Antiangiogenesis: A Role for Natural Therapies
Stephen Holt, MD, LLD(Hon.) ChB., PhD, ND, FRCP (C)
MRCP (UK), FACP, FACG, FACN, FACAM, OSJ
Angiogenesis is a major pathophysiological component of cancer, arthritis, proliferative vascular disease and common skin disease. The increased understanding of the complex cascade of events in angiogenesis has heralded research on inhibitors or modulators of new blood vessel growth. Several drugs or derivatives of natural substances are in clinical trial as angiogenic promoters or inhibitors to combat cancer, cardiovascular and skin disorders. Cartilage and its extracts have been studied in detail and presented with both scientific excellence and unfortunate hype. Other natural substances that modulate angiogenesis include various animal or marine biologicals, the soy isoflavone genistein, colostrum and extracts of fungi and plants. “Natural” antiangiogenesis has great promise in veterinary practice.
The phenomenon of angiogenesis has attracted considerable interest in the scientific community. Angiogenesis play a major role as a determining factor in a variety of diseases, including: cancer, arthritis, skin conditions, eye disorders, and inflammatory disease. The application of methods to manipulate angiogenesis has created fascinating therapeutic options, including the potential use of natural-based compounds that may modify new blood vessel growth.
Angiogenesis and Disease
Angiogenesis can best be defined by dissecting the world to its Greek roots: Angio- and genesis. Angio is Greek for “a vessel, usually a blood vessel” and genesis means “to originate or create.” Angiogenesis is thus the growth of new blood vessel, a process that can happen in normal or disease-state circumstances in the tissues of the body. This process is also called neovascularization (neo means “new”). The phenomenon of angiogenesis has attracted considerable interest in the scientific community. In the normal adult, angiogenesis occurs infrequently. Exceptions are found in the female reproductive system, where it occurs during the development of follicles during ovulation and in the placenta after pregnancy. These periods of angiogenesis are relatively brief and tightly controlled with regard to the extent of new vessels. Angiogenesis is a multi-step event in which endothelial cells, those that form the walls of small blood vessels called capillaries, migrate and proliferate. The capillary formation is triggered by several agents thought to be released largely from tissues near proliferating capillaries. Substances made in the body, called fibroblast growth factors, and other molecules, have the ability to induce all the steps necessary for angiogenesis.
Normal angiogenesis also occurs as part of the body’s repair processes, that is, in the healing of wounds and fractures. Angiogenesis also plays a major role in a variety of diseases, including: cancer (the growth of solid tumors), arthritis, artherosclerosis, skin condition, eye disorders, and inflammatory disease. In these cases, it can often be an unwanted and detrimental phenomenon. By adding the word inhibitors to angiogenesis, we have a descriptive phrase for compounds which prevent or reduce the growth of new blood vessels. The application of methods to manipulate angiogenesis has created fascinating therapeutic options, including the potential of natural-based compounds that may modify new blood vessel growth.
Neovascularization of developing, repairing, or neoplastic tissues is regulated, at least partially, by a family of proteins which can be extracted from certain avascular tissue, such as cartilage. These extractable proteins are known by a variety of terms, such as anti-invasion factors (AIV). They act as local regulators for some of the major mechanisms by which endothelial cells are thought to invade tissues during neovascularization.
The process of forming new blood vessels is essential to tissue repair, ulcer healing, ovulation, and menstruation. Vascularization plays a major role in the propagation of several disease states. Judah Folkman, M.D., of Children’s Hospital, Boston, and Harvard Medical School, is credited with the discovery of importance of angiogenesis in tumor development. His research has offered a unique and promising basis for the therapeutic application of antiangiogenic and proangiogenic compounds. Some natural compounds, such as shark cartilage and soy isoflavones, may modulate angiogenesis in vivo. Whilst the effects of these natural compounds in intact animals or humans has not been explored extensively, such effects are apparent in vitro (in laboratory preparations).
The importance of angiogenesis in the promotion of cancer growth has fueled a considerable amount of research into the control and mechanism of angiogenesis. The growth of most solid tumors depends on the development of a tumor circulation, so it seems logical to inhibit this vascularization, thereby causing the death of neoplasia or limitation of tumor expansion. Many substances are now recognized as exerting a modulating effect on angiogenesis. The initial crude extract of angiogenic factors isolated from neoplasia by Dr. Folkman where referred to as tumor angiogenesis factors (TAFs). A flurry of research has identified inhibitors of TAF and assisted in the characterization of many agents and cofactors that are required for the modulation of angiogenesis.
The simplest way to explain angiogenesis is to consider a four-step process: (1) the localized erosion of the basement membrane in tissues; (2) the migration of activated endothelial cells promoted by angiogenic factors; (3) endothelial cell proliferation; (4) a complex combination of sustaining influences on the angiogenic process. Angiogenic factors and antiangiogenic compounds may play a role in one or more of these four steps.
The complex steps in the process of angiogenesis and its control provide a multitude of sites for the potential application of antiangiogenic or proangiogenic therapy. The large, even increasing, number of identified angiogenic factors makes it unlikely that one discrete, antiangiogenic molecule can be used as a successful treatment. This reinforces the use of potentially more versatile antiangiogenic agents that may have multiple sites of activity. These agents may be used either alone or in combination with other antiangiogenic compounds of natural origin. Recent research shows that combinations of antiangiogenic therapies with other anticancer therapies such as immunotherapy or chemotherapy may be a way of amplifying the killing of cancer cells. Shark cartilage, isoflavones of soyabean origin and other natural agents are candidates for much further investigation as antiangiogenic agents in humans and animals.
Factors Controlling Angiogenesis
New blood vessel growth is normally kept under tight control. This control is exerted by a highly intricate and coordinated production of molecules that both promote and suppress angiogenesis. In brief, the control of angiogenesis is orchestrated by different cell types, molecules or soluble mediators and natural compounds within the supporting matrix of connective tissue.
Certain cytokines, growth factors, matrix proteins and other mediators promote angiogenesis whereas others are angiostatic (Table 1). Angiostatic agents that are involved in the control of new blood vessel growth include thrombospondin (a matrix protein), retinoids, tissue inhibitors of metalloproteinase platelet factor 4 and selected growth factors (Table 1).
We have learned a great deal about the complexity of the cascade of events during new blood vessel formation. To summarize, there is activation of endothelial cells, basement membrane rupture, complex processes of cellular adhesion, cellular migration and proliferation which results in the sprouting of new blood vessels from existing vasculature. Of particular importance is the synthesis of basement membranes which are an essential component of blood vessels. This process is associated with an increase in collagen type IV. The role of basement synthesis in angiogenesis is so important that it has become a key focus for the development of drugs that can alter this process and suppress angiogenesis.
