Folkman (1972) is credited with coming up with the concept of antiangiogenic therapy for cancer. The idea is that after a cluster of cancer cells reaches approximately 2 mm in diameter, they must develop a blood supply, called angiogenesis, to grow further. Because adult humans do not require much angiogenesis, therapy aimed at inhibiting angiogenesis might be
effective against cancer. We now know that there are many angiogenic-promoting agents [e.g., vascular endothelial growth factor (VEGF); fibroblast growth factor (FGF); and secreted protein, acidic and rich in cysteine (SPARC)], which cancers can recruit to stimulate angiogenesis and allow growth. Today there are therapies for cancer with some efficacy such as antibodies against specific angiogenic promoters, such as VEGF.
Copper is required for the activation of many angiogenic promoters (e.g., FGF and SPARC), suggesting that anticopper drugs might be effective antiangiogenic therapies for cancer. Penicillamine was shown to have efficacy in inhibiting a can- cer in rabbits (Brem et al., 1990). Later, TM, discussed earlier in the section on WD, was shown to have excellent efficacy in a series of mouse cancer models (Pan et al., 2002; Cox et al., 2001, 2003; Khan et al., 2002; Pan et al., 2003; van Golen et al., 2002). One particularly impressive example was the HER2/neu mammary cancer mouse model, in which newborn mice are genetically programmed to develop mammary cancer during the first year of life (Pan et al., 2002). TM therapy completely prevented the tumors whereas control mice developed obvious mammary tumors, often multiple. On pathologi- cal examination, the TM mice had small clusters of cancer cells in their mammary glands, which did not grow into visible tumors because of a lack of angiogenesis.
After that there were multiple TM trials in human cancer at the University of Michigan, all of these in bulky advanced cancer and all showing very limited efficacy (Brewer et al., 2000b; Redman et al., 2003; Henry et al., 2006; Gartner et al., 2009; Pass et al., 2008). The way TM was used in the mouse and human studies was to lower copper levels to an inter- mediate status, avoiding clinical copper deficiency, but lowering copper availability, which appears to inhibit angiogenic promoters activated by copper. The proper copper status is obtained by monitoring blood Cp levels. The amount of Cp in the blood is determined by copper availability in the liver. The normal Cp level in the human is 20–35 mg/dL. If the Cp is reduced to 5 mg/dL or lower, then clinical copper deficiency appears, beginning with anemia. To avoid this the target Cp is between approximately 8 and 15 mg/dL.
The University of Michigan licensed the cancer use of TM to a company, which elected to use the choline salt of TM because it had more stability. All of the WD work and the cancer work at the University of Michigan had been done with the ammonium salt, which shows enough air instability that capsules were only used for 60 days after preparation, whereas bulk supplies were protected from air in containers with argon replacing the air. The company essentially repeated the University of Michigan human cancer work with two trials of choline TM against advanced bulky cancer and found very limited efficacy (Lin et al., 2013; Lowndes et al., 2008).
What was forgotten in all of the human work was that the mouse trials that had shown great efficacy were all against what might be called microscopic cancer. The HER2/neu study, which showed such dramatic results, involved small clus- ters of cells. Likewise, the other mouse models involved injections of cancer cells into the mice, again involving micro- scopic cancer. It seems likely that small clusters of cancer cells can use or recruit only one, or a very limited number, of angiogenic promoters, and that the promoter or promoters available are all dependent on copper. In contrast, advanced can- cers cause inflammation and attract all kinds of inflammatory cells, many of which synthesize angiogenic promoters that can be used by the cancer to promote angiogenesis, and many of these are not dependent on copper, limiting TM efficacy against advanced cancers.
The key to TM efficacy against human cancer may be to remember the mouse examples and to use it against micrometastatic disease. An oncologist on the US West Coast has used TM successfully in this manner. He gets the tumor to no evidence of disease (NED) status by conventional therapy. However, the various tumors have close to 100% probability of recurrence because of micrometastatic disease. He treats with TM, keeping the Cp below 15 for 3 years. When the TM is stopped, the disease does not recur, and the cancer is cured. It appears that the micrometa- static clusters of cells die or are killed over that 3-year period. He has successfully cured patients with a variety of tumors. Unfortunately, all of this work is anecdotal because none of it is published. The author has reviewed some of the patient records and is convinced of the authenticity of the findings, but without publication the work is necessarily viewed as anecdotal.
