AIDS TREATMENT NEWS #139, November 22, 1991
John S. James

In addition to the conference presentation, two major papers on anti-HIV activity of melanins in laboratory tests have been published(2,3), both by the same Vanderbilt team led by David C. Montefiori, Ph.D., a well-known researcher who has co-authored dozens of papers and conference presentations on HIV. The melanin work has attracted little attention, however, because of widespread reluctance to believe that these common chemicals, widely found in nature and already present in the body in other forms, could possibly work as an antiviral.

Then about six weeks ago a chemist who had synthesized one of the soluble melanins in a home laboratory began taking a large dose, over 200 mg once per day, for his own HIV treatment. He is enthusiastic about the result and provided the melanin to friends with HIV or AIDS. Today over six people are taking it. They are reporting good results, but almost no objective data is yet available.

Because of the power of the placebo effect, and the hopes and excitement of trying something new, many drugs go through a similar "honeymoon" period of early enthusiasm. Most of them later prove not to be useful. (There is even an old medical saying, "Make haste to use a new remedy, while it still has power to heal.") In view of the many disappointments in the history of AIDS drug development, there is much concern over the risk of a false alarm about something which may not work, and which could be harmful since it has not been through the standard toxicity tests in animals.

Because of this danger, we had mixed feelings about publishing this article now, instead of waiting until more is known. We decided to go ahead, since waiting will gain little; there will never be a "right" time. The peculiar nature of both melanin itself and of the prevailing attitudes toward it almost guarantees that this treatment lead will not be developed rapidly by the research mainstream, if it is developed at all. Therefore the AIDS community itself must take the lead in determining whether the early reports of good human results will be sustained. It is important that more people be involved in this effort.

Yet it is also important to realize that it is much too early to recommend melanins as an HIV treatment.

Background on Melanins

Melanins are pigments responsible for the color of skin, hair, and eyes; they are also found in other organs, including the brain. In the body they are insoluble, however, and do not have an antiviral effect.

There are many different kinds of melanins. But while these chemicals have been familiar for decades (almost 5,000 published papers mentioning melanins since 1966 are listed in the Medline file of the U. S. National Library of Medicine), their chemical structure is still not entirely known. While melanins are generally considered relatively harmless, no medical use for them has ever been found. Therefore, while there are many papers studying the metabolism of melanins, especially in regard to melanoma (which is a cancer of melanin-producing cells), there are few if any published reports about administering these chemicals either to humans or to animals.

The form of melanin of most current interest as a possible HIV treatment is called l-dopa melanin. (Other melanins worked about as well in laboratory tests, but as far as we know, only this one has been tried in humans as an anti-HIV treatment.) In the laboratory, l-dopa melanin is easily synthesized from l-dopa, an amino acid which is found in certain edible beans and is best known as an important treatment for Parkinson's disease; the chemist who first tried taking the melanin used a synthesis procedure based on the one published by Dr. Montefiori(2,3); a similar procedure was published in 1938.(4)

Melanins and HIV

Since there was little or no reason to suspect that any kind of melanin would have anti-HIV activity, we asked Dr. Montefiori why he first thought to try it. He explained that the discovery was in part accidental. He was studying a pigmented compound which showed anti-HIV activity in laboratory tests. Because he was familiar with melanins (having worked with them as a graduate student), because there was a theoretical possibility that they might account for the effect found, and because Dr. Montefiori had excellent facilities for economically screening substances for anti-HIV activity, he tried melanin to see if it might be causing the antiviral effect. It later turned out that the original substance being investigated did not contain melanin, but by then the antiviral activity of the chemical had already been noted.

The first of the two papers published so far on HIV and melanins appeared in April 1990 in Biochemical and Biophysical Research Communications, a journal which specializes in rapid publication of current research. In laboratory tests, melanin synthesized from three starting substances -- dopamine, norepinephrine, and serotonin -- prevented infection of cells at very low concentrations, with 50 percent protection with concentrations as low as 0.09 ug/ml (micrograms per milliliter). [Note: the unreacted starting substances used in this experiment (dopamine, norepinephrine, and serotonin) can be toxic to humans. The starting substances which were used in the later research, described below (L-tyrosine and L-dopa) are safer.]

In another experiment, higher concentrations were required to prevent the formation of syncytia, abnormal giant cells formed when HIV-infected cells merge with each other and with normal cells; concentrations of 5 ug/ml were needed for best results. But if infected cells were pre-incubated with melanin before being mixed with the uninfected cells, syncytia were inhibited at 0.625 ug/ml. By contrast, pre-incubation of the uninfected cells did not help. This difference suggests that melanins work by interacting directly with viral proteins, not by protecting normal cells; a beneficial consequence is that their action is not specific to any particular kind of cell.

The starting substances themselves were tested and found to have no antiviral effect -- even though a small amount of melanin formed spontaneously (by oxidation) while the viral tests were being run.