The body has a natural balance of angiosuppressing and angiopromoting factors. This balance is lost in the presence of unwanted angiogenesis that occurs in a variety of disease states, especially cancer, arthritis and proliferative vascular disorders. It is important to recognize the scope of abnormal angiogenesis in the causation of a wide variety of diseases including, but not limited to: vascular disease, ischemic heart disease, atherosclerosis, wound healing, chronic inflammatory ulcers, maturing burns, peptic ulcer disease, rheumatoid arthritis, gingivitis, psoriasis, chronic variants of eczema, acne rosacea, cancer and metastasis and ocular neovascularization diseases, such as diabetic retinopathy, age related macular degeneration and neovascular glaucoma.
In the mid 1990’s, shark cartilage became the most popular unconventional cancer treatment since the Laetrile controversy of the 1970s. This enthusiasm was manifested by premature reports of the beneficial effects of shark cartilage in cancer therapy (I. William Lane, Ph.D., and Linda Comac, R.N., Sharks Don’t Get Cancer, Avery Publishing Group, 1992). Following major controversies and disciplinary actions by the FTC and the FDA against the illegal act of promoting the sale of shark cartilage as a cancer therapy, interest in shark cartilage as a “general” nutraceutical has waned inappropriately. The commercial organizations that have touted the cancer claims have turned out to be the biggest enemy against the appropriate use and future research of shark cartilage. However, scientifically appropriate studies are under way to investigate the potential safety and efficacy of cartilage for the treatment of cancer and other chronic diseases that depend on angiogenesis. Controlled clinical trials of extracts of shark cartilage for cancer therapy are underway currently at MD Anderson Cancer Center, The Cleveland Clinic, The Mayo Clinic and other centers, sponsored and overviewed by the National Institutes of Health. Commercial companies (Aeterna Laboratoires, Canada and BioTherapies Inc., Fairfield, NJ, USA) are heavily engaged in further research on the use of shark cartilage and its extracts in topical and systemic therapy.
Illogical projections, scientific naivete, and commercial interests in shark cartilage have led to an antagonistic division between basic scientists pursuing the mechanisms of angiogenesis and individuals who are enthusiastic about the clinical uses of natural products with angiogenic properties. The danger of the previous hype about shark cartilage is that the potential benefits of cartilage and other natural based antiangiogenic compounds in certain diseases may be either minimized or overemphasized. In the author’s opinion, research on the use of natural-based antiangiogenic compounds is long overdue, poorly funded, and probably forgotten because of the difficulties in protecting nonproprietary treatments. The manufacturers of nutraceuticals with antiangiogenic potential have an obligation to fund such research, especially if products are going to be promoted or used for assumed antiangiogenic properties. BioTherapies Inc. of Fairfield, NJ has made this commitments in the USA.
Historical Perspective: Cartilage and Antiangiogenesis
To understand the development of the prevailing theory of cartilage therapy, one needs to put angiogenesis into historical perspective. The term angiogenesis was coined approximately 60 years ago. In the 1960s and 1970s, experimental animal models were designed to study tumor growth and the importance of neovascularization as a rate-limiting step in tumor growth was identified.
Several articles in the lay press have traced the history of the early rejection of Dr. Folkman’s theories of angiogenesis by the scientific community and their current acceptance, together with an account of his “vindication”. A writer for the Boston Globe newspaper (1997) states: “Dr. Judah Folkman’s work illustrates the slow pace of progress in cancer research but his visionary ideas have led to new ways of understanding this pernicious disease and to renewed hope that it can be vanished”.
The emergence of interest in shark cartilage as a source of angiogenic inhibitors came out of the observations of Drs. Lee and Langer. They discovered a substance in bovine cartilage with potent angiogenic properties, but recognized that cartilage is present in only relatively small quantities in mammals. Drs. Lee and Langer noted that the shark’s skeleton was composed entirely of cartilage and focus sed on the knowledge that cartilage does not have a network of blood vessels. There are several protein fractions in shark cartilage that prevent angiogenesis. Crude extracts of shark cartilage have been shown to strongly inhibit tumor-induced neovascularization whereas bovine cartilage and other cartilage has to be highly purified by chromatography before angiogenic activity became apparent. Recently, patients have been filed on various fractions of the protein content of shark cartilage for their inhibitory effects on angiogenesis. Drs. Lee and Langer (1983) estimated that sharks may contain about 100,000 times more potential antiangiogenic activity per animal that cattle. These observations are some of the compelling reasons to favor shark over bovine or chicken cartilage as a potential natural source of inhibitors of vascularization.
In addition, shark cartilage appears to be nontoxic, even in very large doses. Over many years of research and thousands of human doses, no significant metabolic toxicity has been reported from using shark cartilage (in the form of Cartilade®), that can be ascribed to administering the compound. When administered orally or rectally, it has shown no evidence of local or systemic reactions in several clinical trials. The only possible problem is excessive calcium intake when very large doses of cartilage are used, of the order of 1gm/kg or greater. The author does not believe that cartilage compounds can be administered rectally as a reliable route to access the systemic circulation. Injection of crude cartilage in humans presents an antigenic load and is not to be recommended; even though this approach was taken by John Prudden, M.D. of Columbia University, New York with the application of bovine cartilage to disease treatment (arthritis, inflammatory bowel disease, wound healing and cancer).
Use of Antiangiogenic Therapy in Cancer
The rationale for antiangiogenic therapy in neoplastic disease rests upon the hypothesis that tumor growth and metastatic dispersion of malignant disease are angiogenic-dependent processes. Considerable indirect and direct evidence has accumulated during the past two decades to support this hypothesis and confirm the angiogenic dependence of neoplasia. The onset of angiogenic activity appears to occur as a definable event in tumor formation, and most tumors progress from a prevascular to a vascular stage.
A promising level of antitumor activity of solid dosage formats of shark cartilage has been demonstrated in two small clinical trials, one conduced in Mexico on eight patients and another in Cuba involving about forty patients (Jose Menendez, M.D., and J. Fernandez-Britto, M.D., personal communication, 1994). The Cuban study demonstrated that shark cartilage induced histologic changes in tumors with notable changes in vascular pattern, and these changes were not considered to be explained by chance alone. The author had the opportunity to review the histologic slides of tumors that were obtained from shark cartilage-treated patients in the Cuban trial. The author feels strongly that the observations of fibrous encapsulation of the tumors and evidence of cell death within the tumors are extremely interesting. These observations are significant and need further study.
James Lott, Ph.D., Professor of Physiology and Biophysics at North Texas State University, Denton, has performed experiments in mice bearing transplanted tumors (data presented at the First International Congress on Alternative and Complementary Medicine, May 1995). After administering shark cartilage, the tumor-bearing mice lived longer and Dr. Lott found definite histologic changes in transplanted tumors that resemble some of the histologic changes observed in tumor specimens examined in the Cuban clinical trial. However, the author has been unable to substantiate any conclusive clinical outcome concerning the benefit of shark cartilage therapy in the Cuban or Mexican studies and Dr. Lott’s studies had protocol problems. Any contemporary claims about the benefit of solid dose shark cartilage in the treatment of cancer must be considered to be still somewhat speculative.