There is one study, with breast cancer, in which this kind of TM work has been published. Breast cancer offers a good chance to test TM against micrometastatic disease. After removal of the primary (if there are multiple) positive lymph nodes, micrometastatic disease is present, and these cancers have a very high probability of recurrence. Jain et al. (2013) from Vahdat’s group have recently published very encouraging preliminary data on TM treatment of breast cancer. They have found a type of circulating cell, called endothelial progenitor cells (EPCs), which are important in initiating angio- genesis and are a key to predict relapse in breast cancer. Forty-one breast cancer patients were enrolled: 29 stage 2/3 and 12 stage 4 NED. TM (100 mg) was given orally to maintain Cp at less than 17 mg/dL for 2 years or until relapse. Seventy- five percent of patients achieved the copper depletion target within 1 month. In copper-depleted patients, but not in those patients who did not achieve copper depletion, there was a significant reduction in EPCs. Of six patients who relapsed, only one had EPC levels below baseline. Eighty-five percent of the patients were still relapse free at 10 months, a remarkable
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time in this group of patients with severe disease, and the study is ongoing. They concluded that TM is safe, can maintain EPC levels below baseline if copper depletion is achieved, promotes tumor dormancy, and ultimately may prevent relapse.
A study to further evaluate the efficacy of TM against micrometastatic cancer has been initiated by the author and his colleagues and involves a double-blind study of osteosarcoma in dogs. After removal of the primary, most of these dogs have micrometastatic cancer in the lungs. Endpoints are time to macroscopic lung metastases and survival.
In summary, it appears there is much promise for TM therapy for the cure of many cases of human cancer, in which the remaining cancer is micrometastatic, or the disease can be brought to NED status, with only micrometastatic disease remaining. It would appear these patients can often be cured by TM. Unfortunately, the early human trials were all against bulky, advanced cancer, and the minimal efficacy caused pharmaceutical companies to turn away. That is extremely unfor- tunate because the promise of TM to cure many cancers is very real.
There is also great promise that many diseases in addition to cancer can be treated by copper-lowering therapy. These include diseases of fibrosis, inflammation, and autoimmunity. Discussing fibrosis first, there are many serious, often fatal, diseases of fibrosis such as liver cirrhosis, idiopathic pulmonary fibrosis, fibrotic disease of the kidneys, and others. Fibrosis results from pathological activation of the cytokines of the fibrotic pathway, which operate physiologically for wound heal- ing, as an example. This pathway involves activation of transforming growth factor-β (TGF-β), which activates connective tissue growth factor (CTGF), which turns on many genes involved in fibrosis, such as collagen genes. Many steps in this pathway including activators of TGF-β, and perhaps CTGF itself, are dependent on copper.
The bleomycin mouse model of pulmonary fibrosis is a reasonably good model of human idiopathic pulmonary fibro- sis. Bleomycin is injected via the trachea of mice. In a study using TM versus placebo, TM strongly prevented the fibrosis (Brewer et al., 2003b) and prevented the large increase in TGF-β and an inflammatory cytokine, tumor necrosis factor-α (TNF-α; Brewer et al., 2004). TM also was very effective in preventing cirrhosis in mice from carbon tetrachloride injec- tion (Askari et al., 2004). In addition, TM prevented cirrhosis from bile duct ligation in another mouse model of cirrhosis (Song et al., 2008). TM also speeded up recovery from carbon-tetrachloride–induced cirrhosis in mice offering hope for treatment of human cirrhosis (Hou et al., 2009). The concept is that the fibrotic process and the repair process are going on simultaneously, and TM inhibits the fibrotic process, allowing recovery to go on much faster.
In summary, from the mouse work it appears that TM therapy offers promise of substantial benefit in human fibrotic conditions. There are no known treatments for fibrotic diseases. In particular, cirrhosis is very common—from alcoholic liver disease, hepatitis C, primary biliary cirrhosis, and others—and there are no treatments that offer recovery from any of these.
Excess inflammation is part of many disease processes, including fibrotic and autoimmune diseases. The inflammatory process begins with some type of tissue injury, the accumulation of inflammatory cells at the site, and the release of excess damaging inflammatory cytokines, such as TNF-α, interleukin (IL)-1β, IL-2, and nuclear factor kappa B, a transcription factor that causes release of inflammatory cytokines, which can further damage tissue cells. A mouse model of immune- modulated tissue damage is injection of concanavilin A (Con A) into mice. The Con A binds to liver cells, which causes an immune attack on the liver cells. The tissue damage can be measured by the release of the transaminase enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), from the damaged liver. TM strongly prevents the release of AST and ALT after Con A injection in mice and inhibits the usual elevation of TNF-α in the blood (Askari et al., 2004).
