regard to hyperfibrinolytic conditions. Most binding inhibitors (except lysine) are used clinically to treat such conditions. As a result, monitoring of the subject's coagulation and fibrinolytic sysem is recommended before and during treatment for occlusive cardiovascular disease. Long-term administration of binding inhibitors will require formulations in which the dosages of binding inhibitors are in the lower ranges of the dosages given in Table 1.

Prevention, as contrasted with treatment, of cardiovascular disease may be accomplished by oral or parenteral administration of ascorbate alone. Table 1 gives a range of ascorbate concentrations sufficient to lower the serum Lp(a) concentration.

Preferably the prevention of the occlusive cardiovascular disease according to the invention is accomplished by use of a physical mixture of ascorbate and one or more binding inhibitors, or by use of a compound comprising covalently linked ascorbate with one or more of the binding inhibitors, which inhibit binding of Lp(a) to the arterial wall. A binding inhibitor or mixture of binding inhibitors may also be administered without ascorbate to prevent Lp(a)-associated occlusive cardiovascular disease.

To optimize the therapeutic effect of the release of Lp(a) from the blood vessel walls, the ascorbate and the binding inhibitors described above may be separately administered. Further optimization of therapeutic effect can be gained by using a time release composition to achieve relatively constant serum concentrations of the agent through time.

TABLE 1
DOSAGES OF COMPONENTS IN THE DRUG COMPOSITIONS OF THE PRESENT INVENTION
Oral Parenteral Administration



EXPERIMENTAL

Having disclosed the preferred embodiment of the present invention, the following examples are provided by way of illustration only and are not intended to limit the invention in any way.

EXAMPLE 1

Because of its metabolic similarity to man, with resepct to the metabolism of ascorbate and Lp(a), the guinea pig was used in this example.

No study has been previously reported in the guinea pig to identify the lopoprotein involved as risk factors in plasma and as constituents of the atherosclerotic plaque.

Three female Gartly guinea pigs with an average weight of 800 g and an approximate age of 1 year wer stuided. One animal received an extreme hypoascorbic diet with 1 mg ascorbate/kg body weight/d. Another animal received 4 mg/kg BW/d. The third animal served as a control receiving 40 mg ascorbate/kg/BW/d)

Blood was drawn by heart puncture from the anesthetized animals and collected into EDTA containing tubes at the beginning, after 10 days, and after 3 weeks, when the animals were sacrificed. Plasma was stored at -80 DEG C. until analyzed. Lp(a) was detected in the plasma of the guinea pigs by use of SDS-polyacrylamide gels according to Neville (J. Biol. Chem., 246, 6328-6334 (1971)) followed by Western blotting (Beisiegel, et al., J. Biol. Chem., 257, 13150-13156 (1982)). 40 .mu.l of plasma and 20 mg of arterial wall homogenate were applied in delipidated form per lane of the gel. The immunodetection of apo(a) was performed using a polyclonal anti-human apo(a) antibody (Immuno, Vienna, Austria) followed by a rabbit anti-sheep antibody (Sigma) and the gold-conjugated goat anti-rabbit anti-body with subsequent silver enhancement (Bio-Rad). The determinations of cholesterol and triglycerides were done at California Veterinary Diagnostics (Sacramento) using the enzyme assay of Boehringer Mannheim. Plasma ascorbate was determined by the dinitrophenylhydrazine method (Schaffer, et al., J. Biol. Chem., 212, 59 (1955)).

Vitamin C deficiency in the diet led to an increase of Lp(a) in the plasma of the guinea pig indicated by a clear band in the immunoblot of the plasma after 10 and 20 days of a hypoascorbic diet (FIG. 1). At necropsy the animals were anesthetized with metophase and were exsanguinated. Aorta, heart and various other organs were taken for biochemical and histological analysis. The aorta was excised, the adventitial fat was carefully removed, and the vessel was opened longitudinally. Subsequently the aorta was placed on a dark metric paper and a color slide was taken. The picture was projected and thereby magnified by an approximate factor 10. The circumference of the ascending aorta, the aortic arch and thoracic aorta as well as the atherosclerotic lesions in this area were marked and measured with a digitalized planimetry system. The degree of atherosclerosis was expressed by the ratio of plaque area to the total aortic area defined. The difference in the 3 one-year old animals of the experiment was significant and pronounced lesions were observed in the ascending aorta and the arch of the vitamin C deficient animal (FIG. 2B).

EXAMPLE 2

To confirm the data obtained in Example 1, a second guinea pig experiment was conducted, using 33 male animals with a mean weight of 550 g and an approximate age of 5 months. One group of 8 animals served as a control and received 40 mg ascorbate/kg BW/d (group A). To induce hypoascorbemia 16 animals were fed 2 mg ascorbate/kd/d (group B). Group A and half of the animals of group B (progression sub-group) were sacrificed after 5 weeks as described above. Half of group B was kept for 2 more weeks, receiving daily intraperitoneal injection of 1.3 g sodium ascorbate/Kg BW/d as a daily intra peritoneal injection with the intention to reduce the extent of atherosclerosis lesions. After this period these animals also were sacrificed.

Plasma ascorbate levels were negatively correlated with the degree of the atherosclerotic lesion. Total cholesterol levels increased significantly during ascorbic acid deficiency (Table 3).

The aortas of the guinea pigs receiving a sufficient amount of ascorbate were essentially plaque free, with minimal thickening of the intima in the ascending region. In contrast, the ascorbate-deficient animals exhibited fatty streak-like lesions, covering most parts of the ascending aorta and the aortic arch. In most cases the branching regions of the intercostal arteries of the aorta exhibited similar lipid deposits. The difference in the precentage of lesion area between the control animals and the hypoascorbic diet animals was 25% deposition of lipids and liporpoteins in the arterial wall.

TABLE 3
MEAN PLASMA PARAMETERS OF THE DIFFERENT GROUPS IN RELATION TO THE AREA OF AORTIC LESIONS



EXAMPLE 3

Human arterial wall was obtained post mortem from the aorta ascendens. The tissue showed homogeneous intimal thickening (early atherosclerotic lesion). It was cut into pieces, with 100 mg of the cut up tissue homogenized in a glass potter for a 1 minute in 2.5 ml of the following solutions:

PBS (Dulbeco) + 50 mg/ml
NaAscorbate
PBS + EACS 50 mg/ml
PBS + Tranexamic Acid
50 mg/ml
PBS + NaAscorbate +
50 mg/ml
Tranexamic Acid

Results of this treatment are given in Table 4 and show that, compared to the control solution, a considerable amount of Lp(a) was released from the interior arterial wall.

TABLE 4
Lp(a) RELEASED FROM HUMAN AORTA IN RELATION TO SPECIFIC BINDING INHIBITORS
 


By now it is apparent that the methods and compositions of the present invention meet longstanding meeds in the field of prevention and treatment of occlusive cardiovascular disease. Although preferred embodiments and examples have been disclosed, it is understood that the invention is in no way limited by them, but rather is defined by the claims that follow and equivalents thereof.



Use of ascorbate and tranexamic acid solution for organ and blood vessel treatment prior to transplantation
US5230996


RATH MATTHIAS; PAULING LINUS

A method and pharmaceutical agent are provided for the prevention and treatment of cardiovascular disease, particularly cardiovascular disease in the context of diabetic angiopathy, by-pass surgery, organ transplantation, and hemodialysis, by administering ascorbate and substances that inhibit the binding of lipoprotein (a) to blood vessel walls. The use of ascorbate and lipoprotein (a) binding inhibitors such as tranexamic acid in a temporary storage solution for blood vessels and organs prior to transplantation is also demonstrated.