The data from these studies, although subject to contention, had provided the rationale for further human testing in patients with advanced malignancies. Charles Simone, M.D., of the Simone Protective Cancer Institute in New Jersey, received a recommendation from the Office of Alternative Medicine of the National Institutes of Health in the mid 1990’s to conduct clinical trials using shark cartilage in advanced cancer, but these studies were placed on hold and Dr. Simone has never published his results in detail. Dr. Simone, however, reported favorable effects of shark cartilage cancer at several meetings. Unfortunately, Dr. Simone’s studies may confuse the assessment of shark cartilage as a cancer therapy because his protocol added a ten-point “Anticancer Lifestyle Program” that could have confounded the results. One of the alleged benefits of shark cartilage therapy has been the suggestion that this therapy improves quality of life measures in cancer patients. Therefore, clinical outcomes in studies of shark cartilage need to be clearly identified in clinical protocols. Protocols should not be constructed that confuse clinical outcome variables. This is one of the most important limitations of research in the field of alternative and complementary medicine.
Shark Cartilage and Cancer: The Odds Were Stacked Against a Favorable Outcome?
The most significant finding to date in relation to the treatment of cancer with shark cartilage has been reported by researchers at the Cancer Treatment Research Foundation (in cooperation with the Cancer Treatment Centers of America and BioTherapies Inc., owners of Cartilage Technologies). This study attempted to assess the safety and efficacy of shark cartilage in the treatment of advanced cancer of various types (breast, colon, lung, and prostate) in more than fifty patients. Dr. Dennis Miller presented the results of the trial at the Thirty-third Annual Meeting of the American Society of Clinical Oncology in Denver in May 1997. Overall, shark cartilage exerted no measurable benefit in the treatment of cancer as measured by lack of a reduction in tumor size or significant improvement in measures of quality of life in the recipients of the shark cartilage therapy. Two patients, however, showed improvement in quality of life as measured by a quantitative scale. Disease stabilization was noted in one in five patients but the trial supervisor, Dr. Miller, was guarded in his interpretation of the significance of this potentially beneficial effect.
The author believes that Dr. Miller, was too guarded in his conclusions and his colleagues could have seriously underestimated the significance of the one in five (20%) disease stabilization in terminal cancer patients that had failed all previous therapies. A further Phase II trial of shark cartilage in cancer was conducted by Dr. Rothkopf and his colleagues at St. Barnabas Hospital in New Jersey which also failed to show significant benefit.. All the patients in the Miller study had advanced cancer and were terminal and it could be argued, strongly, that the protocol was not fair appraisal of the treatment. The odds were stacked against showing “degrees” of benefit. In addition, the effect of pretreatment on the outcome of shark cartilage therapy could not be assessed. It should be noted that in all trials to date, shark cartilage has been used only in patients who have failed all acceptable cancer therapy and in some who have subsequently failed other alternative medical options. Is not any benefit, no matter how small, significant in these studies?
Although at first sight the results of both of these controlled, open-label observations of shark cartilage as a cancer cure are disappointing, the authors believe that the observations of disease stabilization in the Miller study are worthy of careful scrutiny and follow-up. Shark cartilage should not be dismissed as a promising potential cancer therapy; in the authors’ opinions, it should be subjected to much further research. Bear in mind, sold dose shark cartilage was used and not the biologically active fractions of shark that have been isolated by BioTherapies inc. Fortunately, some serious scientists have picked up the gauntlet. Aeterna Laboratoires (Quebec, Canada) are actively researching hydrosoluble (watersoluble) fractions of shark cartilage as cancer therapy with some early but very promising results. Aeterna Laboratoires market a “liquid extract” of shark cartilage called Car-T-Cell but it is not certain what use they sell this product for although they claim it is anti-angiogenic. BioTherapies Inc. has liposome coated, antiangiogenic, hydrosoluble, fractions of shark cartilage in Cartilade L.E.D.
Negative Clinical Trials With Shark Cartilage for Cancer
In brief, the researchers reached an unduly negative conclusion in the face of reporting disease stabilization in approximately one in five patients. There is no question whatsoever that the Miller Study (Cancer Treatment Centers of America Inc.) and the Leitner Study (St. Barnabas Hospital) cannot be considered a fair appraisal of the ability (or lack thereof) of shark cartilage to benefit cancer patients. Added to this picture are the negative results of very limited studies of shark cartilage therapy, using solid dosing, performed by Dr. Rosenbluth and his colleagues at Hackensack University Medical Center in New Jersey.
New studies are underway using both solid dose shark material and hydorsoluble extracts of shark in larger numbers of patients. One problem is the number of patients required in a prospective study in order to evaluate outcome. Despite the continuing controversy there are a significant number of commercial companies, scientists, patients and practicing physicians who strongly support the promise of shark cartilage for cancer and other disease treatment.
Further FDA “approved”, Phase II Studies of Shark Cartilage
Impressed sufficiently with early promising data on the use of solid dosage forms of shark cartilage in cancer therapy, the FDA awarded a phase II study approval for research on cartilage and cancer to continue. Researchers at St. Barnabas Medical Center in New Jersey reported the outcome of this abandoned study as not showing benefit in twenty patients with breast cancer and twelve with prostate cancer. This study was terminated, perhaps ill-advisedly, because of the negative outcome of the Miller Study, as well as concerns about the ability of the protocol to adequately answer the scientific questions that were posed.
Clinical Trials of Other Antiangiogenic Modalities
Increasing knowledge of the factors that “switch on” angiogenesis and the upregulation of positive stimulators has led to several phase I and phase II clinical trials of a variety of antiangiogenic therapies besides cartilage. Trials of the potent antiangiogenic compound pentosan polysulfate have shown some benefit. This is one of a group of polysaccarides that are capable of interfering with the function of heparin-binding growth factors that promote angiogenesis. Other antiangiogenic compounds in clinical trials include alpha interferon, platelet factor IV, and AGM 1470. At least twenty angiogenic agents are in early clinical trials, with some impressions of benefit emerging from these pilot studies.
Not enough attention has been focused on the modulators of angiogenesis from natural sources. Several such compounds have been discovered in addition to those in cartilage. One of the potential angiogenesis inhibitors isolated from cartilage is a collagenase inhibitor. Other natural antiangiogenics include vitamin D3-analogues, fumigallin, herbimycin A, and soy isoflavones.