Another example of tissue damage from excess inflammation is cardiac damage from the chemotherapeutic drug, doxo- rubicin, widely used as a treatment of cancer, but with a side effect of heart damage. A toxic dose of doxorubicin in mice causes release of the enzymes lactic dehydrogenase (LDH), creatinine kinase (CK), and troponin I into the blood from the damaged heart. TM therapy was able to prevent heart damage as measured by preventing the release of LDH, CK, and tro- ponin I into the blood and preventing the marked increases in the blood of TNF-α, IL-1β, and IL-2, an indicator of cytotoxic T cell activation (Hou et al., 2005).
Another example of tissue damage from excess inflammation is the liver damage produced by excess doses of acetamin- ophen (ACAP), also known as Tylenol. Overdoses of ACAP, accidental or with suicide intent, are the most common cause of acute liver failure seen in the emergency room. TM therapy was able to almost completely prevent liver damage from a toxic dose of ACAP in the mouse with a very strong inhibition of AST, ALT, and IL-1β appearance in the blood (Ma et al., 2004). Furthermore, it was shown that TM could be given by injection some time after ACAP injection and still prevent much of the liver damage, indicating that TM might be effective in the emergency room for ACAP poisoning.
There are multiple examples of TM therapy in mouse models greatly mitigating immune-modulated disease, model- ing human autoimmune diseases. The Con A immune-modulated model for hepatitis was discussed earlier (Askari et al., 2004). There is also an immune-modulated arthritis mouse model involving the injection of bovine collagen II. TM therapy greatly inhibited swelling and redness of joints, and it markedly reduced histologic abnormalities (McCubbin et al., 2006), suggesting that TM should be evaluated in the very common human autoimmune disease, rheumatoid arthritis. TM also
markedly reduced the great increase in TNF-α, IL-1β, and IL-2 seen in controls. TM also markedly protected against the great increase in urinary isoprostanes (a marker of oxidant damage) seen in controls.
Multiple sclerosis (MS) is a human autoimmune disease involving an immune attack on myelin of the central nervous system. A mouse model of MS involves injection of a protein, myelin proteolipid protein, which causes clinical symptoms and lesions in the mouse brain and spinal cord that can be counted and graded. TM therapy strongly prevented clinical symptoms; reduced the number of central nervous system lesions; inhibited elevated IL-2, TNF-α, and interferon-γ; and prevented the marked increase in urine isoprostane levels seen in controls (Hou et al., 2008).
Summarizing, it appears that TM therapy has great promise in the treatment of human diseases of excess inflamma- tion and autoimmunity. Some of these diseases are very common, and treatments currently available are far from optimal.
Symptomatic treatments include aspirin and nonsteroidal antiinflammatory drugs, which can help alleviate pain but do not inhibit inflammation and the resulting tissue damage, at least not very much. Corticosteroids are the best treatment, but they usually are only partially effective and have a long list of serious side effects, particularly if used long term. In general, it appears that anything steroids can do, TM can do better and without all of the side effects.
Given how promising for human medicine TM would appear to be based on mouse models of fibrosis, inflammation, and autoimmunity, it is surprising that only one human clinical trial has been conducted. This was a double-blind clinical trial in primary biliary cirrhosis (Askari et al., 2010). This disease involves all three causation elements under discussion. It is an autoimmune attack on the bile ducts, which leads to inflammation and fibrosis (cirrhosis). The study was designed to be a 2-year double-blind trial of TM versus placebo. In TM patients the approach was similar to that used in the cancer trials to keep the Cp between 8 and 15. Endpoints were improved blood levels of AST, ALT, and TNF-α in TM-treated patients versus controls. The study ended prematurely because of a cutoff in funding. Nevertheless, 13 TM patients and 15 controls were followed for an average of 13 months. Endpoints were reached in that AST, ALT, and TNF-α levels were significantly improved in TM-treated patients versus controls (Askari et al., 2010). There was a trend in a favorable direction for bilirubin levels that did not reach statistical significance. Serum-free copper was also significantly reduced by TM.
In summary of this section, there is great promise in TM use for curing cancer if the remaining disease is micrometa- static, or it can be reduced to micrometestatic, NED, status. There are a plethora of mouse studies that indicate TM should be evaluated in human fibrotic, inflammatory, and autoimmune diseases. There is even one positive human trial in such a disease, primary biliary cirrhosis. As the title of this section indicates, there is much promise of copper-lowering therapy with TM in cancer and other diseases.