TECHNICAL FIELD

The present invention relates generally to the prevention and treatment of cardiovascular disease arising as a complication from surgery or a pre-existing, unrelated disease, and more particularly to methods and compounds for that inhibit the binding of lipoprotein (a) to components of the arterial wall.

BACKGROUND OF THE INVENTION

Lipoprotein (a) ("Lp(a)") was first identified by Blumberg, B. S., et al. (1962) J. Clin. Invest. 41: 1936-1944 and Berg, K. (1963) Acta Pathol. 59: 369-382. The structure of Lp(a) resembles that of low-density lipoprotein ("LDL") in that both share a lipid/apoprotein composition, mainly apolipoprotein B-100 ("apo B"), the ligand by which LDL binds to the LDL receptors present on the interior surfaces of arterial walls. The unique feature of Lp(a) is an additional glycoprotein, designated apoprotein(a), apo(a), which is linked to apo B by disulfide groups. The cDNA sequence of apo(a) shows a striking homology to plasminogen, with multiple repeats of kringle 4, one kringle 5, and a protease domain. The isoforms of apo(a) vary in the range of 300 to 800 kDa and differ mainly in their genetically determined number of kringle 4 structures. McLean, J. W., et al. (1987) Nature 300: 132-137. Apo(a) has no plasmin-like protease activity. Eaton, D. L., et al., (1987) Proc. Natl Acad. Sci. USA, 84: 3224-3228. Serine protease activity, however, has been demonstrated. Salonen, E., et al. (1989) EMBO J. 8: 4035-4040. Like plasminogen, Lp(a) has been shown to bind to lysine-sepharose, immobilized fibrin and fibrinogen, and the plasminogen receptor on endothelial cells. Harpel, P. C., et al. (1989) Proc. Natl. Acad. Sci. USA 86:3847-3851; Gonzalez-Gronow, M., et al. (1989) Biochemistry 28: 2374-2377; Miles, L. et al. (1989) Nature 339: 301-302; Hajjar, K. A., et al. (1989) Nature 339: 303-305. Furthermore, Lp(a) has been demonstrated to bind to other components of the arterial wall like fibronectin and glycosaminoglycans. The nature of these bindings, however, is poorly understood.

Essentially all human blood contains lipoprotein (a); however, there can a thousand-fold range in its plasma concentration between individuals. High levels of Lp(a) are associated with a high incidence of cardiovascular disease. Armstrong, V. W., et al. (1986) Atherosclerosis 62: 249-257;Dahlen, G., et al. (1986) Circulation 74: 758-765; Miles, L. A., et al. (1989) Nature 339: 301-302; Zenker, G., et al. (1986) Stroke 17: 942-945 (The term cardiovascular disease will be used hereafter as including all pathological states leading to a narrowing and/or occlusion of blood vessels throughout the body, but particularly atherosclerosis, thrombosis and other related pathological states, especially as occurs in the arteries of the heart muscle and the brain.)

It has also been suggested that Lp(a), the concentration of which increases markedly in the blood during pregnancy, may be linked to cardiovascular disease in woman. Zechner, R., et al. (1986) Metabolism 35: 333-336. It has also been observed that diabetics, many of whom suffer in some degree from atherosclerotic diseases, display greatly elevated serum levels of Lp(a). Bruckert, E., et al. (1990) JAMA 263: 35-36.

Low levels of ascorbate have also been associated with high incidences of cancer (Wright, L. C. et al. (1989) Int. J. Cancer 43: 241-244) and atherosclerosis in diabetes mellitus patients (Som, S. et al. (1981) Metabolism 30: 572-577). In all instance, serum concentrations of Lp(a) were elevated.

In addition to atherosclerosis and thrombosis in arteries Lp(a) has also been linked to stenosis of vein grafts after coronary bypass surgery. Hoff, H., et al. (1988) Circulation 77: 1238-1244. Similar problems of rapid occlusion of vessels have been observed in heart transplant patients.

For some time, general medical practice has focused on the role of LDL, the so called "bad cholesterol," in cardiovascular disease. A great many studies have been published ostensibly linking cardiovascular disease with elevated levels of LDL. As a result, most therapies for the treatment and prevention of arteriosclerosis rely on drugs and methods for the reduction of serum levels of LDLS. Such therapies have had mixed results. The efficacy of such approaches to the problem of cardiovascular disease continues to be major source of debate.

There exists a need, therefore, for a drug and therapy for reducing the binding of Lp(a) to vessel walls, for reducing the overall level of Lp(a) in the circulatory system and for promoting the deposition of existing deposits of Lp(a) on vessel walls.

SUMMARY OF THE INVENTION

The foregoing needs in the treatment and prevention of cardiovascular disease are met by the methods and compositions of the present invention.

A method is provided for the treatment of cardiovascular disease, particularly atherosclerosis, induced or promoted by kidney failure, diabetes, transplant surgery and the like, comprising the step of administering to a subject an effective amount of ascorbate and one or more binding inhibitors, as a mixture or as a compound comprising ascorbate covalently linked with binding inhibitors, which inhibit the binding of Lp(a) to blood vessel walls, such as arterial walls and vein grafts used in by-pass surgery. This effect may also be obtained by administering an effective amount of one or more inhibitors, without ascorbate. The term binding inhibitor throughout the specification and claims is intended to include all substances that have an affinity for the lysine binding site present on the interior walls of blood vessels, particularly arteries, the site of Lp(a) binding. Most of these substances compete with plasmin for the lysine binding site and some of these compounds, in high doses, are in clinical use for the treatment of hyperfibrinolytic states.

A method is further provided for the prevention of atherosclerosis related to or as a complication of surgery, a preexisting disease or a therapy such as hemodialysis. comprising the step of administering to a subject an effective amount of ascorbate and one or more binding inhibitors as previously discussed but further comprising one or more antioxidants. The term antioxidant throughout the specification and the claims is intended to exclude ascorbate, which itself is a powerful antioxidant.

It is thus an object of the invention to provide a method for treatment of induced cardiovascular disease by administering to a subject an effective amount of ascorbate and one or more binding inhibitors, or an effective amount of one or a mixture of binding inhibitors.

It is another object of the invention to provide a method for prevention of induced cardiovascular disease, by administering to a subject an amount of ascorbate effective to lower the amount of Lp(a) in the plasma of the subject.

Yet another object of the present invention is to provide a method for prevention of induced cardiovascular disease by administering to a subject an effective amount of ascorbate and one or more binding inhibitors, or an effective amount of one or more binding inhibitors.

A further object of the present invention is to provide a pharmaceutically acceptable agent for the treatment of induced cardiovascular disease.

Still another object of the present invention is to provide a pharmaceutically acceptable agent for the prevention of induced cardiovascular disease.

Yet another object of the present invention is to provide a method for preservation of explanted tissues and organs that reduces the risk of occurrence of cardiovascular disease in the tissues and organs after implantation.

It is also an object of the present invention to provide a pharmaceutically acceptable agent to assist in the preservation of explanted tissues and organs prior to implantation.

Still another object of the present invention is to provide a pharmaceutical compound and method for treating cardiovascular disease arising from a preexisting condition of diabetes mellitus.