Isoflavones found in soya beans are very exciting compounds. They have direct tumoricidal properties against several tumor types and regulate key enzyme expression, that is, a process involved in tumor growth. Isoflavones are also antioxidants and play a role in apoptosis.
The principal soy isoflavones genistein, daidzein and glycetein are very interesting polyphenolic phytochemicals with versatile and potential biological effects. Most research has focused on the properties of genistein which is known to induce significant antiangiogenic effects in animals. Less well known, is work from researchers at Yale University that has shown the isoflavones to exert antiangiogenic effects in humans.
Joshua R. Korzenik, M.D., in colaboration with Stephen Barnes, Ph.D., have preliminary results on the use of soy protein containing isoflavones in the treatment of an uncommon condition called hereditary hemorrhagic telangiectasia. This disease runs in families and is characterized by the occurrence of nosebleeds, hemorrhage from the gastrointestinal tract, and the variable occurrence of migraine headache. Individuals with this disorder bleed from the clumps of dilated blood vessels (telangiectasis) that can occur in the mouth, nose, and gut.
This study is an ideal human model in which to study the antiangiogenic effects of isoflavones. Eight of nine patients who took soy protein containing isoflavoens in their diet had variable but positive responses to soy. Six of the patients had nose bleeds, and three of these individuals had complete – or near complete – cessation of nasal hemorrhages. One of three of the people with hereditary hemorrhagic telangiectasia had a marked positive response to be soy in the diet, associated with a diminished need for blood transfusion and a partial correction of their severe anemia. It is notable that four patients with migraine had relief of their headaches. Unfortunately, the cause of the headaches in this disease is not completely understood, but this observation may justify the study of isoflavones as an adjunct to the management of the very common and distressing migraine disorder.
The importance of these preliminary observations rests in the fact that soy protein with isoflavoes seems to be causing measurable antiangiogenic effects in humans. These are truly exciting observations, and the angio-modulating effects of soy isoflavones in wound healing and tissue repair deserve more study.
However, a word of caution is required concerning the use of isoflavones in veterinary practice. Evidence exists that several species of animal, notably felines and some birds (parrots) experience toxicity from chronic isoflavone ingestion. Studies on captive cheetahs in North America zoos reveals sporadic death from soy and isoflavone intake, due to liver failure. Death has also been recorded in parrots. There appears to be great interindividual tolerance for soy isoflavones among certain animal species – a phenomenon not shared by humans.
The Holt Hypothesis
“The author has” taken it on the chin for issuing press releases that shark cartilage in solid dosing is not to be perceived as being of any consistent and reliable benefit for cancer therapy, given the current status of our knowledge. However, I still believe, in an impervious manner, that shark cartilage contains substances that are antiangiogenic that can be used in cancer or other angiogenic dependent diseases with further knowledge about their characterization and use.
Having been involved in shark cartilage research for a decade, I am impressed by the reports of prolonged survival or stabilization of some cancer patients in anecdotal observations, but anecdotes do not satisfy our necessary burden of proof. I am certain that there is a response in some cancer patients in anecdotal observations, but anecdotes do not satisfy this burden of proof. Although I believe that there is a response in some patients (and so are others) the response is inconsistent, infrequent and unreliable with the use of solid dosage formats of shark cartilage. If I am correct, how are these phenomena explained ?
I believe that the answer rests in part in the issue of intestinal permeability and systemic bioavailability of the demonstrated antiangiogenic fractions (proteins) that are found in shark cartilage. Enhanced intestinal mucosal permeability (“leaky gut”) is associated with a variety of disease including intestinal mucosal disease, arthritis (rheumatoid disease), psoriasis and, of course, cancer. However, the presence of significant enhanced gut permeability is highly variable – about as variable as “responder” with an angiogenic dependent disease is to solid dose shark cartilage therapy! Bear in mind, that enhanced intestinal mucosal permeability (leaky gut) tends often to be associated with angiogenic dependent disease – perhaps this is more than a coincidence ?
I postulate that the variable response of individuals with angiogenic dependent diseases to shark cartilage administration is due to the variable access that antiangiogenic protein may have to the systemic circulation. There are several proteins in hydrosoluble extracts of shark cartilage with variable molecular weights (sizes), some of which may gain access through the gut wall into the body but many of which may not. The human gut is impervious to molecules much greater than 50,000 Daltons in size and some of the antiangiogenic proteins in shark exceed this size (molecular weight). Thus, I propose the following for consideration.
1) The individuals who have stabilized or improved the status of their angiogenic dependent disease with shark cartilage may be those who have a leaky gut that lets in macromolecules or fractions of antiangiogenic protein.
2) Systemic access of the antiangiogenic components of shark cartilage is the rate limiting factor in achieving any benefit in angiogenic dependent disease.
3) Factors or formulations that enhance the access of antiangiogenic protein fractions of shark cartilage may be valuable e.g. liposomes, but immunological consequences are unknown. .
I have isolated hydrosoluble fractions of shark cartilage (and so have others) that have demonstrable in vitro antiangiogenic activity in both CAM assays (chorio-allantoic membrance of the chick embryo) and bovine endothelial cell proliferation assays. These hydrosoluble extracts have been encapsulated in liposomes for delivery. The liposome is created from an essential fatty acid to create in “microbubble” that may partially protect the proteins in the hydrosoluble fractions of shark cartilage from acid hydrolysis or enzymatic digestion in the gut. In addition liposomes can be shown to cross epithelial barriers efficiently as “carriers”. Whilst complete bioavailability cannot be assured with liposome encapsulated hydrosoluble extracts of shark cartilage – facilitated absorption may occur. These issues are waiting to be tested before firm conclusions can be drawn. There are obvious drawbacks because of the variable efficiency of liposome transfer in the gut, but this approach is novel and exciting.
Hydrosoluble extracts of shark cartilage with antiangiogenic activity are available strictly as dietary supplements only from BioTherapies Inc, Fairfield, NJ in the form of Cartilade L.E.D. where L denotes liposome, E denotes extract and D denotes delivery. Dietary supplements by law cannot be used to prevent or treat disease – an enigma.
Other Antiangiogenesis Research
Interest in being shown in the use of vascular targeting agents which can bind specifically to vascular endothelial cells (e.g. monoclonal antibodies). These target agents can be linked chemically with different types of antiangiogenic agents or effector molecules such as coagulants, anticoagulants, drugs, toxins or radioisotopes. This approach is an extension of disciplines such as radioimmunotherapy or photoimmunotherapy and other means for the selective localization of disease. These approaches have afforded great promise since the 1980’s, but they have swallowed up large amounts of research dollars without great success.
One examples of vascular targeting technology is the use of combretastatin A4 prodrug (CA4P) which acts to reduce the blood supply to tissues through a tubulin binding mechanism. It is believed that CA4P is capable of attacking pre-existent tumor vasculature, unlike other antiangiogenic agents. This research is licensed to Bistol-Meyers Squibb of Princeton, New Jersey.
