It is perhaps testimony to HIV's resilience and versatility that physicians celebrate drugs that do no more than slow the virus's progress in the human body. HIV hides and mutates in order to dodge the medications sent to stop it. And while these agents are limited in their therapeutic value, so far they have provided the only hope in the treatment of AIDS.
Researchers at Yale have played key roles in developing two of these compounds, the reverse transcriptase inhibitor d4T, known commercially as Zerit, and 3TC, known as Epivir. Both are key ingredients of the so-called drug cocktail that has fundamentally changed the nature of AIDS therapy during the past three years. They not only inhibit the reverse transcriptase enzyme, but more importantly, by terminal incorporation into the growing proviral DNA, a precursor to the virus, they prevent the reproduction of the virus. These compounds are frequently combined with other inhibitors which inhibit the viral enzyme protease, whose activity is essential for the formation of an infectious virus.
In addition to contributing to traditional, small-molecule pharmaceutical development, Yale scientists are exploring new avenues in the fight against AIDS, in particular the potential of biological agents to effect a cure. One novel approach has resulted in a genetically engineered virus that attacks and kills HIV-infected cells in culture while leaving other cells unharmed. Whether it will do so in humans, or even animals, remains to be evaluated.
William Prusoff, Ph.D., professor emeritus of pharmacology, has spent a 45-year career at Yale investigating potential antiviral and anticancer compounds, part of the traditional, small-molecule approach. In the late 1950s he synthesized idoxurine, an analog of thymidine, which was the first antiviral compound approved by the FDA for therapy in humans. It was used to treat herpes infection of the eye. Dr. Prusoff and his long-time collaborator, the late Tai-Shun Lin, Ph.D., discovered in the 1980s that a thymidine analog, reported in scientific literature as a poor anticancer agent, was very effective in slowing the production of HIV. This compound is known as d4T or stavudine. How does d4T work? “It gets incorporated into the growing viral DNA chain and synthesis of proviral DNA is stopped,” Dr. Prusoff says. HIV's mechanism for reproducing is simply turned off.
Bristol-Myers Squibb developed the drug under the trade name Zerit and brought it to market in 1994. Almost single-handedly, d4T has boosted Yale's annual patent royalty income nearly tenfold, to $34 million for the year ending June 30. [AZT, the first medication shown to slow the progress of HIV, was created by a Burroughs Wellcome team led by Yale alumnus David W. Barry, M.D. '69, HS '69-72. (Yale Medicine, Fall 1997)]
“They are very potent in decreasing the viral load in the bloodstream,” says Dr. Prusoff, referring to the limitations of the currently available anti-viral medications, “but you don't really eliminate the viral load in the cell where the virus is hiding. This is critical for an eventual cure.”
The combination approach
Two hurdles in AIDS pharmacology are toxicity and drug resistance. As with cancer chemotherapy, AIDS medications can make people very sick. For example, d4T can cause peripheral neuropathy, tingling, numbness and pain in the extremities; ddI can cause lethal pancreatitis; and AZT is toxic to the bone marrow and can cause anemia, headaches and nausea. In addition, HIV often mutates when challenged by a single therapeutic agent, creating drug-resistant strains of the virus that foil treatment. By prescribing AIDS drugs in combinations, the physician can reduce individual dosages and minimize both side effects and the potential for resistance. “There are dozens of different combinations,” Dr. Prusoff says. “Resistance develops to all of those compounds.” Resistance to d4T has been found with laboratory strains of HIV, but mutant forms resistant to d4T have not been observed in patients being treated with d4T, Prusoff says.
Yung-Chi (Tommy) Cheng, Ph.D., the Henry Bronson Professor of Pharmacology, has worked on a parallel course to Dr. Prusoff. While Dr. Prusoff found drugs that work against AIDS, Dr. Cheng has sought ways to reduce their toxicity. Long-term usage, Dr. Cheng says, leads to a decline in the mitochondrial DNA of certain organs, affecting their ability to function properly. After a month or two of use, anti-retroviral agents such as AZT, ddI and DDC can cause problems in nerves, the pancreas, muscles and the liver.
“If we know the mechanism for the toxicity, we may be able to prevent the toxicity by either making a new compound or by combination with other drugs,” says Dr. Cheng, whose laboratory team studies the behaviors of virus-specific proteins in order to exploit them. “The next question is, 'Can we find a drug that will be active against the virus but will have no toxicity to the mitochondrial DNA?' ”
That drug turned out to be 3TC, a compound that has positive and negative forms that mirror one another. Originally synthesized by a Canadian researcher and identified as an antiviral agent, samples were sent to Dr. Cheng for study of its toxicity. He found that 3TC's negative form reduced side effects when used in combination with AZT. The combination increases 3TC's efficiency at inhibiting reverse transcriptase, an enzyme HIV uses to reproduce its genetic material. Dr. Cheng identified 3TC as an agent that would be less toxic to mitochondrial DNA than DDC, ddI or d4T.
“We are increasing the antiviral effects, decreasing the side effects. This may be a key mechanism of the combination protocol against HIV,” he says. “In the meantime we also discovered the same compound is active against the hepatitis B virus. The results were very promising.” So far, 3TC has been approved for HIV treatment and is in clinical trials for use against hepatitis B.
Search and destroy
A new approach to AIDS may grow out of work led by John K. Rose, Ph.D., professor of pathology and cell biology. The agent he developed, based on a common virus found in cattle, has killed HIV-infected cells in culture. Scientists in the Rose lab are trying to develop a form of the engineered virus that will work against SIV, the simian form of HIV, for use in animal trials. If it is proven safe and effective in animals, human trials could follow.
Dr. Rose's discovery was the result of efforts to determine how viruses are constructed. “Once we know that, it becomes possible to change the components,” he says. The strategy was to trick HIV-infected cells into binding with another virus that would destroy them. He exploited the affinity between an HIV protein and its human targets–molecules on the surface of T-cells that allow HIV to enter and infect them. Infection begins when the HIV protein, gp120/41, is drawn to the receptor molecule CD4 and the co-receptor molecule CXCR4. “What we did was to reverse that process,” Dr. Rose says.
Dr. Rose deleted glycoprotein genes from the cattle virus known as vesicular stomatitis virus (VSV) and replaced them with the human genes that produce CD4 and CXCR4. With gp120/41 on human cell surfaces as its prey, the receptor-laden virus becomes a hunter drawn to HIV-infected cells like a magnet. “It doesn't completely eliminate HIV,” Dr. Rose says. “It reduces the viral load 10,000-fold in tissue culture cells. In the best of all possible worlds it might prevent people from progressing to AIDS.”
He also sees the possibility of developing an AIDS vaccine, using recombinant VSV as a vaccine vector. “You can create a live virus vaccine, which might express protein from a virus that you want to immunize against.” Dr. Rose's group has already shown that VSV vectors expressing influenza virus proteins can protect against influenza.
“The ideal situation,” says Dr. Prusoff, “would be to develop a vaccine to prevent infection and at the same time find drugs which not only decrease the virus in the blood stream, but also get in to the hiding places of the virus in the various parts of the body. Eventually the source of the virus will be killed and you prevent new infections from taking place.”