These and other objects will be more readily understood upon consideration of the following detailed descriptions of embodiments of the invention and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immunoblot of the plasma of guinea pigs from the test described in Example 1. Increase of Lp(a) in plasma of guinea pigs with a hypoascorbic diet. Immuunoblot with anti apo(a) antibodies. Lane 1: human control plasma. Lane 2: guinea pig plasma at the start of experiment. Lane 3: guinea pig plasma after 10 days of hypoascorbic diet. Lane 4; guinea pig plasma after 20 days. HMW: high molecular weight standard.

FIG. 2A is a photograph of the aorta of a guinea pig receiving an adequate amount of ascorbate from the test diet in Example 1.

FIG. 2B is a photograph of an aorta of a guinea pig receiving a hypoascorbic diet after three weeks from the test diet in Example 1.

FIG. 3 is an immunoblot of plasma and tissue of guinea pigs from the test shown in Example 2. Plasma and tissue of guinea pigs. Immunoblot with anti apo(a) antibody. HC: human control plasma; L: liver tissue; B: brain tissue; A: aortic tissue, homogenate of plaque area from FIG. 2B.

FIG. 4 is a diagrammatic representation of the action of Lp(a) binding inhibitors on Lp(a) to cause release of Lp(a) from fibrin fibers of an arterial wall.

FIG. 5 is a diagrammatic representation of the action of ascorbate to prevent association and reassociation of Lp(a) to an arterial wall.

   

DETAILED DESCRIPTION OF THE INVENTION

Our invention is based in part on our discovery that animals which have lost the ability to produce ascorbate, such as higher primates and guinea pigs, uniformly produce Lp(a), whereas most animals which possess the ability to synthesize ascorbate generally do not produce Lp(a). Further, we have found that ascorbate deficiency in humans and guinea pigs tends to raise Lp(a) levels and causes atherosclerosis by the deposition of Lp(a) in the arterial wall, from which we conclude that ascorbate administration lowers plasma Lp(a) levels.

We have also discovered that substances that inhibit the binding of Lp(a) to components of the arterial wall, particularly to fibrinogen, fibrin and fibrin degradation products herein identified as binding inhibitors, such as lysine or .epsilon.-aminocaproic acid. Thus, ascorbate and such binding inhibitors are not only useful for the prevention of cardiovascular disease, but also for the treatment of such disease.

Some beneficial effects of ascorbate in the prevention and treatment of cardiovascular disease have been established (see FIG. 2). Our invention reveals the relation to and therapeutic use of ascorbate for Lp(a), one of the most atherogenic lipoproteins, directly related to the development of atherosclerotic plaques. The beneficial effects of ascorbate suggest that ascorbate therapies would be useful in a variety of situations where occlusion of blood vessels by Lp(a) deposition is a problem. For instance, ascorbate may be useful in transplantation of blood vessels and whole organs, where a combination of tissue damage to the transplant, such as by oxidation, and high serum Lp(a) in the transplant recipient results in rapid occlusion of blood vessels in the transplant. Ascorbate may also be useful in the area of hemodialysis, where loss of ascorbate and other vitamins and trace elements from the blood of hemodialysis patients can result in increased serum levels of Lp(a) and thus increased risk of cardiovascular disease. Finally, it appears that ascorbate alone and in combination with binding inhibitors, specifically with plasmin competitors, may be therapeutically useful for treatment of the pathogenic effects of diabetes which is associated with elevated serum concentrations of Lp(a).

The present invention provides methods and pharmaceutical agents for both the treatment and prevention of cardiovascular disease in vivo; methods and agents for the preservation of damage linked vessel occlusion in explanted tissues and organs, as well as methods and agents for the prevention of hemodialysis-linked cardiovascular disease. Each of these embodiments is discussed in turn below.

GENERAL APPLICATIONS

The present invention provides a method and pharmaceutical agent for the treatment and prevention of cardiovascular disease generally, particularly atherosclerosis, by administering to a subject an effective amount of ascorbate and one or more binding inhibitors which inhibit the binding of Lp(a) to blood vessel wall components, particularly to fibrin or fibrinogen. As used herein, the term "ascorbate" includes any pharmaceutically acceptable salt of ascorbate, including sodium ascorbate, as well as ascorbic acid itself. Binding inhibitors include, but are not limited to .epsilon.-aminocaproic acid (EACA), lysine, tranexamic acid (4-aminomethylcyclohexane carboxylic acid), p-aminomethylbenzoic acid, p-benzylamine sulfuric acid, and .alpha.-N-acetyl-lysine-methyl ester and PROBUCOL (a compound comprised of 2 butyl hydroxy tocopherol groups linked together by a disulphide group), Aprotinin, trans-4-aminomethylcyclohexanecarboxylic acid (AMCA), and benzamidine derivatives such as amidinophenylpyruvic acid (APPA) and 1-naphthyl-(1)-3-(6-amidinonaphthyl-(2))-propanone-1 HCl (NANP). An effective amount of a binding inhibitor or a mixture of binding inhibitors may also be used, without ascorbate. Other substances used in the treatment of cardiovascular disease may be co-administered, including: antioxidants, such as tocopherol, carotene and related substances; vitamins; provitamins; trace elements; lipid-lowering drugs, such as hydroxy-methyl-glutaryl coenzyme A reductase inhibitors, nicotinic acid, fibrates, bile acid sequestrants; and mixtures of any two or more of these substances.

Although ascorbate can be used alone or in varying combinations with one or more representative constituents of the above classes of compounds, we prefer when treating a pre-existing cardiovascular condition to combine ascorbate with at least one each of the binding inhibitors, antioxidants and lipid lowering drugs elements in the dosages (per kilogram of body weight per day (kg BW/d)) provided in Table 1. It should be noted that Table 1 provides differing concentration ranges of each constituent, depending upon whether the agent is to be administered orally or parenterally. The variance in dosages is reflective of variation in disease severity. It will be realized therefore that if the subject has been diagnosed for advanced stages of atherosclerosis, dosages at the higher end of this range can be utilized. However, if only prevention of an atherosclerosis condition is the object, dosages at the lower end of this range can be utilized.

As an alternative, a pharmaceutical agent identical to the one just described, but omitting ascorbate, may be employed.

Where ascorbate and binding inhibitors are utilized in the same agent, they may simply be mixed or may be chemically combined using synthesis methods well known in the art, such as compounds in which ascorbate and the inhibitor are covalently linked, or form ionically bound salts. For example, ascorbate may be bound covalently to lysine, other amino acids, or .epsilon.-aminocaproic acid by ester linkages. Ascorbyl .epsilon.-aminocaproate is such an example. In this form the ascorbate moiety may be particularly effective in preventing undesirable lipid peroxidation.

In the case of oral administration, a pharmaceutically acceptable and otherwise inert carrier may be employed. Thus, when administered orally, the active ingredients may be administered in tablet form. The tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid, and/or a lubricant such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring agents may be used. If administration is by parenteral injection, in isotonic saline, a phosphate buffered solution or the like, may be used as pharmaceutically acceptable carrier.

The advisability of using binding inhibitors in treating cardiovascular disease will depend to some extent on the subject's general health, particularly with regard to hyperfibrinolytic conditions. Most binding inhibitors (except lysine) are used clinically to treat such conditions. As a result, monitoring of the subject's coagulation and fibrinolytic system is recommended before and during treatment for cardiovascular disease. Long-term administration of binding inhibitors will require formulations in which the dosages of binding inhibitors are in the lower ranges of the dosages given in Table 1.

