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HIV Protease Inhibitors -- Background Information Bioengineering and Environmental Health Acquired Immune Deficiency Syndrome (AIDS). AIDS appeared as a clinical entity in 1981 and, within two years, it became clear that the causative agent was likely to be a virus. Two viruses have been implicated in AIDS. The first is human immunodeficiency virus I, or HIV-1, which is responsible for most cases of the disease worldwide. HIV-2 is now a serious concern in sub-Saharan Africa and is spreading into Asia. It is estimated that in excess of 22 million people are HIV positive (note that being HIV positive does not mean that you have AIDS; it is, however, a necessary pre-requisite). Over 90% of those who are HIV positive live in areas of the world where they either do not have access to or cannot afford the various therapies available to slow the development of the syndrome. In the US, the number of people who are HIV positive is approximately 1 million. People become exposed to HIV mainly by unsafe sex with HIV positive persons of either sex, sharing contaminated needles during recreational drug use, or by transfusions with virally contaminated blood. HIV selectively attacks white blood cells called CD4-positive T-cells and, with less efficiency, macrophages and neurons. T-cells are components of the immune system that are critical to our ability to fight off diseases such as tuberculosis, parasitic infections, fungal infections and infections by viruses. These immune cells also help prevent certain cancers, such as Kaposis sarcoma, non-Hodgkins lymphoma and Burkitts lymphoma as well as primary malignancies of the brain. When the virus interacts with a T-cell, it triggers a cascade of events that results in the death of the cell. The exact mechanism(s) for cell death are not known precisely but one proposal is that the interaction causes a natural process called programmed cell death, or apoptosis, to be turned on at the wrong time. Another proposal is that the virus marks the T-cell for destruction by other white blood cells. By either or both mechanisms, the initial symptom following HIV exposure is a flu like syndrome accompanied by a sharp drop in CD4 T-cells. Typically, the levels of CD4 T-cells drop from the normal level of about 1,200 cells per ์l to less than 400. There is then a period of rebound, followed by a slow reduction in T-cell count to the point that the infected person cannot fight off opportunistic infections and endogenous cancers. At this time, the person has full blown AIDS. Finally, until recently, all patients with AIDS eventually died. They could live with their disease for 2-15 years, but AIDS was essentially a death sentence. Interestingly, a small fraction of the population carries a specific mutation in a chemokine receptor that makes them immune to developing AIDS, even though they are HIV positive. The life cycle of HIV. To design a drug to treat a disease, it is of great value to know as much as possible about the biology of the disease. Many years ago, it was believed that most cancers were caused by viruses (actually, few cancers have a viral origin). As a consequence, the public health community around the world had invested much effort toward probing the how viruses get into cells, replicate, and transform cells to malignancy. This platform of information on retroviruses (RNA viruses) became particularly valuable as the story slowly unfolded that the retrovirus, HIV, was the likely cause of AIDS. HIV infects T-cells and macrophages by docking with a cell surface receptor protein called CD4 (macrophages have less CD4 than T-cells and are less easily infected). The component of the virus that snares the receptor is the glycoprotein (a protein decorated with carbohydrates) termed gp120. The virus is engulfed and thereby introduces its single stranded RNA genome (two copies are carried per virus) into the cytosol of the target cell. The viral RNA has a closely associated protein called reverse transcriptase (RT), a polymerase that copies RNA into a complementary DNA (cDNA) strand. The RNA parental genome is degraded and a second replication event by RT results in the initially single stranded DNA genome being made into a duplex. The duplex DNA is shuttled from the cytosol of the cell into the nucleus, where the enzyme HIV integrase facilitates its random insertion into the genome of the host T-cell (or macrophage). The cDNA inserted into the host genome is called a provirus. Once the HIV genome is in the proviral state, it can be transcribed into RNA, an event that is promoted by a transcription factor known as NF-์B. The process of transcription produces an RNA copy of one DNA strand. Early in the replication cycle some of the transcript is exported to the cytosolic compartment where it is translated into a long polyprotein -- i.e., a tandemly arranged, covalently connected, series of proteins. These are cleaved by the HIV protease to give rise to the various proteins of the virus. Another portion of the RNA transcript is exported to the cytosol where it becomes the genome of the next generation of viruses. The RNA and the proteins co-mingle and matured viruses begin to bud from the cell. Those viruses then go and infect other cells. An infected Tcell eventually dies, so there is a steady reduction in the number of T-cells and, therefore, immune competence. Treatments for AIDS. Coming up with a treatment for AIDS is complicated by numbers -- there are billions of viruses produced per day even in persons who are not showing the symptoms of the disease. In addition, the virus hides out in T-cells that are themselves often hidden away in out-of-the-way places in the body, primarily in the lymph nodes. Moreover, the virus is really only vulnerable to todays therapies when it is invading cells or replicating -- when it is in the provirus state, it is invulnerable to the countermeasures the body or conventional pharmacology can throw at it. Finally, the virus is error prone in its replication and that fact, combined with its enormous proliferative capacity, makes it evolve quickly in the host to drug resistant forms. The obstacles indicated above notwithstanding, some therapies have been known for a decade to slow the spread of the virus and thus extend the lives of people with AIDS. The bold and underlined words in the previous section are the principal targets of these therapies. The earliest therapeutics were the HIV reverse transcriptase inhibitors. These are agents such as AZT (3'-azidothymidine; Glaxo-Wellcome) that are selectively incorporated into the growing viral DNA molecule but, once incorporated, they are not extendable by RT. Thus, they block viral replication. There are no integrase inhibitors on the market, but some are in the pipeline and represent opportunities for the future. Protease inhibitors have only been on the market for several years, but they have shown remarkable success, especially when used in combination with RT inhibitors, to block the polyprotein cleavage reaction that is essential for production of mature viral proteins. It is also important to note that many efforts are underway to develop a vaccine against HIV. Vaccine trials are underway in Thailand, or will be soon, through the efforts of a company named VaxGen. There is an economic risk for companies that emphasize vaccines, although immunization would be without question the way to curb this epidemic. The risk is owed to the fact that the virus mutates quickly and hence the virus may evolve to a form against which the vaccine is ineffective. There are several protease inhibitors1 on the market, including Saquinavir (Roche), Ritonavir (Abbott) and Indinavir (Merck). Several years ago market for therapies aimed at HIV specifically was currently slightly ~$600 million (which is about half the cost of AIDS, with the balance of the cost directed toward treatment of the cancers and infections that attend the disease). The protease inhibitors have not been on the market long enough for their full market potential to have been realized. One estimate predicts that 275,000 HIV positive and AIDS patients in the US in this year, 2000, will produce a market of $755 million divided among about six competing protease inhibitors. Each inhibitor will cost the patient about $4,000-6,000 per year; they must be used in a triple combination chemotherapeutic regimen (which would include typically two RT inhibitors) costing the patient a total of about $12-15,000 per year. The upper estimate of the annual cost is $25,000. The doses of the protease inhibitors used to achieve a pharmacologically active level of drug are high: typically several grams per day. Because the drugs must be given frequently, and several different drugs must be taken, patients take between 9 and 36 pills per day. Patient compliance with such a complicated regimen is problematic. The side effects of AIDS therapy may also be severe. The question for our enterprise is the following. Can we develop a means to make Western AIDS therapy work in the East? Can we make protease inhibitors cheaply enough to make this therapy feasible? What are the possibilities for vaccination? Are there other drugs that will work, and if they are less effective, will they be effective enough to have an impact on this disease? 1 Science, 272: 1876-1890 (1990).