Research Initiative Treatment Action (RITA!); Vol 5, No. 2 April 1999
L. Joel Martinez
HIV's ability to survive and thrive depends entirely on its ability to penetrate and use the mechanism of a human cell. Given that scientists estimate of the rate of viral replication to be in the vicinity of 10 billion particles per day, it is natural to assume that entering a cell is a walk in the park for the motivated virus.
Not so. The cell presents a formidable barrier for the invading virus. The cell's defenses are not a thick membrane or a set of tightly bound molecules on its surface. The cell's primary defense is its structuremdash;a structure with natural impediments against invasion, a structure that does not give easy access to any antigen.
The membrane of the human cell consists of two sheets of densely packed, electrically charged, hydrophilic (water-loving) chemicals that sandwich a hydrophobic (water-hating) lipid layer. Carbohydrate chains projecting out and a film of tightly bound water surrounding the cell further shield this bilayer construction. (Blakeslee, JAMA, HIV/AIDS Information Center Online, 1999)
HIV may come close to the cell yet not be close enough to bring the virus in contact with the cell's membrane and even docking with the CD4 protein of the CD4 T cell is merely the first step in a complex process of entering the cell.
To fully understand this entry process of HIV into human cellsmdash;a process referred to as "fusion"mdash;it is important to understand a bit of the structure of HIV.
HIV has spikes emanating from its envelope coat. The spikes consist of two parts. The outermost portion is made of molecules, collectively called glycoprotein 120, or gp120. These molecules are attached to a bundle of protein chains tightly bound together. These chains are called glycoprotein 41, or gp41 and are imbedded through the virus membrane at one end and connected, though not tightly, to gp120 at the other end. (See Figure 1.)
The process of fusion begins with a viral particle attaching its gp120 molecules to the CD4 protein of the CD4 T cell. Once anchored, HIV attaches again to a coreceptor on the cell, generally either CCR5 or CXCR4. (See Figure 2.) This double attachment releases gp41 and permits it to unfold like Louganis coming out of a perfect jackknife dive.
Once extended, gp41 harpoons its newly freed end into the cell's membrane. (See Figure 3.) Inserted, gp41 once again folds back on itself like those bobby pins that opened and then through tension came together again to hold your sister's hair in place.
Glycoprotein 41's refolding occurs as a result of the structural affinity that two sections of the protein strands have for each other. The coils of half of gp41 fit themselves neatly and perfectly into the groves created by the other half of the gp41 coil. Suddenly what was a longer three-stranded structure becomes a much shorter six-stranded structure. (See Figures 4 & 5.)
These actions of gp41 unfolding, harpooning the CD4 T cell membrane and folding back bring the virus and the cell membranes close enough to establish contact. Fusion of the two membranes is as simple as two bubbles merging to form a single, larger bubble. Once the membranes are merged the virus spills its contents into the cell and the process of replication within the cell begins. (See Figure 5.)
How does one keep Louganis from making that perfect score on his jackknife dive? One solution would be to attach a big block of styrofoam to his feet so that his hands could not properly touch his toes and his dive would be incomplete. Similarly, a pencil inserted between the prongs of a bobby pin would keep your sister, your mother or your friendly drag queen, for that matter, frustrated and her hair a mess.
Could interfering with the actions of gp41 be as simple as that? The short answer appears to be "yes."
The synthesis of the peptide effectively able to block gp41's conformational changes occurred almost by accident. While working on an HIV vaccine, Dr. Thomas Matthews of Duke University created a chain of 36 amino acids that could somehow prevent HIV from infecting human cells and also prevent the cell-to-cell infection that often occurs in HIV disease.
Later it was confirmed that this 36 amino acid peptide chain, called DP178 at the time, had a great affinity for a section of gp41. Its attachment to that portion of gp41 kept gp41 from successfully refolding back onto itself. If this motion is interrupted HIV does not get close enough to the cellular membrane to complete fusion. (See Figure 6.)
The next significant step in exploring the therapeutic possibilities of this peptide was to find out if it could inhibit fusion in humans. By this time the compound had acquired new names, "T-20" or "pentafuside" and a pharmaceutical sponsor, Trimeris, Inc.
The first issue was of administration of the new drug. T-20 is a large peptide. Scientists determined that oral administration would not work, since it was likely that the stomach would view the drug as food and break it down.
Thus, the first study of the drug involved the continuous intravenous administration of four different doses to patients. The trial lasted fourteen days and involved 16 participants. The doses ranged from 3 mg to 100 mg twice a day. In the four patients given the 100 mg twice a day dose viral load declined to below 500 copies by the bDNA assay at the end of the study. When their plasma viral load was measured by a more sensitive assay measuring down to 40 copies the patients had not reached unquantifiable levels but there was evidence of a 1.96 log drop. No significant side effects were noted in this study.