Prevention, as contrasted with treatment, of cardiovascular disease may be accomplished by oral or parenteral administration of ascorbate alone. Table 1 gives a range of ascorbate concentrations sufficient to lower the serum Lp(a) concentration. Preferably the prevention of the cardiovascular disease according to the invention is accomplished by use of a physical mixture of ascorbate and one or more binding inhibitors, or by use of a compound comprising covalently linked ascorbate with one or more of the binding inhibitors, which inhibit binding of Lp(a) to the arterial wall. A binding inhibitor or mixture of binding inhibitors may also be administered without ascorbate to prevent Lp(a)-associated cardiovascular disease.

To optimize the therapeutic effect of the release of Lp(a) from the blood vessel walls, the ascorbate and the binding inhibitors described above may be separately administered. Further optimization of therapeutic effect can be gained by using a time release composition to achieve relatively constant serum concentrations of the agent through time.

CORONARY BYPASS APPLICATIONS

As discussed above, recurrence of cardiovascular disease after bypass surgery is a frequent problem. Physicians often observe that the veins used to replace occluded arteries become rapidly occluded themselves after implantation, often requiring the patient to undergo successive surgical episodes to replace clogged bypasses. While not wishing to be bound to any theory, we believe that the rapid occlusion observed in many individual's results from a combination of the patient's pre-existing elevated levels of Lp(a) and injury to the bypass veins, during transplantation, particularly as a result of oxidative damage during explantation. This damage makes binding of Lp(a) to the vessel interior easier. Further, Lp(a) has been detected in abundance in reoccluded by-pass veins after coronary bypass surgery. See, Cushing, et al. (1989) Atherosclerosis 9:593-603. Lp(a) is now known to be the most significant factor for reocclusion of bypass veins. See, Hoff, H, et al. (1988) Circulation 77:1238-1244. Thus, a further embodiment of this invention includes using the pharmaceutical agent of the present invention to lower the bypass patient's Lp(a) before, during and after surgery while at the same using a solution containing the agent to rinse and store the bypass veins until such time as the veins are implanted into the recipient, thereby reducing oxidative damage that can make Lp(a) binding more likely after implantation.

The treatment protocols for the bypass patient generally follow those described above for the treatment of pre-existing cardiovascular disease. The composition of the pharmaceutical agent will generally include ascorbate, one or more binding inhibitors, one or more antioxidants and one or more lipid lowering drugs as enumerated and in the dosages given in Table 1. Of course, the level of dosage will depend on disease severity. Further, the constituents of the agent can be combined just as described above, can be administered either orally or parenterally and can be combined with a pharmaceutically acceptable carrier.

TABLE 1
DOSAGES OF COMPONENTS IN THE DRUG COMPOSITIONS OF THE PRESENT INVENTION
Parenteral / Oral Administration



Turning now to vessel treatment and storage, it is important to provide an in vitro environment which minimizes vessel injury. We conclude that vessel injury can be reduced by the addition of a combination of ascorbate, binding inhibitors and antioxidants to the solution in which the vessels are normally stored. A range of effective concentrations of these constituents in solution is given in Table 2. The general aspects of live vessel preservation and storage prior to implantation are well known in the art.

TABLE 2
CONCENTRATION OF COMPONENTS IN THE SOLUTION OF THE PRESENT INVENTION

Ascorbate 50.5000 mg/l
Binding Inhibitors:
EACA 2-2000 mg/l
Tranexamic Acid 1-300 mg/l
Para-aminomethyl 1-200 mg/l
benzoic acid
Lysine 10-5000 mg/l
Antioxidants
Tocopherol 1-1000 mg/l
Carotene 0.1-100 mg/l

APPLICATIONS IN ORGAN TRANSPLANTS

We have also found that the solution and method of the present invention are effective in preventing cardiovascular disease from occurring in transplanted organs that have been otherwise successfully implanted in an organ recipient, particularly in the case of the heart.

As with occlusion of transplanted veins after bypass surgery, a transplanted heart free of any substantial arterial occlusion may suffer accelerated atherosclerosis after implantation. We believe that the mechanism described for occlusion of transplanted vessels applies equally to the heart itself as a whole, namely that the heart muscle itself, as well as the interiors of the arterial walls become damaged, making the arteries of the heart more prone to binding with Lp(a). Because the organ recipient often presents elevated serum concentration of Lp(a), particularly after surgery (see, Maeda, S. et al. (1989) Atherosclerosis 78: 145-150), atherosclerosis can proceed at an accelerated rate.

Treatment follows along the same line as that described for bypass surgery. Damage to the organ itself is minimized by placing the organ in a solution containing a mixture of ascorbate, binding inhibitors, and antioxidants in an otherwise standard storage solution. Concentration ranges for the various components in the final solution are given in Table 2. Because of the oxidative cellular damage during extended periods of explantation, the concentration of antioxidants should be in the higher range of dosages disclosed in Table 1. The standard storage solution itself is well known in the art. Storage of the organ in this solution will tend to minimize damage to arterial walls, thereby providing fewer places for Lp(a) to bind.

Of course, patient treatment is also desireable. If the organ recipientsuffers from some degree of atherosclerosis at the time of organ transplant, the protocol and drug described above generally for the treatment of atherosclerosis should be employed. If, however, the patient does not suffer from atherosclerosis, use of the drug and protocol described above for prevention of atherosclerosis is desired. In all cases, the lowest dosages of ascorbate should be employed in the drug composition since ascorbate has an immune stimulatory effect.

APPLICATIONS IN HEMODIALYSIS TREATMENT

It is well known that patients who suffered renal failure and require regular dialysis treatment to cleanse the blood of metabolic waste products are also at an increased risk for cardiovascular disease. We believe that the reason for this may be a depletion of ascorbate, vitamins in general and other essential substances from the blood supply during the hemodialytic process. As described more fully above, the loss of ascorbate would result in greater injury to the interior of the artery walls over time and may also result in the production of elevated Lp(a) levels in the blood serum.

As can appreciated, the solution and method of the present invention can be applied both to the patient and the hemodialysis solution to prevent and control hemodialysis-related cardiovascular disease. Turning first to the hemodialysis solution, it is desired to add a combination of ascorbate, binding inhibitors and antioxidants to the solution to produce concentrations of these compounds in solution in the range of concentrations provided in Table 2.

In order to achieve the best results, treatment of the dialysis patient should be carried out in addition to modification of the hemodialysis solution. Treatment should follow the drug and protocols set forth in detail above for the treatment of a preexisting atherosclerotic condition.

APPLICATIONS IN TREATMENT OF DIABETES

The composition and method of the present invention are also useful in the treatment of the pathological effects of diabetes mellitus. In diabetes mellitus, pathological charges in the arteries frequently lead to clinical symptoms or complete failure in various organs such as the kidney, eye and peripheral circulation system. Therefore, one therapeutic focus in diabetes mellitus is the treatment of diabetic angiopathy.

It appears that glucose competitively inhibits the physiologic uptake of ascorbate in different cell systems of the body, including the arterial wall. Kapeghian, et al. (1984) Life Sci. 34: 577. Such damage to arterial walls creates binding sites for Lp(a). Further, Lp(a) has been found to be elevated in the blood serum of diabetic patients. The atherogenic process is perhaps therefore accelerated by the combination of damaged arteries and elevated Lp(a). Therefore, we propose that ascorbate alone or in combination with at least one binding inhibitor has therapeutic value in treating diabetes-related atherosclerosis.