The other approaches to antiangiogenesis involve the use of hydrosoluble fractions of shark cartilage. The lead use patent 5,075,112 and its recent applied for extensions are owned by Dr. Stephen Holt, M.D. of BioTherapies Inc., Fairfield, NJ. This technology is combined with a proprietary liposome delivery system in the form of the research product and dietary supplement Cartilade® L.E.D. Following on from this original use patent are developments of hydrosoluble shark cartilage extracts by Aeterna Laboratories Inc. (Quebec, Canada).
Whilst the original use patent 5,075,112, owned by Stephen Holt, M.D. was filed with solid dose cartilage, all of the in vitro development work recorded in the patent was performed using hydrosoluble fractions. Aeterna has manufacturing patents on compound AE-941 (Neovastat), a shark cartilage extract. Cartilade L.E.D (BioTherapies Inc.) is a shark cartilage extract which uses the advantage of liposome delivery. Sequential patients on shark cartilage extracts 5,618,925 and 5,985,839 and 6,025,334 and 6,028,118 on topical use and other areas are owned by Aeterna Laboratories Inc., Canada.
Aeterna has moved to phase III clinical trials of AE-941 (Neovastat) in patients with lung cancer and progressive renal cancer – conditions for which Cartilade L.E.D. is potentially useful is research. The proposed, but yet to be completely defined, actions of Neovastat (extract of shark cartilage) includes both the inhibition of matrix metalloproteinases and interaction with vascular endothelial growth factor receptors, which are centrally involved in new blood vessel budding and growth.
An interesting approach, again using vascular targeting, is the attachment of “killing agents” to vascular targeting molecules. The early promise of this approach rests with the possibility of the local induction of thrombosis in new blood vessel to cause occlusions and eradicate their benefits to cancer. A number of coagulants can be targeted to blood vessel.
EntreMed Inc. of Rockville, Maryland has developed Angiostatin and Endostatin for potential cancer therapy and therapy of atherosclerosis respectively. In contrast, Boston Life Sciences, is further developing the antiangiogenic compound Troponin 1. Troponin 1 can exert angioinhibitory effects by interacting with basic fibroblast growth factor (bFGF) receptors and it may inhibit VEGF by a similar mechanism. Fragments of Troponin 1 have been isolated that account for the principal antiangiogenic actions of the complex molecules.
Several genes control angiogenesis and some scientists have focused attention on VEGF – 145 gene – which is now patented. This gene can potentially induce angiogenesis and blood supply to tissues that are starved of blood as a consequence of coronary heart disease or peripheral vascular disease. The matter of genes and angiogenesis is extraordinarily complex. Viral agents (e.g. adenovirus subtype) have been produced in a recombinant manner to express genes that cause favorable treatment effects (proangiogenesis) in animal models of coronary heart disease (FGF-4).
Perspectives on Angiogenesis Research
The use of agents that modulate angiogenesis marries the nutraceutical, biotechnology and pharmaceutical industries – even though many representatives from these branches of medicine would deny this vehemently! I stress the term modulation and introduce the advantage of natural antiangiogenesis inhibition as very attractive because it is often more gentle and perhaps sometimes safer than using “drugs” or “synthetics” with very potent actions (all or none effects). Potency of angiogenesis inhibition has become a problem with some antiangiogenic agents which are so powerful that they may precipitate unwanted cardiovascular events. After all, as much as biotechnology dreams about the selective localization of therapeutic agents to diseased tissue, non selective actions of chemotherapy, radiation, drugs and even “targeted” agents have a history of progress that has been plagued.
For these reasons, I see some of the most important advances in angiogenesis research in the field of natural therapies. Farsighted companies have stuck with natural approaches and shark cartilage still stands out, as likely to fulfill its promise, even though “marketing scoundrels” have inflated the anticancer promise of shark cartilage prematurely.
Arthritis and Angiogenesis
Various types of arthritis may be amenable to therapy with antiangiogenic compounds. Shark cartilage has been well recognized and it has been characterized as having a major potential application in the treatment of pain and inflammation associated with arthritis in animal studies. John Prudden, M.D., and colleagues at the Columbia-Presbyterian Medical Center administered bovine cartilage to humans by both mouth and injection to treat osteoarthritis, rheumatoid arthritis, psoriasis, and regional enteritis with alleged significant therapeutic benefit. However, the group did not subscribe to the notion that the observed beneficial effects were caused by antiangiogenic activity. Several other researchers have reported a clear association between neovascularization and osteoarthritis, adding weight to the rationale to use cartilage and other antiangiogenic compounds to treat arthritis. Shark cartilage is also a rich source of calcium, which is beneficial for patients with osteoporosis who may require calcium supplementation. It has other values added components.
“Value Added” Components of Shark Cartilage
Shark cartilage in the form of Cartilade® has been used by hundreds of thousands of people with bone and joint problems. Studies have been aimed at assessing the safety and efficiency of Cartilade for the management of arthritis in animals and humans. The administration of oral Cartilade or Cartivet (dogs and cats) Cartequine (horses) manufactured by BioTherapies Inc., Fairfield, NY, to seven humans Table 2 and seven horses (respectively) Table 3 with osteoarthritis or traumatic arthritis in open label, pilot clinical studies showed a beneficial overall effect. Cartilade was well tolerated without adverse effect. Numerical scores of disability and clinical signs of arthritis showed statistically significant improvement with Cartilade therapy in patients for whom conventional medical treatment had generally failed. The findings, together with other observations in the literature, dictated the need for further prospective controlled clinical trials of Cartilade as promising therapy for arthritis in humans and animals.
Considerable interest has focused on the use of cartilage for the treatment of arthritis in animals and man. The rationale for the use of shark cartilage as therapy for arthritis and osteoporosis relates to both its biological effects and general nutrient composition. Cartilage has a putative effect on angiogenesis in vivo. It is composed of calcium, phosphorous, chondroitin sulfate and other elements that are required to maintain optimal skeletal health. While the in vitro demonstration of an antiangiogenic affect of cartilage is clear, questions remain about a consistent demonstration of an in vivo effect.
Animal Trials: Shark Cartilage (Cartilade®) in Joint Problems
To determine the tolerability and efficacy of 100% pure cartilage in the treatment of common joint disease in the athletic horse, seven thoroughbred racehorses (three females and four males, age range 3-6 years) were entered into an open label trial of Cartequine® for arthritis. The horses were selected from three different training stables in California and all were in active daily training and periodic competition. Although the horses were in good general health and were deemed “racing sound”, each suffered from mild to moderate degrees of osteoarthritis or degenerative joint change in two or more joints (Table 3).