The second trial of T-20 (TRI003) was a 28-day trial designed to assess the antiviral activity, safety and plasma pharmacokinetics of the drug in six treatment arms. The doses of T-20 ranged from 12.5 to 200 mg per day and were given either by continuous subcutaneous infusion with a portable pump or subcutaneous injections given twice a day. Seventy-eight patients participated in this trial, all having HIV RNA viral loads greater than 5,000 copies and either on a stable antiretroviral regimen or no antiretroviral therapy. Seventy-seven of seventy-eight patients had taken previous antiretroviral therapy. The mean number of antiviral therapies was 9 per patient. Only 2 patients withdrew from the study due to injection site complications. T-20 was able to suppress HIV viral load, with greater suppression occurring in persons at the higher doses. The average maximum drop in viral load varied from 0.3 to 1.6 logs.
In addition, this protocol established that subcutaneous injections were comparable to continuous infusions, eliminating the need to use a portable pump for drug administration.
While the antiviral activity of T-20 has been established, the duration of this activity is still not certain. Many of the patients in the TRI003 trial had virological rebounds from the suppression achieved with T-20. The question of resistance to the compound is still not completely answered.
In vitro studies of the drug indicate that two mutations of gp41 at residues 36 and 38 are all that is required to establish resistance to T-20. (See Rimsky, Journal of Virology, 72:2, p.986, 1998.) It is unclear whether some of the patients in the TRI003 protocol may have developed resistance to the drug. Trimeris has undertaken phenotypic testing to establish whether patients have reduced sensitivity to the drug.
Many of the participants in TRI003 were on T-20 monotherapy and this may account for the early virological breakthroughs. If this is the case then T-20 may have to be used in combination with other antiretrovirals.
In the interim, Trimeris has begun another trial (T-20-205) as a rollover protocol for persons who have participated in previous trials with the drug. In this 48-week trial T-20 experienced participants will be given 50 mg of the drug twice a day in combination with two antiretrovirals specifically chosen for the individual following genotypic testing. The primary purpose of this study is to look for long-term toxicities of the drug and for effects on the CD4 T cells and viral loads of the participants.
Curiously, Trimeris has chosen to test a lower dose than the dose that produced the best results in earlier studies.
T-20 is unique in that it is the only drug to prevent infection of the target cell. It performs its job outside the cell, whereas all current therapies require entry into the cell to perform their inhibition. Further, in theory this approach should work against a wide variety of viruses, even those viruses that have become resistant to existing therapies and T-20 and other fusion inhibitors may have fewer side effects because they perform their inhibition outside the cell. (See "Body Changes and Blood Abnormalities: The Lipodystrophy and Fat Redistribution Conundrum" on page 22 of this issue.)
In a commentary that accompanied publication of some initial T-20 results Douglas D. Richman of the University of California San Diego expressed concern about the "distribution of a large hydrophobic molecule into critical compartments of HIV replication such as the genital tract and central nervous system" (Richman, Nature Medicine, 4:11, p. 1232). If T-20 is unable to penetrate some sanctuary sites it will have to be used in combination with other drugs capable of penetrating the barriers of these compartments.
In this same commentary Richman speculates whether "a smaller molecule with more desirable pharmacologic characteristics (oral bioavailability and central nervous system penetration)" might be able to be designed. (Ibid, p. 1233) Now that the target and its mechanism have come into better focus perhaps new and better drugs are on their way.
As this newsletter goes to press, Trimeris, Inc. issued a press release announcing that the review period for its Investigational New Drug application with the U.S. Food & Drug Administration for its second fusion inhibitor, T-1249, has passed and the company intends to initiate a phase I clinical trial in the second quarter of 1999.
This new compound has shown activity against clinical isolates. Also, it has demonstrated activity against pentafuside-resistant isolates.
The phase I trial of T-1249 will be a multicenter, dose-ranging study designed to evaluate the safety, pharmacokinetics and antiviral activity of the peptide. Sixty HIV-infected patients will receive T-1249 monotherapy by subcutaneous injection for 14 days. Once a day dosing will be explored in the trial.
Development of a drug that is active against pentafuside-resistant virus is good news for the participants in earlier trials who may have become resistant to pentafuside. It is unclear whether those patients will be rolled over to this new compound and whether Trimeris may be creating yet another generation of drug-resistant patients with their monotherapy strategy.
Antigen: a foreign substance, usually a protein that stimulates an immune response.
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Copyright © 1999 - Research Initiative Treatment Action (RITA!). Reproduced with permission. RITA! is published by The Center for AIDS. Contact Thomas Gegeny, MS, ELS, Editor, RITA! for permission to reproduce RITA!. tom@centerforaids.org. http://www.centerforaids.org
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