Thus, another embodiment of the present invention is the use of a composition and method in treating the pathogenic effects of diabetes mellitus, particularly with regard to atherosclerotic conditions.

The treatment protocol involves the oral or parenteral administration of a pharmaceutical composition comprised of ascorbate, one or more binding inhibitors and one or more antioxidants. Dosages for a course of treatment are provided in Table 1. The dosage of ascorbate should preferably fall within the higher range, thereby increasing its chance of cellular uptake in the presence of high serum levels of glucose.

EXPERIMENTAL

Having disclosed the preferred embodiment of the present invention, the following examples are provided by way of illustration only and are not intended to limit the invention in any way.

EXAMPLE 1

Because of its metabolic similarity to man, with respect to the metabolism of ascorbate and Lp(a), the guinea pig was used in this example.

No study has been previously reported in the guinea pig to identify the lipoprotein involved as risk factors in plasma and as constituents of the atherosclerotic plaque.

Three female Hartly guinea pigs with an average weight of 800 g and an approximate age of 1 year were studied. One animal received an extreme hypoascorbic diet with 1 mg ascorbate/kg body weight/d. Another animal received 4 mg/k BW/d. The third animal served as a control receiving 40 mg ascorbate/Kg BW/d.

Blood was drawn by heart puncture from the anesthetized animals and collected into EDTA containing tubes at the beginning, after 10 days, and after 3 weeks, when the animals were sacrificed. Plasma was stored at -80 DEG C. until analyzed. Lp(a) was detected in the plasma of the guinea pigs by use of SDS-polyacrylamide gels according to Neville (J. Biol. Chem., 246, 6328-6334 (1971)) followed by Western blotting (Beisiegel, et al., J.Biol. Chem., 257, 13150-13156 (1982)). 40 .mu.l of plasma and 20 mg of arterial wall homogenate were applied in delipidated form per lane of the gel. The immunodetection of apo(a) was performed using a polyclonal anti-human apo(a) antibody (Immuno, Vienna, Austria) followed by a rabbit anti-sheep antibody (Sigma) and the gold-conjugated goat anti-rabbit antibody with subsequent silver enhancement (Bio-Rad). The determinations of cholesterol and triglycerides were done at California Veterinary Diagnostics (Sacramento) using the enzyme assay of Boehringer Mannheim. Plasma ascorbate was determined by the dinitrophenylhydrazine method (Schaffer, et al., J. Biol. Chem., 212, 59 (1955)).

Vitamin C deficiency in the diet led to an increase of Lp(a) in the plasma of the guinea pig indicated by a clear band in the immunoblot of the plasma after 10 and 20 days of a hypoascorbic diet (FIG. 1). At necropsy the animals were anesthetized with metophase and were exsanguinated. Aorta, heart and various other organs were taken for biochemical and histological analysis. The aorta was excised, the adventitial fat was carefully removed, and the vessel was opened longitudinally. Subsequently the aorta was placed on a dark metric paper and a color slide was taken. The picture was projected and thereby magnified by an approximate factor 10. The circumference of the ascending aorta, the aortic arch and thoracic aorta as well as the atherosclerotic lesions in this area were marked and measured with a digitalized planimetry system. The degree of atherosclerosis was expressed by the ratio of plaque area to the total aortic area defined. The difference in the 3 one-year old animals of the experiment was significant and pronounced lesions were observed in the ascending aorta and the arch of the vitamin C deficient animal (FIG. 2B).

EXAMPLE 2

To confirm the data obtained in Example 1, a second guinea pig experiment was conducted, using 33 male animals with a mean weight of 550 g and an approximate age of 5 months. One group of 8 animals served as a control and received 40 mg ascorbate/kg BW/d (group A). To induce hypoascorbemia 16 animals were fed 2 mg ascorbate/kd/d (group B). Group A and half of the animals of group B (progression sub-group) were sacrificed after 5 weeks as described above. Half of group B was kept for 2 more weeks, receiving daily intraperitoneal injection of 1.3 Na-ascorbate/kg BW/d as a daily intra peritoneal injection with the intention to reduce the extent of atherosclerosis lesions. After this period these animals also were sacrificed.

Plasma ascorbate levels were negatively correlated with the degree of theatherosclerotic lesion. Total cholesterol levels increased significantly during ascorbic acid deficiency (Table 3).

The aortas of the guinea pigs receiving a sufficient amount of ascorbate were essentially plaque free, with minimal thickening of the intima in the ascending region. In contrast, the ascorbate-deficient animals exhibited fatty streak-like lesions, covering most parts of the ascending aorta and the aortic arch. In most cases the branching regions of the intercostal arteries of the aorta exhibited similar lipid deposits. The difference in the percentage of lesion area between the control animals and the hypoascorbic diet animals was 25% deposition of lipids and lipoproteins in the arterial wall.

TABLE 3
MEAN PLASMA PARAMETERS OF THE DIFFERENT GROUPS IN RELATION TO THE AREA OF AORTIC LESIONS




A possible inhibitor may be identified first by adding molar amounts of the possible inhibitor at a little larger, by approximately 5 times, the amount of .epsilon.-aminocaproic acid found in the earlier study. If, at this concentration, a possible inhibitor is found to inhibit the agglutination, studies are made at lower concentrations, to determine the concentration that has a 50% initiatory effect.

EXAMPLE 3

Human arterial wall was obtained post mortem from the aorta ascendens. The tissue showed homogenous intimal thickening (early atherosclerotic lesion). It was cut into pieces, with 100 mg of the cut up tissue homogenized in a glass potter for 1 minute in 2.5 ml of the following solutions:

PBS (Dulbeco) + 50 mg/ml
NaAscorbate
PBS + EACS 50 mg/ml
PBS + Tranexamic Acid
50 mg/ml
PBS + NaAscorbate +
50 mg/ml
Tranexamic Acid

Results of this treatment are given in Table 4 and show that, compared to the control solution, a considerable amount of Lp(a) was released from the interior arterial wall.

TABLE 4
Lp(a) RELEASED FROM HUMAN AORTA IN RELATION TO SPECIFIC BINDING INHIBITORS



By now it is apparent that the methods and compositions of the present invention meet longstanding needs in the field of prevention and treatment of induced cardiovascular disease. Although preferred embodiments and examples have been disclosed, it is understood that the invention is in no way limited thereby, but rather is defined by the claims that follow and the equivalents thereof.



US5639787
Therapeutic method for the treatment of cancer


Inventor(s):     RIORDAN NEIL H [US]; RIORDAN HUGH D [US] +

A method of treating cancer in a patient by raising and maintaining the concentration of ascorbic acid, or ascorbate, in the patient's plasma to at least the level expected to be toxic to an in vitro culture of cells of the type of cancer being treated, the required plasma ascorbate levels being achieved and maintained using long term intravenous infusions of large amounts of ascorbate, with or without ascorbate cytotoxicity effectiveness enhancing or tumor site delivery and absorption enhancing agents.



http://www.paulingtherapy.com/

This web site was established in 1996. The methods described here for treating heart disease were invented by the great American scientist Linus Pauling, and his German associate Matthias Rath, MD. These methods and protocol have been proven in practice over the past twenty years. At the required high dosages, the protocol is called The Pauling Therapy in Linus Pauling's honor. Unfortunately, heart patients are not going hear about this safe and apparently very effective treatment (even cure) for heart disease from their doctors. -- Owen Fonorow



http://vitamincfoundation.org/suppress.htm

Chronic Scurvy: The Suppression the Real Nature, Cause, and Cure for Heart Disease, (2005)

Owen Fonorow


The leading killer in the United States, the condition that those in medicine call heart disease, or occlusive cardiovascular disease, is really a low-grade form of scurvy. This fact is becoming increasingly more difficult for modern medicine to deny.