Results of confirmatory and followup studies(3) were published in January 1991 in Antiviral Research. Melanin synthesized from L-dopa provided 100 percent protection against two HIV strains, at 0.31 and 0.62 ug /ml respectively. It was also tested against HIV-2, a different AIDS virus found mostly in parts of Africa, and was found to be less effective; a 3 ug/ml concentration provided 50 percent protection.

Other research reported in the same paper explored the possible mechanisms of the antiviral activity of melanins. These substances seem to work early in the viral replication cycle; they need to be present with the virus (or viral proteins), since treating uninfected cells and then washing the melanin off does not protect the cells.

Another test showed that melanin inhibits binding of the viral protein gp120 to the CD4 receptor on T-cells -- the mechanism by which virus attaches to uninfected cells and enters them. Free gp120 or gp160, which bind to the CD4 receptor, were added to cell cultures; later, the unbound portion was washed away, and that which remained on the cells was measured. Melanin blocked this binding process; complete blockage of gp160 binding was achieved at 10 ug/ml, although gp120 binding was only partly blocked. Another experiment, with monoclonal antibodies, suggested that melanins specifically blocked the binding of HIV proteins to the CD4 receptor, not binding to receptors in general.

Melanins were also found to have no effect on the enzyme reverse transcriptase. Therefore their antiviral effect is due to a different mechanism of action than that of AZT or other reverse-transcriptase inhibitors. It also appears that soluble melanins do not get inside the cells, which is consistent with the theory that they work by direct interaction with free virus, or with viral gp120 on the cell surface or elsewhere.

We asked Dr. Montefiori why the mouse-urine result presented at the Sixth International Conference on AIDS was not included in the published article. He told us that there was nothing wrong with the work, but that all the animal results had been deleted by the reviewer assigned by the journal, due to the preliminary nature of the experiments. There was also a prevailing skepticism in the scientific community about melanins as an antiviral. (In fact, three other journals had rejected the paper, before it was accepted by Antiviral Research.) The animal work is important, because it shows that melanin given orally (or some antiviral component or product of it) could be absorbed by mice and appear in their urine, which then inhibited HIV even when diluted many times. Unfortunately this evidence that soluble melanins may be orally bioavailable has not yet been published in the scientific literature. Additional pharmacological studies are urgently needed.

One reason for the skepticism about melanins is that no one knows their mechanism of action as an antiviral. Montefiori and Zhou(3) suggest that certain chemical sites in melanin might bind to gp120 or another substance needed for viral entry into cells. Melanins "have been shown to contain substituted hydroxyindoles, indolequinones, pyrroles, free carboxylic acid groups, phenolic hydroxyls, carbon-based free radicals, and uncyclized aliphatic chains" (page 22). Another possibility suggested in the same paper is that part of the melanin molecule might resemble a peptide sequence in gp120 and bind specifically to it.

Insoluble melanin in the body is "known to bind certain chloroquine and phenothiazine antibiotics" (Montefiori and Zhou, page 22). Whether soluble melanins would affect these antibiotics is unknown.

Biosource Genetics Corporation in Vacaville, California is developing melanins as an absorber of ultraviolet light in sunscreens, and also for possible medicinal uses. Bob Erwin, president of Biosource Genetics, told AIDS TREATMENT NEWS that the company's animal tests had found little or no toxicity, whether the chemicals were given orally or by injection -- and that another potential advantage is that melanins can be produced at low cost. Biosource Genetics specializes in developing methods to manufacture high-purity melanins and related indole chemicals economically; its scientists recently published an article on using genetically engineered bacteria to produce new kinds of melanins.5 Dr. Erwin also noted that melanins occur in many foods.

Anecdotal Reports

The chemist who first tried the L-dopa melanin has been taking 200 mg per day for two months now. A total of eight people people are now using it, with the same dose. Four of them have taken it for several weeks and reported back about their experience. (The chemist himself has tried a larger dose, up to 200 mg four times a day for a week, to look for any toxicity. He did not find any problem, but also did not find any additional benefit over the dose of 200 mg once per day.)

The most uniform result reported is that people felt much better, with energy, motivation, and appetite coming back, starting very quickly, usually the day after the first dose. (The only exception is one person who was already feeling well, who did not notice any change). The chemist said that he felt better after starting the drug than he had since becoming ill. His myopathy is gone, neuropathy which he has had for two years has improved, and he has gained 10 pounds after losing weight during his illness.

After three days his wife, who also has symptomatic HIV disease, started taking the melanin, also with good results. Both have taken it continuously for two months.

A third person who had fevers was feeling good and back at work a day after starting the melanin. He once ran out of the drug for about a week and fevers returned; they went away after he restarted the treatment.