Each horse underwent an examination prior to entry into the study and on a weekly basis thereafter for eight weeks. The animals received 10 g daily of 100% pure shark cartilage for 10 days followed by 5 g daily for a further 50 days. During the study the horses remained on a daily training regimen and each animal was monitored daily for general well-being, appetite and any adverse effects of the study medication. The clinical status for each horse is summarized in Table 4.
Results Human trial
The overall results of the mean objective measures of improvement are summarized in Table 5. There was a statistically significant improvement in joint pain and stiffness and visual appearance of the joints (p < 0.05, rank-sum test). Changes in the range of movement of the joints were favorable and there appeared to be a trend for overall improvement in this small sample of patients (p<0.05, Table 5). Animal trial The results of Cartequine® treatment in the seven horses are summarized in Table 4. The outcome data are based on daily clinical examination and subjective assessments of physical status. Overall the results indicate a beneficial therapeutic effect for Cartequine®. Discussion and Review of Cartilage use in Arthritis Therapy There are several published and unpublished observations that support the use of the cartilage in the treatment of arthritis and other inflammatory disorders that are angiogenesis dependent. The present human study in Dr. Greenberg's practice is the only study of cartilage in any disease state that has been subjected to statistical analysis and shows benefit (Table 5). In William Lane's overview of the subject (1991, 1992), he describes personal communications with Orloff (1985), who is credited with the successful use of oral Cartilade (9g/day) in the alleviation of pain from degenerative joint disease in a 49-year-old female patient. The successful use of shark cartilage in the treatment of osteoarthrits or degenrative joint disease in six patients underwent nine outpatient visits during which 9 g/day of shark cartilage were administered orally for the first four weeks of therapy and 4.5 gm/day were administered orally for the first four weeks of therapy and 4.5 gm/day were administered for the second four-week period. Assessments were undertaken of the patient symptoms and the tolerability of the shark cartilage. Of the six patients in the study, three completed the entire assessment, whereas one patient attended for several visits and two patients were seen on only one occasion. Lane reports Orloff’s observations as showing an approximate 50% decrease in pain in the three patients completing the study and varying degrees of amelioration of symptoms in others. In this study, there was a confirmed reduction of pain during physical exercise in two patients. Studies in animals have shown quite promising results with the use of cartilage for the treatment of arthritis of diverse form. Rauis presented important data to the British Small animal Veterinary Association Congress in 1991 on the beneficial effects of a prototype preparation of Cartilade for the treatment of secondary arthritis in the dog. Rauis (1991) utilized Cartilade brand of shark cartilage in 10 dogs with lameness due to the following disorders alone or in combination: joint fracture, hip dysplasio, joint dislocation, spondylopathy and rupture of the cruciate ligament. The presence of osteoarthritis was confirmed by x-ray in all animals. In this study, each dog received one capsule of Cartilade (740 mg) per 5 kg of body weight per day for three weeks and other treatments were withdrawn for the duration of the study. Evaluations of the dogs were made at days 0. 8, 15, 21 and 36 of the study and clinical scores of disability were made between 0 and 56 days for each of several clinical parameters in the dogs. These parameters included local swelling, atrophy of regional muscles, joint crepitation and/or pain, lameness before action, lameness after action and difficulty in negotiation and obstacle. Lameness was clearly defined as difficulty to walk or run after several hours of immobility (lameness before action), involving the climbing of stairs and/or the capacity to get over an obstacle that had not been previously overcome by the animal. Rauis considered the number of animals in the study to be insufficient to reach global conclusions, but several beneficial outcomes were noted in these animal studies. No significant side effects of the administration of the prototype preparation of Cartilade administration were encountered in the study, and the study compound was considered very easy to administer to the animals, since it mixed readily with dog meals and nine out of 10 animals were reported to “like” the dietary supplement very much. Rauis reported that in all cases the owners indicated that their dogs were much more active and even apparently “happy”. This status was ascribed to the apparent relief of the pain that had been experienced by the animals. The main beneficial effect in the study seemed to be reduction in the local swelling and inflammation in the joints of the dogs. Rauis described the overall effect on functional parameters in the dogs as “impressive”. In this non-label, non-blinded study, Cartilade appeared effective and safe to administer in the treatment of canine osteoarthritis. Dr. John F. Prudden (1985) is to be credited with pioneering studies of the use of cartilage in the treatment of arthritis of varying type and degree of severity. Early observations of the clinical effects of bovine cartilage by Prudden an Balassa led to the use of bovine cartilage by both parenteral and oral administration to treat osteoarthritis and inflammatory arthritis in humans. These studies were based on the reasoning that abnormalities of the polysaccaride component of cartilage was a key abnormality in the joints of patients with osteoarthritis. This notion is supported to some degree by the findings that a stimulation of protein-chondroitin sulfate synthesis occurs as a consequence of the administration of articular cartilage. Prudden and Balassa reasoned that by supplying the building blocks of cartilage, they could promote resynthesis of healthy cartilage. This is an example of the widely practiced, but clinically unproven, concept of “protomorphogenesis”. In these studies, sterilized bovine cartilage solution (Catrix-S) was administered by subcutaneous injection to 26 patients with long-standing osteoarthritis. These patients had varying degrees of functional disabilities (1), and most of these patients had evidence of marked joint degeneration, which had been confirmed by x-ray. Prudden and Balassa reported an excellent result from bovine cartilage therapy in terms of improvement in pain and disability in 17 cases, good improvement in six cases, marginal benefits in two cases and no benefit in one case. Despite the parenteral administration of cartilage in this study, no toxicity was observed. The parenteral administration of cartilage may be considered potentially dangerous because of the introduction of the antigenic load of foreign protein. Although the work of Prudden and Balassa was not in the form of a controlled clinical trial, it would appear that the beneficial effects observed by them may not be explicable by chance alone. Prudden (cited by Kirchhof and Kirchhof, 1995), performed studies on parties with rheumatoid arthritis and reported favorable responses on subcutaneous administration of bovine cartilage suspension for periods up to 35 days. Thereafter, “booster” doses of cartilage were given at intervals of approximately three to four weeks in a manner determined by the therapeutic response of the patient. In these studies, there were nine patients with severe rheumatoid disease who had marked joint swelling and/or immobility. Sic of these patients were described as having a “good” result from cartilage treatment whereas three were described as having an “excellent” result. One out of the nine patients in the study was particularly notable, since she was a 