Chronic scurvy: Heart disease is a misnomer. The disease is characterized by scab-like build-ups that slowly grow on the walls of blood vessels. The underlying disease process reduces the supply of blood to the heart and other organs resulting in angina ("heart cramp"), heart attack and stroke. The correct terminology for this disease process is chronic scurvy, a sub-clinical form of the classic vitamin C deficiency disease.

The true nature of the disease was identified in the early 1950s by a Canadian team led by G. C. Willis, MD. This finding was confirmed in the late 1980s, by the world's then leading scientist, Linus Pauling, Ph.D.

Pauling alerted the world in lectures, in writing and on video after he and his associates conducted experiments that confirmed the Willis findings. To date, this alert has never made its way into a mainstream media outlet. Moreover, cardiologists are taught, and routinely tell their patients, that there is no connection between vitamin C and heart disease, and also that there is no value in vitamin C in amounts much higher than the minuscule RDA.

From a scientific standpoint, if a medical doctor, or anyone, tries to challenge the true nature of cardiovascular disease, they must be able to cite experiments that refute the Pauling/Willis chronic scurvy hypothesis. Such experiments have never been published.

Its been twelve years since Pauling issued the alert. Pharmacology professors Steven Hickey and Hilary Roberts in their recent book ASCORBATE: The Science of Vitamin C (2004) document, incredibly, that there have been no independent experiments published that were designed to test the Pauling hypothesis (except one at much lower doses that was conducted by Pauling’s close associate Dr. Matthias Rath.)

We are aware of only one clinical study in humans that has been carefully designed to test the Pauling high-dose hypothesis. The study was performed in the UK, with 200 men, over a period of three years, and the data confirmed Pauling’s theory and therapy. Yet, so far, Dr. Kale Kenton’s study has not appeared in a medical journal.

Will the giant pharmaceutical industry facing these facts survive or will it collapse in 2005? The end of the suppression of vitamin C will reveal the CODEX restrictions for what they really are, a means to prop up an industry that has little reason to exist in its present form. The public is beginning to realize that the world’s most profitable industry is really a house of cards. Its most profitable products are at best useless, at worst dangerous. Prescription drugs beget more drugs. The secret that dooms Big Pharma is that the best of health is achieved by taking high doses of vitamin C and avoiding toxic prescription medications as if your life depended on it.

History of the Great Suppression

The 700,000 people who die needlessly every year are those who heed their cardiologists advice. The American Heart Association estimates that 63 million Americans suffer cardiovascular disease. More than one million undergo some form of heart operation, and over 15 million are taking statin cholesterol lowering drugs on the advice of their doctor. These popular statin drugs are known to deplete CoQ10 and probably cause heart failure.

The pioneering research into the relationship between vitamin C and heart disease began in the late 1940s, not long after the structure of vitamin C was determined. Canadians doctors proved that a vitamin C 'deficiency' causes the condition, commonly called atherosclerosis. These doctors found that the condition will arise in 100 per cent of vitamin C-deprived animal test subjects that don't make their own vitamin C. Furthermore, these Canadian pioneers demonstrated that vitamin C alone reverses atherosclerosis in laboratory animals. [THE REVERSIBILITY OF ATHEROSCLEROSIS, G. C. Willis, Canad. M. A. J., July 15, 1957, Vol 77., Pg 106-109 ]

The team performed similar studies in humans. The results, while not conclusive, showed reversals of atherosclerotic plaques in one third of the human subjects. Notably, these studies were of low doses, no more than 1500 mg per day. [SERIAL ARTERIOGRAPHY IN ATHEROSCLEROSIS, G. C. Willis, A. W. Light, W.S. Cow, Canad. M. A. J. Dec 1954, Vol 71, 1954, p. 562-568 ]

The knowledge that heart disease is a form of scurvy has been suppressed from the time that the first series of Willis articles were published in the Canadian Medical Journal in the early 1950s. Inexplicably, since the 1950s, no articles favorable to vitamin C and its connection with atherosclerosis have appeared in a reputable medical journal that is widely read by medical doctors.

Cardiologists to-be are taught that there is no relation between vitamin C intake and heart disease, and that it is quackery to suggest otherwise. These assertions seem justified because reports of such studies are lacking. But, as vitamin C expert and pharmacological professor Steve Hickey points out, every cardiologist could have performed these studies on his/her own.

"Time has moved on and the medical profession has FAILED over the past 50 years to produce the required experiments. The budget of say the NIH alone is over 27 billion but over the past 50 years no one has replicated the early vitamin C and heart disease research, which could be done by almost any cardiologist from petty cash.

Since Pauling, and others, have promoted ascorbate as a cure for heart disease, it seems silly that a potential cure for the worst killer in the developed nations (atherosclerosis) has not been refuted. To a scientist from any other discipline this lack of interest would be bizarre.

Its a fact that the experiments have been done in animals and the results show that ascorbate protects against atherosclerosis and may reverse it. There is some additional evidence from human studies that is consistent with this interpretation. So why have the human studies not been performed? Or we may ask if they have been performed, was the data withheld? The enemies of Pauling, as well as the drug companies, would love to see the Pauling hypothesis discredited. Why have the experiments not been reported? -- Dr. Steve Hickey email correspondence, Dec 2004

The Lecture Video

In a 1992 lecture recorded on video, Linus Pauling explained the reason atherosclerosis forms on the walls of arteries when vitamin C is deficient. This leading scientist explained how a specific form of cholesterol that compensates for low vitamin C causing plaques and why his discovery of a rapid cure for chronic scurvy includes the amino acid lysine. [*]

There is no doubt that the news of this cure has been suppressed, otherwise most of the public would have learned that twice Nobel prize winner Linus Pauling had suggested it. Millions are dying needlessly for lack of disseminated knowledge of the Pauling discovery which amounts to the suppression of it.

Is Linus Pauling's high-dose ascorbic acid and amino acid therapy the cure for heart disease? The crime is that no one knows!

Pauling and his former associate, Dr. Matthias Rath, did their part by running the experiments and attempting to publicize these discoveries. Now it is up to other researchers in the medical scientific community. If there is the mere chance that Drs. Willis, Pauling and Rath are correct, it is truly criminal to fail to run experiments under fair conditions. Apparently, medical science is controlled by the drug industry. Even the United States National Institutes of Health gives all appearances of being under the control of the drug industry and it seems that not even members of Congress can overcome this obstacle.

Gross Negligence at the National Institutes of Health (NIH)

In the years 1998 and 2002 the Vitamin C Foundation submitted grant requests for government funding to study Dr. Pauling theories. These requests were formally submitted to the new Office of Alternative Medicine at the NIH. [http://vitamincfoundation.org/NCCAMgrant]

The reasons for these requests were twofold: The Foundation sought funding so that Linus Pauling's recommended therapy would be fairly investigated in humans. All previously known tests have been performed with less than adequate amounts of vitamin C. These grant applications also put the United States government on notice that Linus Pauling had in fact made the claim of an outright cure for heart disease.

