By David N. Leff
Try this not-so-trivial, trivial-pursuit question: What tissues in the healthy human body don't need a blood supply?
The answer: three for sure - cartilage, cornea and the eyeballs' vitreous humor, plus perhaps tendons.
How these bloodless structures fend off the circulatory system's perpetual payload of flowing veins and pumping arteries is a question of concern to oncologists. Malignant tumors can grow and metastasize only if fueled with oxygen and nutrients, delivered by a constantly spreading web of blood-bearing venules, arterioles and capillaries.
Efforts to nip this process of angiogenesis in its bud have led of late to the burgeoning research field of anti-angiogenesis. Most current strategies target the endothelial cells that line the smooth, seamless inner walls of the blood vessels.
Two proteins - endostatin and angiostatin - aimed at stultifying the growth of endothelial cells, became celebrated last year for their anti-angiogenic effects on mice. Since then they remain in the preclinical limbo of research and development. (See BioWorld Today, April 7, 1998, p. 3.)
Meanwhile, other anticancer investigators were reasoning that blood-free cartilage must be warding off angiogenesis by its own constituent anti-endothelial molecules, to preserve its own biomechanical smoothness.
One such protein, Troponin I (Tn I), has just been identified as a potent anti-angiogenic factor derived from avascular cartilage. A paper in the current Proceedings of the National Academy of Sciences (PNAS), dated March 16, 1999, reports: "Troponin I is present in human cartilage and inhibits angiogenesis."
The article's senior author is chemical engineer Robert Langer, at the Massachusetts Institute of Technology (MIT). "In 1976," Langer told BioWorld Today, "Judah Folkman and I reported in Science the first angiogenesis inhibitor."
It was Folkman who recently constructed the twin anti-endothelial entities, angiostatin and endostatin. He heads the surgical research laboratories at Boston Children's Hospital, where Langer now collaborates with the PNAS paper's first author, biochemist Marsha Moses.
Moses told BioWorld Today, "We're very interested in understanding the mechanism by which Troponin I is inhibiting angiogenesis in cartilage, where it had never been found before. My lab is now doing some structure-function studies to try to determine that. Tn I is an interesting molecule with interesting domains," she observed, "so we're trying to determine whether or not there's a specific anti-angiogenesis sequence or domain in that protein."
She added that Tn I is "about a 21,000-Dalton protein, containing 191 amino-acid residues. We expressed and purified it in our own lab here."
In her Tn I work, Moses, an assistant professor at Harvard Medical School, is funded by a research grant from Boston Life Sciences Inc. (BLSI). That company's chief scientific officer, immunologist Marc Lanser, pointed out, "Troponin I can't be patented, because it's been in the public domain for many years. What we have is a patent issued in November 1998. It covers the therapeutic use of Tn I for all angiogenic diseases, of which there's a long list, headed by cancer, macular degeneration and diabetic retinopathy." Moses and Langer are inventors of the patent, assigned to Children's Hospital. BLSI has the exclusive license.
"In both macular degeneration and diabetic retinopathy," Lanser pointed out, "you have this neovascularization. People think it's stimulated by VEGF - vascular endothelial growth factor - which in turn is stimulated by some unknown primary, inciting insult. That causes this florid, abnormal overgrowth of blood vessels, which end up destroying the retina and its light-detecting cells." (See BioWorld Today, March 16, 1999, p. 1.)
Lanser added: "We've shown in preliminary, unpublished experiments that we can inhibit that retinalneovascularization with an intraocular injection of Troponin I into the vitreous humor."
Troponin Checked Melanoma Metastases
After Moses and her lab crew isolated the active anti-angiogenic component in cartilage, the co-authors performed four experiments, ranging from inhibition testing in culture to the growth of angiogenesis networks on a chick's egg yolk and in the corneas of mice, to ultimately measuring the efficacy of Tn I against metastasizing melanoma tumors in vivo.
First the co-authors injected a culture of fast-growing, aggressive melanoma cells into the veins of mice. A day or two later, they began treating the tumor-doomed animals with frequent systemic injections of Tn I, at varying doses and frequencies, After four weeks on this regimen, they sacrificed the animals, and compared the clearly visible brown melanoma metastases on their lungs with those of untreated controls.
"Because of the biological variation," Lanser recounted, "the best we could achieve in this particular test was 60 to 70 percent tumor inhibition. To my knowledge, no other group has attained any higher inhibition. If you look at it from that perspective," he pointed out, "Tn I could be interpreted to be anywhere from 10 times to 50 times more potent than other anti-angiogenic that's been reported in this exact same preclinical model. So we're going to bracket the clinical dose in human trials around that 1mg/kg in the mouse, twice a week. If you extrapolate to humans on the basis of body surface area of a 70-kg man - what's usually done - you get a dose of anywhere from 5 mg to 50 mg per person, twice a week, a phenomenally low dosage.
"Ultimately," he predicted, "if this kind of dose extrapolation holds up, it could actually be self-injected by patients twice weekly, which would be an incredible advantage.
Lanser made the point that "basically we have enough evidence of efficacy to move into the clinic." He hopes to apply to the FDA for an investigational new drug permit in the second half of this year, "and then start clinical trials in cancer patients who have had their primary tumors removed, and are at extraordinarily high risk of developing metastases. The goal," he added, "would be to show that after a year's treatment the number of individuals who develop tumor metastases is far less than those who received conventional chemotherapy."
Moses, at Children's Hospital, concluded: "The major motivation for our Tn I research is that perhaps we can isolate a highly anti-angiogenic amino-acid sequence. This suggests that we can discover a second and third generation of drugs, which may end up being more potent or more bioavailable than existing ones."