57-year-old female who had progressive rheumatoid disease with resultant severe immobility. The response of this patient to cartilage administration was considered dramatic by Prudden. After an initial three-month period where the patient reported that her joints were becoming more swollen, there was a progressive improvement with restoration to reasonable activity. This improvement was sustained for at least three and one half years following one cartilage treatment course. One of the most important studies of cartilage preparations in the treatment of osteoarthritis was performed by Rejholec who undertook a five-year, double-blind study of the effect of a bovine cartilage preparation in 147 patients with osteoarthritis who were divided into three study groups. One study group received placebo and the other two received an extract of bovine cartilage. In the cartilage treated-groups, pain scores were reduced by more than 85% compared with only 5% reduction of pain scores in the placebo-treated group. At the end of five years, joint degeneration was noted to be significantly less in the cartilage treated group compared with the control group. Rejholec produced limited outcome data but indicated that significantly less time was lost from work in the subjects who received cartilage with the control group. One year following the studies of Rejholec, Brown and Weiss published their findings of angiogenic stimulators in the joint fluid of patient with osteoarthritis. These findings added weight to the rationale for the use of cartilage and other “antiangiogenic” compounds in the treatment of osteoarthritis. This review of studies performed in seven humans and seven horses and antecedent experiences have produced a favorable outcome and they provide further supportive evidence for a potential role of shark cartilage in the treatment of arthritis. The mechanism of the observed effects on arthritis may be mediated in part by the antiangiogenic properties of shark cartilage. These current studies, together with previous data, support the need for future large-scale prospective, controlled trials of the use of cartilage in the treatment of arthritis in humans and animals. Cartilade® or Cartivet® or Cartequine® may be highly useful alternative therapies of natural origin for the improvement of osteoarthritis and sport injuries Whilst antiangiogenesis is an important potential mechanism of action of shark cartilage, well prepared Cartilade is a holistic mixture of several natural agents for which there is much credible support for efficacy in the promotion of bone and joint health. Shark cartilage contains glycoaminoglycans (glucosamine-like molecules), Chondroitin (especially Type A and C), calcium and phosphorus in an ideal 2:1 ratio and type II Collagen. It is notable that the oral administration of type II collagen induces immune tolerance and has favorable effects on experimental arthritis. Skin Disorders Angiogenesis play a major role in several types of skin disease, such as psoriasis and eczema, and it is one of the pivotal steps in wound healing. Bovine cartilage preparations have been shown to have beneficial effects on wound healing, and topical application of cartilage has accelerated healing of wounds in some circumstances. The tensile strength of wounds has also been significantly enhances by administering cartilage. Many studies have been conduced as a follow-up to this promising research but proprietary interests have not permitted widespread publication of this important research. Shark cartilage is a key cosmetic ingredient in anti-aging skin formulae (e.g. Estee Lauder Inc.). Several clinicians have suggested the use of topical or systemic administration of cartilage, especially shark cartilage, as a potential treatment for such skin diseases as psoriasis, contract dermatitis, eczema, pruritis, angiofibroma, hemangioma, Kaposi's sarcoma, and even burns. Because angiogenesis may play an important role in the pathogenesis of these diseases, they may be amenable to antiangiogenic treatment. However, without controlled studies, the result of such treatments become susceptible to illogical projection and misrepresentation of the potential benefits of antiangiogenic compounds. Eye Disease Many eye diseases are associated with angiogenesis, including neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, and subtypes of macular degeneration. Considerable basic scientific research, as well as anecdotal use of shark cartilage in patients with eye disease, seem to support the need for further human clinical trials. Researchers in Israel reported studies using shark cartilage in the favorable treatment of diabetic retinopathy and neovascular glaucoma, but the work has not been published in detal. Angiogenesis: A Double-Edged Sword There are circumstances where angiogenesis is necessary, including pregnancy, ovulation, and the need to develop a collateral circulation, such as in coronary artery disease. These circumstances are examples of conditions where antiangiogenic therapy is best avoided. Several pathologic states result directly from reduced vascularity. Tissue necrosis or ulcertaion, fistulae, and avascular atrophic changes cause organ damage. The question that remains unanswered is: It there a circumstance in neoplastic proliferation where angiogenesis is a beneficial phenomenon? This question arises from observations where avascularity of neoplasia can be associated with resistance to treatment by chemotherapy and radiation. Attempts to induce angiogenesis have helped to identify many potentially applicable proangiogenic cytokines, including acidic fibroblast growth factor, epidermal growth factor, transforming growth factors alpha and beta-1, tumor necrosis factor alpha, vascular endothelial growth factor, platelet-derived endothelial cell-growth factor, angiogenin, and angiotensin. Angiogenic cytokines show promise in plastic and reconstructive surgery. Pre-treatment of donor and receptor skin graft sites with angiotropin has prevented tissue necrosis in skin flaps. Cytokines have also been used to promote angiogenesis during surgical procedures. Administering agents that promote angiogenesis may even be a means of overcoming radiation-induced effects in tissues that result from impaired angiogenesis. The intriguing role that angiogenesis may play in the amelioration of disease has led to speculation that angiogenic promoters may be useful in treating such disorders as peptic ulcers, fistulae, and hypoxia-induced resistance of neoplasia to repeated irradiation treatments and chemotherapy. In addition, other patients with disorders may benefit from inducing neovascularization, including those with aseptic bone necrosis and vascular occlusion of organs, notably peripheral vascular disease and ischemic heart disease. In Inflammatory bowel disease neovascularization may occur. This angiogenesis has been reported to show some favorable response to bovine cartilage treatments. Several trials of angiogenic compounds in the treatment of peptic ulcer have described variable outcome. Although antiangiogenic agents should be avoided in peptic ulcer disease, no clinical exacerbations of peptic ulcer have been recorded in humans taking shark cartilage orally. Angiogenesis has been associated with life-threatening pathologies, such as cancer, and contributes to the pathology of disease, such as atherosclerosis, psoriasis, and arthritis. New drugs and compounds that inhibit angiogenesis are under intense research and development. Once proper clinical trials are completed, antiangiogenic compounds, especially shark cartilage and isoflavones, may well become the first new class of anticancer compounds and afford great promise for the treatment of arthritis and many other disease. Table 1 Selected Inhibitors of Angiogenesis Under Investigation Strategy / Compound Extracts of Shark Cartilage Soy isoflavones Colostrum? Selected herbs? Cytokine/Growth Factor Inhibitors VEGF Mab, soluble receptors VEGF antisense oligonucleotide Soluble FLT-VEGF receptor VEGF receptro-gyrosine kinase