The NIH was free to design, sponsor and run their own study with their own choice of scientists. Had such studies been conducted, millions of lives and billions of dollars might now have been saved. Unfortunately for Americans, the NIH, Office of Alternative Medicine, rejected both grant requests, and failed to run their own studies.

Both requests were turned down by the United States Government, not because the reviewers had any objection to the science or the protocols, but because the scientists and medical doctors that the Foundation recruited to run the study were "inexperienced." Apparently, investigators that run studies for the NIH have to be members of "The Club." This travesty is a matter of public record. - Mike Till, Sr.

The Cholesterol Lowering Drug Debacle

Cardiology has been on the wrong path for a long time. The result has been that heart disease is still the leading cause of mortality in the United States, and statin cholesterol-lowering drugs have become the top-selling class of prescription drug. Statin drugs generate more than $7 billion in annualized sales, but these drugs have significant side effects. Vitamin C, with annualize sales close to $180 million, has the very same cholesterol-lowering property as the popular statin drugs.

Vitamin C Even Lowers Cholesterol - Naturally

In 1985, two years prior to the introduction of the popular statin cholesterol-lowering drugs, the scientists who were investigating the enzymes that help the body produce cholesterol, made an important discovery: Vitamin C is a powerful anti-cholesterol agent. The vitamin C molecule inhibits the same enzyme, HMG CoA Reductase, that the statin cholesterol-lowering drugs inhibit. [*]

Individuals using home cholesterol-monitoring devices, such as the LifeStream® monitor which is available in 55,000 retail outlets, report that 6000 mg to 10,000 mg of vitamin C may be required for maximum cholesterol lowering effect.

Statin Drugs May Cause Heart Failure Leading to Heart Transplants

The structure of Coenzyme Q10 (CoQ10 or ubiquinone) was determined by the Merck scientist Karl Folkers after its discovery in 1957. [*] There have been at least 35 clinical studies showing CoQ10's massive benefits for heart patients, especially patients in heart failure. [*] And in Japan, until last year, CoQ10 was a heart medication only available by prescription.

The drug giant, Merck, learned during their cholesterol-lowering research that statin drugs block the body's production of its own CoQ10. This blockage of CoQ10 synthesis is a serious action of statins that causes fatigue, muscle pain and skeletal myopathy, (a grave deterioration of muscle). Drug advertisements in Canada must carry the CoQ10 statin-depletion warning, but the American FDA does not require these important warnings, keeping U.S. medical doctors in the dark and putting their patients at risk. [*]

Merck has more than one 1990 patent for adding CoQ10 to statins as a means of circumventing the issue of blocking CoQ10 biosynthesis. [United States Patent 4,933,165 ] Their having these patents since 1990 is proof that members of Merck corporation have been aware that statins cause muscle deterioration. (The Merck Patents were never implemented, probably because the world supply of CoQ10 is far too limited to supply all statin drug users).

It is sad and truly frightening that today's hottest selling class of prescription drugs, statin cholesterol-lowering drugs, are known to deplete CoQ10 synthesis, yet these drugs are routinely prescribed to heart patients!

Another Statin Side-Effect? Transient Global Amnesia

Former NASA astronaut Duane Graveline, M.D., believes that the statin drug Lipitor® caused his own case of Transient Global Amnesia (TGA) [http://www.spacedoc.net/Statins_flyer.html]. Graveline believes that these drugs are the cause of a recent epidemic of TGA to hit emergency rooms, and fearing the dire possibilities with airline pilots who take statins, Dr. Graveline has begun a crusade to educate the medical profession and public about the potential danger of cholesterol lowering drugs. The vitamin C foundation has posted its collection of concerns at www.vitamincfoundation.org/statinalert.

Chronic Scurvy Verified by CardioRetinometry

It has long been known that human arteries weaken without vitamin C and other necessary nutritional support. Atheromas, or soft atherosclerotic plaques, are the names given to abnormal formations that appear in arteries. Dr. Pauling and associates theorized with Willis that such plaque formations serve to strengthen arteries, because they appear most often where the blood pressure is highest. Sometimes a weak artery ruptures and the resulting clot causes a heart attack or stroke. This condition is most properly characterized as chronic scurvy.

Atheromas in the microscopic arteries in the retina have been clearly visible to eye doctors, but until recently, they did not believe that such build-ups were reversible. Dr. Sidney Bush, DOpt, of the United Kingdom, accidentally discovered that atheromas can be reversed in those patients instructed to take from 3,000mg to 10,000mg of vitamin C. Dr. Bush made his discovery while studying eye infections in contact lenses wearers. Vitamin C was being tested as a preventive measure for these infections and, serendipitously, Dr. Bush noticed that atheromas disappeared in the study patients taking vitamin C.

Dr. Bush reports that some patients require as much as 10,000mg daily to reverse soft atheromas.

Dr. Bush has invented a new diagnostic technique that he calls CardioRetinometry. He believes this method of diagnosis will revolutionize cardiology.

A new diagnostic technique can access coronary heart disease risk (CHD) suggested by universal retinal arterial atheroma, previously unsuspected as reversible. Physicians have overlooked, and Optometrists/Opthamologists were not expecting that vitamin C would have this effect. This effect was accidentally found and linked to the vitamin C that contact lens wearers had agreed to take. We have increasingly noticed it from 1999 using Retinometry in the Hull Contact Lens and Eye Clinic. Such a discovery requires urgent evaluation."

Dr. Bush has also promoted the idea that chronic scurvy not only exists, but can be accurately measured. Eye doctors can now easily diagnose this condition by examining the microscopic arteries behind the eye before any symptoms of heart disease manifest.

Thanks to Dr. Bush we now know that vitamin C will reverse the condition, in short order at the optimal dosage determined by Cardioretinometry.

People today are under the extremely seriously mistaken impression that nobody dies of scurvy any more! These studies may prove that we are all dying faster from scurvy than hitherto suspected

The pericorneal vasculature, studied frequently by contact lens practitioners, shows that Scurvy affects all humans some of the time and most of us most of the time.

The largely unrecognized chronic subclinical form can best be diagnosed (and cured) by Optometrists using sequential electronic retinal artery images and highly variable amounts vitamin C, occasionally with other nutrients. . - Dr. Bush

Cardioretinometry clearly demonstrates the relationship between vitamin C intake and "atheromas" - plaques forming on the arteries that serve the retina in the eye. Dr. Bush has published before and after pictures taken with his new method and advocates the need for rigorous studies.

The atheroma of the retinal arteries is a virtually perfect surrogate outcome predictor of coronary heart disease and will continue to be so as long as the eyes are connected to the rest of the system. The modern electronic eye camera/microscopes with high definition magnification facility show the impacting of the cholesterol beautifully and also its redissolving into the bloodstream when the system is restored to balance. And this is seen in arterioles too small to be seen with the naked eye!

Whilst day to day variations in the pericorneal vessels are a relatively easily readable ‘barometer’ of ‘ephemeral’ scurvy especially when viewed via the slit lamp biomicroscope of the contact lens practitioner, little attention has been paid to it except by a few dedicated medical practitioners.