The fourth person had been hospitalized before starting melanin, and was in such poor condition that his physician, a leading AIDS clinician, discontinued the patient's conventional drug treatments, believing that he had less than one week to live. He had starting taking melanin four days before the other treatments were discontinued. Recovery began several days after he started the melanin, and at his family's request, the regular medications were restarted. According to what we have heard both from the chemist and from a friend of ours who knows the patient well, this person greatly improved after starting melanin, becoming ambulatory and lucid; serious neuropathy had entirely disappeared by the sixth day (and has not returned since), and his skin was described as looking much better. The patient was discharged from the hospital one week after he had been expected to die, and a week later he was able drive his car. He is still seriously ill with MAC, despite treatment with clarithromycin.

The only bloodwork now available from anyone who has used melanin is from the chemist himself. His T-helper count had been declining very rapidly, losing about 30 percent a month for several months. The last count before starting the melanin was 100; the only count since, with blood drawn about three weeks after the treatment began, was 111. The increase of 11 is well within the range of error of the test. It is possible that the melanin treatment stopped or reversed the ongoing decline -- but it is also possible that the apparent reversal was coincidence.

The melanin dose has usually been taken in the evening, usually with an antacid. The antacid might or might not be useful. Melanin is stable in acid, but it precipitates out of solution. It would likely dissolve again in the intestines, but this is not known. The antacid may help to keep it from coming out of solution in the stomach.

Speculation: Mechanism of Action

What is surprising about these reports is how rapidly a substantial improvement has been seen -- usually within one day of starting treatment. It seems unlikely that so rapid a change would result from reducing viral replication. The two theories which seem most consistent with the available information are (1) a placebo effect, and (2) that the drug may block harmful effects of gp120, a toxic protein produced by HIV which may be responsible for immune dysregulation and other pathogenic effects of HIV infection. Melanin did block the binding of gp120 to the CD4 receptor of cells (which is probably how gp120 causes harm) in the laboratory tests cited above.

If melanin does work by blocking gp120 (in addition to any effect it may or may not have on viral replication), then it might be especially useful late in the course of illness, when the gp120 levels are presumably high. It would not be the whole answer, however, as it apparently does not get into cells, so the virus in infected cells would still remain active.

If melanin does work this way, then theory suggests that viral resistance to it may be slow to develop. For if a drug blocks the harmful effect of gp120 on the body, without directly inhibiting the virus, then there would be no selective pressure on the virus to develop resistance. If the drug also works as an antiviral, then there would be such pressure. But the binding of gp120 to the CD4 receptor may be critical, and if so, viruses which changed the binding region would be unlikely to be able to infect cells.

Comment

It is too early to know for sure if the melanin is helpful, or if the reports from the human experience so far result from placebo effect and lucky coincidence. But clearly this potential treatment deserves serious attention immediately. The above reports of patients' experiences came to us from three different people; all three are well known to this writer, very knowledgeable about HIV, and skeptical of new-drug claims. All three learned what they told us from personal observation, not by hearing it from others.

A research organization could begin by sending a medical team follow up the information available from the people who have used the treatment so far, interviewing and examining them and checking their medical records; this could be done without the ethical or legal complications (and resulting delays) of giving someone an untested drug. Also, it would take advantage of the two months of human experience already available. Perhaps the next step would be for other patients to start take L-dopa melanin under the supervision of a research physician, who would make sure that the appropriate baseline and other research tests were done. A formal trial should begin as soon as possible; but at this time, no such trial is being planned.

For More Information

DATA (Direct Action for Treatment Access), a new research and advocacy organization (see AIDS TREATMENT NEWS #137, October 18, 1991), is collecting information on treatment uses of melanins. For the latest available information, send a self- addressed stamped envelope with a note requesting melanin information to: DATA, P. O. Box 60391, Palo Alto, CA 94306, or phone 415/323-6051.

References

1. Candia AF, Modliszewski M, Shaff DI, and Montefiori DC. Inhibition of HIV replication and cytopathicity in vitro by synthetic soluble melanins. Sixth International Conference on AIDS, San Francisco, June 20-24, 1991 [abstract number Th.A. 228].

2. Montefiori DC, Modliszewski A, Shaff DI, and Zhou J. Inhibition of human immunodeficiency virus type 1 replication and cytopathicity by synthetic soluble catecholamine melanins in vitro. Biochemical and Biophysical Research Communications. April 16, 1990; volume 168, number 1, pages 200-205.

3. Montefiori DC and Zhou J. Selective antiviral activity of synthetic soluble L-tyrosine and L-dopa melanins against human immunodeficiency virus in vitro. Antiviral Research. January 1991; volume 15, pages 11-25.

4. Arnow LE. The preparation of dopa-melanin. Science. Volume 87, page 308.

5. Della-Cioppa G, Garger SJ, Sverlow GG, Turpen TH, and Grill LK. Melanin production in Escherichia coli from a cloned tyrosinase gene. Biotechnology. July 1990; volume 8, pages 634-638.

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