antagonsits, Tiel, Tie2… CA2 anti- TNF a Mab IL1b and TNF a inhibitors GM-1474, sulfate oligosaccaride (bFGF) CAMs/Martirx Proteins Humanized forms of LM-609, Vitaxin (anti-avb3) Cycline peptide avb3 ligands Small molecule avb3 antagonists Other Angiogenesis Inhibitors PF4 TNP-470 Thalidomide / Angiostatin Receptor tyrosine kinase inhibitors (ZD-1893) Matrix metalloproteinases (GM-6001) Urokinase receptor antagonist Antioxidant nitrode-related therapeutics BB-94 (Batimastat); MMPI;BB-2516 DS-4152 (Tecogalan) Patient Arthritis Site/Severity Comment 75 y old white male Right knee; severe loss of 5 year history function, referred for knee without sustained replacement relief from NSAID physical therapy 41 y old white female Moderately severe 3 y history un osteoarthritis, both feet responsive to treatment with NSAID by orthopedic surgeon and physical therapy 49 y old white female Moderate osteoarthritis 2 y history un- with osteoporosis affecting responsive to NSAID, midthoracic spine and left heel massage and physical therapy 32 y old white female Moderate to severe 1 y history of severe osteoarthritis of thoracic and pain unresponsive to lumbar spines NSAID 54 y old white female Severe osteoarthritis of both 2 y of treatment with hips and hands severe debilitating pain 82 y white male Left shoulder frozen with Loss of movement in osteoarthritis the left shoulder with 22 y history of progression despite NSAID 56 y old black female Total spinal osteoarthritis, left 40 y progressive sacroiliac osteoarthritis history of osteoarthritis unresponsive to NSAID of late Table 2 Human Trial Seven patients (five females, two males, age range 32 to 82 years) with moderate to severe symptomatic osteoarthritis received 100% pure shark cartilage for 90 days. Each patient had failed conventional medical therapy and in some circumstances alternative therapy (Table 1). All existing therapies were discontinued prior to treatment. Each patient received 4440 mg daily of 100% pure shark cartilage in divided doses (1480 mg in 2 capsules, three times daily). Objective and subjective assessments of the clinical course of the patients arthritis were made prior to the therapy and at intervals up to 90 days (Days, 30, 60 and 90). The extent and nature of the assessments are shown in Table 2. Table 3 Objective assessments of the seven patients receiving cartilage for arthritis made at days 0, 30, 60 and 90. On Examination Areas of joints affected by arthritis. Relative movement of the afflicted joint, calculated in degrees. Elicitation of pain on palpation. Signs of joint swelling, heart or redness. By Standardized Questionnaire Restriction of activity due to arthritis. Degree of stiffness and joint swelling. Extent that symptoms limited specific movement oriented tasks. Overall level of incapacity that could be directly attributed to the pain of arthritis. The clinical features of the seven patients in the clinical trial are summarized in Table 1. The tolerability of the medication was monitored by direct questioning and compliance was assessed by tablet counting. Statistical analysis of the clinical outcomes was undertaken using the non-parametric, rank-sum test. Table 4 Clinical Status Prior to Study Clinical Progress and End Point Comment 4 y old stallion in continuous training for 12 Slight improvement in clinical appearance of his month prior to study. A "stakes-class" race- fetlock joints and training performance by the third horse who suffered from OA in both front week of the end study, continued to steadily improve fetlocks with moderate to severe boults over the second month of the investigation and had of capsulitis. fewer non-training days during this period. Periodic treatments with phenylbutazone which were necessary before the shark cartilage therapy began, were discontinued. 4 y old gelding in continuous training for No improvement during the first month of study, the seven months, in "claiming races", had inflammation and distension in fetlock joints worsened undergone carpal surgery to remove an during the first 2-3 weeks of the trial; by second month ostoechrondial fracture one year prior to this animal began to show steady improvement. Shark the study . The animal had a moderate cartilage therapy had no effect, on post exercise amount of joint distention and osteo- myositis ("tying up"). arthritis in both carpi. 4 y old filly in training for 10 months prior Remarkable improvement by the second week to the study, raced in "allowance" type of study, chronic carpal inflammation and joint races, a slight to moderate amount of joint distention disappeared and training soundness inflammation as a result of osteoarthrits, also improved. By the third week of the study also intermittent bouts of "tying up" removed from daily regimen of oral butazoliden, continued to improve clinically over the course of the investigation and it was unnecessary to dose with NSAID, in spite of more intensified training. 4 y old filly which had been in continuous Only slight improvement in fetlock inflammation training for 9 months prior to the study. Until the fourth week of investigation. At that time Capsulitis of both front fetlocks due to began to show market decrease in joint effusion and Chronic osteoarthritis in both joints, a capsulitis, improved steadily during the next 4 "stakes" class individual. Weeks A six y old stallion in continuous training Improvement began to become apparent by the third For 9 months prior to the clinical investi- week of the program, exhibited a moderate reduction Gation, had a history of a surgical repair of joint distention and pain at the time and maintained Of a non-displaced lateral condylar that state throughout the rest of the study period. Fracture of the distal metacarpus 17 Months prior to the study, a moderate Amount of joint distention and capsulitis as a result of chronic osteoarthritis of both front fetlocks, an "allowance" level competitor. 5 y old gelding in continuous training for During the first 10 day period of 10g/day of 10 months prior to the trial, had a history Cartequine, this animal showed immediate of carpal surgery to remove an osteo- improvement, improved state lessened when dosage chrondial fracture 18 months prior to was decreased to 5 g/day, but joint inflammation investigation. This joint showed no did again show improvement in the fourth evidence of chronic inflammation, week and continued to improve thereafter. exhibited problems of osteoarthritis of both front fetlocks with capsulitis and joint effusion, "stakes class" competitor Table 5 Results of clinical outcomes in the seven patients treated with Cartilade® for osteoarthritis. Data are mean + standard deviation. Asterisk "*" denotes significant differences between interval data: Day ), the start of the study is compared with each successive interval, based on the rank-sum statistical test and using the level of significance of p < 0.05. Mean: Day 0 Day 30 Day 60 Day 91 Mean numerical 2.86+-0.82 2.29*+-1.39 1.43*+-0.65 1.14* +-0.78 Composite score Of visual appearance Of joints, 0-4, represents normal, mild, moderate and severe edema and or erythema, respectively Mean numerical compo- 2.25+-0.57 1.23*+-0.45 0.96*+-0.33 1.61*+-0.88 site score of joint pain and or stiffness derived from questionnaire Overall mean percentage 100 30.00*+-20 56.43*+-22 45.71*+-34 level of improvement based on composite of symptoms, signs and subjective responses Table 6 Angiogenesis Models In vitro Models In Vivo Models Cultured EC on different substratum Matrigel inmice Matrigel Chick chorioallantoic membrane Collagen / Fibronectin CAM assay Laminin Rabbit cornea Fibrin / Geletin Hypoxia / ischemia-induced Sprouting from aortic rings Retinal / iris NV in rats, mice and primates Laser-induced choroidal NV Human skin / human tumor transplant on SCID mice Tumor metastatic models in mice References The Power of Cartilage, Holt S., Barilla J., Kensington Publishing, NY, NY, 1998 (available 1-800-700-7325) The Shark Cartilage Alternative, Holt S., Fuerst MA, Gagliardi J., Keats Publishing, New Canaan, CT.