The pericorneal arterioles and capillaries can and are graded in my system of practice into ten degrees of scurvy allowing the accurately prediction to patients of how much or little vitamin C they have been eating. The highest mark anybody has had is 94% When I started this grading, c.1997, I confounded my nursing staff by being able to correctly identify patients who ate no or few greens. But the same ease of observation does not attach to identification of the chronic subclinical variety. It cannot identify dietary faults in the most recent past. In a similar way to slow build-up of vitamin E in the body fat and cell walls of the brain, it takes over a month to be sure what is happening to the cholesterol in the retinal arteries.

Dr. Bush now has evidence that even calcified "hard" plaques can be reversed over the course of 2-years on a high vitamin C intake. This development throws a hammer into the Government/CODEX 'recommended' daily allowance of 60 mg and the 2000mg maximum tolerable allowance.

The moral of the story is to have regular examinations of the retinal arteries by a patient, suitably equipped optometrist trained in CardioRetinometry. This is in my opinion, after five years of observation of my patients' health, the most valuable safeguard of one's cardiovascular and probably many other systems as they do not act in isolation.

Our Path to the World of Vitamin C and Other Discoveries

In 1994, no one knew whether Linus Pauling's high vitamin C/lysine protocol really worked. Our company, Intelisoft Multimedia, Inc., had obtained the rights to the Pauling video on heart disease [*] and tried to promote it. At that time, the author had no financial interest in any nutritional product. Years later, we realized that Pauling's close associate, Matthias Rath, apparently had not fully appreciated what Pauling had been advocating. For this reason we took on the task of promulgating Pauling's discoveries. Tower Laboratories Corporation was willing to promote a new product with sufficiently high doses of vitamin C and lysine, which matched Pauling's dosage recommendations.

From the beginning, the Pauling therapy began to absolutely cure the incurable - miracle after miracle. Many of these testimonials have been written and are posted at the video web site PaulingTherapy.com. Yet, none of the old media printed a word about this phenomenon.

Kevin Trudeau has written a new book entitled Natural Cures 'They' Don't Want You to Know About ( naturalcures.com). Most of the information in this book dovetails with our experience advocating the Pauling therapy for heart disease during the past decade. Trudeau points out that it is against Federal law to tell people that a particular product cures a disease without consent from the Government. Kevin is mad, and I feel the same way.

Almost everyone in authority concerned with heart disease knows, or has been advised, except patients, that Pauling's theory is undeniable and that his recommended therapy works quickly.

The safe and effective answer to the most common form of heart disease - plaques forming over weak arteries - is 6000mg to 18000mg vitamin C to strengthen the arteries. And Dr. Pauling invention of administering high-dose lysine resolves existing plaques. This combination appears to work in most individuals within 10 days, with the correct dosage

The general attitude of traditional medicine is wrong and self-serving on these matters. Instead of depriving patients of a potentially life-saving therapy until the 'necessary' (and, as yet still unplanned) studies are run, doctors should be recommending Dr. Pauling's therapy to all heart patients until there is evidence this non- toxic therapy doesn't work. We have heard reports of cardiologists becoming livid after having been briefed on Pauling's theories by respected sources. These doctors expressed verbal frustration at their medical journals for not informing them of these developments.

The Solution to Other Forms of Heart Disease

Heart Failure

Many people experience a remission from heart failure after they adopt Pauling's vitamin C and lysine therapy. However, there is much evidence that the cause of heart failure in most people is a Coenzyme Q10 deficiency. This vitamin-like coenzyme is required in our fuel-cells, the mitochondria, in order to manufacture the body's fuel Adenine Triphosphate (ATP).

Several other vitamins are required for the human body to produce its own CoQ10, and humans are known to synthesize less CoQ10 as we age. Scores of prescription drugs, and in fact all the statin cholesterol-lowering drugs, block the body's production of CoQ10! Therefore, it can be accurately stated that these drugs, given to most heart patients, cause a form of heart disease - heart failure. The rate of heart failure has tripled, and CoQ10 experts cite studies which attribute this increase to higher dosages of statins. [*]

The only recognized cure for heart failure is heart transplant.

Forget VIOXX, CELEBREX, ALEVE, etc., the statin drugs are a bigger scandal.

High Blood Pressure/Hypertension

Normally, blood pressure elevates during times of stress (fight-or-flight) for short periods. The higher blood pressure ensures that glucose and other nutrients enter the cells in order to aid response to the stress. It is also normal for high blood pressure to normalize after the stressful event passes. Generally, doctors measure blood pressure because a small narrowing of the artery has an exponential effect on hypertension. This blood pressure reading is considered an indicator of (weak) arterial plaque. According to discussions in the British Medical Journal, opthamologists have noticed that the plaques form in microscopic retinal arteries before the onset of elevated blood pressure.

Pauling's therapy has been an effective treatment for hypertension, as are other nutrients, such as magnesium, vitamin B6, the amino acid arginine and several other orthomolecular approaches. Health journalist Bill believes that 200 mg of vitamin B6 is more effective than many prescription drugs for hypertension. [http://www.askbillsardi.com/sdm.asp?pg=hyper_1 ]

Calcified Arteries

Many heart patients have hard or calcified arteries. This condition makes heart attack more likely because blood vessels are unable to dilate properly in the event of a clot or blockage. The most probable cause of excess calcium building up-in the arteries of heart patients is the use of prescription blood thinners. These prescription medications either simulate or block vitamin K and they are routinely prescribed. There are over 200 MEDLINE studies as evidence of our suspicion that this is a fact. [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Display&dopt=pubmed_pubmed&from_uid=9743228 ] High dose vitamin K reduces calcium in soft tissues and is considered a standard treatment for osteoporosis in Japan. The vitamin acts as a hormone and helps move calcium from soft tissues into bone. [http://www.lef.org/magazine/mag2000/feb00-report.html]

Chelation doctors deserve great respect. Their EDTA detoxification therapy benefits many patients, but EDTA will not cure heart disease by itself. The Chelation treatments that are effective incorporate a supplement program which includes vitamin C.

Heart Attack

Strong vitamin C/lysine-fortified arteries are less likely to rupture. If there is no rupture, there will be no clot. If there is no clot, there will be no heart attack caused by a blockage of blood to the heart.

World Health Organization (WHO) researchers have discovered that low serum vitamin E is a 70% better predictor of heart attack than either hypertension (high blood pressure) or high cholesterol. Also, Teo and other researchers have discovered that a magnesium injection immediately after a heart attack saved 55% of those who would have died. (Placebo controlled trial) [http://www.internetwks.com/pauling/jon.html ].

Congenital Heart Defects and Heart Damage

We have documented extraordinary cases of patients whose damage hearts, as measured by EKG, have returned to normal. Harvard medical researchers found that vitamin C was the only one of 880 substances tested that caused heart muscle cells to regenerate from stem cells. [ http://www.sciencedaily.com/releases/2003/04/030401073122.htm]

It is our experience that a good natural vitamin E with mixed tocopherals and tocotrienols (such as 2000 IU Unique-E from A. C. Grace), in conjunction with high vitamin C, as ascorbic acid, is required for EKG reversals. Such a reversal occurred in 3 months. [http://www.internetwks.com/carolsmith]

Cardiology: A Real Nightmare Scenario

Heart patients have the right to be highly skeptical or even fearful of their cardiologist. With the exception of the nitroglycerine patch [*], there are no standard heart medications or treatments that do help heart patients. In our opinion, all prescription treatments, of which we are aware, make patients worse by blocking CoQ10, or by causing rapid calcification of soft tissues, by making blood clots more likely, or by raising


 


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