Day 81: What to do with maraviroc?

I read in the Wall Street Journal today that Pfizer is going to try and reformulate maraviroc (Selzentry) as a microbicide—something I have been saying they should do ever since I saw the efficacy data. Maraviroc is exciting because it is a new class of drug. It’s an entry inhibitor that works by blocking HIV’s interaction with one of the co-receptors, called CCR5. 

For those of you who don’t know, HIV predominantly uses two different co-receptors. The CD4 molecule is the primary receptor. HIV binds CD4, which causes a change in the viral entry proteins, allowing them to bind the co-receptor and enter the cell. Along with CCR5, CXCR4 acts as a major co-receptor (there are other minor co-receptors that HIV can use, but I am not sure how important these players are). So, any cells that HIV is going to infect need to have CD4 and either CXCR4 or CCR5. These are mainly CD4 cells, which carry CXCR4 and sometimes CCR5, and macrophages, which have CD4 and CCR5. 

A virus can be CCR5-trophic, CXCR4-trophic, or dual trophic. These used to be called M-trophic and T-trophic (for macrophage and T-cell, respectively), but these names changes when the co-receptors were discovered. As you might have figured out, M-trophic virus is also CCR5-trophic and T-trophic is CXCR4-trophic. When someone is infected with HIV via sexual contact (I am not sure about intravenous transmission), the virus that is almost always transmitted is CCR5-trophic. (One day I need to write about the viral genetic shift to baseline during transmission).  For some reason, the predominant strains in the body shift to CXCR4 as disease progresses. I don’t know this for fact, but I had always assumed that the reason why the transmitted virus is CCR5-trophic is because of virus-macrophage interaction in the genital tract. This could be totally wrong, though, so please don’t take this as fact. 

Getting back to Maraviroc, you might understand how a CCR5-inhibitor might have some limitations. First, it would only be effective for patients that have predominantly CCR5-trophic virus. This means you have to test the patients for their predominant strain, which can be expensive. Second, viral trophism changes and patients at later stages of disease progression tend to have the CXCR4 virus. Therefore, maraviroc would probably be useful for patients at earlier stages of disease progression, like first- or maybe second-line. The problem is that we already have good regimens for first line therapy, so maraviroc would have to be pretty efficacious to crack the line-up. The other problem is that most medications start as salvage therapy (multiple failures); maraviroc is probably not going to help these patients because it likely won’t be active against their viral strains. So you can see where Pfizer might have a problem with this drug. It is approved for human use, though, so why not try to use it in another manner? 

It seems to me like a good candidate for a microbicide. It’s a small molecule, so it should be fairly stable. Formulation is always an issue, but the real test is going to be what it does to the genital tract. We know it has antiviral activity—that’s not the issue. But there are a lot of compounds that have antiviral activity, which doesn’t mean they’ll work as microbicides. One of the more famous cases is nonoxinol-9, the spermicide. It can kill virus and was thought to be a potential microbicide. The problem was that it increased the infection rate, instead of decreasing it (sound familiar?). It turns out that N-9 also causes inflammation, and HIV loves inflammation. So, any compound that works as a microbicide cannot be inflammatory. Another issue is that microbicide trials are essentially like vaccine trials; that is, they are big and expensive. Last year a promising microbicide was halted early during Phase III trials because it increased the transmission rate. This trial had over 3,000 patients and was at something like eight trial centers on three or four continents. Lastly, it may be that a microbicide is going to need to be used in combination with another microbicide, similar to HAART. This would be to prevent the development of strains that are resistant to the microbicide. Unfortunately, we don’t have any microbicides right now, so that would be a problem. 

I hate to end on a pessimistic note, though. I think it is great that Pfizer is taking my advice and doing this (well, they never consulted me, but they should have). Not only do I hope it is successful, I hope maraviroc makes oodles of money as a microbicide and spurs other pharmaceutical companies to look at microbicide development as a viable business plan. 

M. Linde

Day 72: The first new NNRTI in a decade

The FDA approved Tibotec’s Etravirine (TMC 125) on Friday—the first new non-nucleoside reverse transcriptase inhibitor approved in almost 10 years. Just thought you might be interested. Apparently, it will cost about $8,000 a year.

M. Linde

Day 72: What’s on the virus and what’s needed to make the virus

I’ve just spent the last hour comparing what’s found in HIV virions with what host factors the virus needs to produce virions. Chertova et al. published a great paper a few years ago in the Journal of Virology where they used mass spectrometry to determine what proteins are found in HIV particles produced in macrophages (primary cells). Brass and colleagues recently published a study in Science in which they used an RNAi knockdown screen to determine what host proteins are needed for viral production in HeLa and TZM-Bl cells (cell lines). It is very important to note that HeLas and TZM-Bls (which I believe are derived from HeLas, if memory serves) are very different cells from primary macrophages, so you might expect that HIV might have different requirements for each cell type and that some of the proteins found in one cell type may not even be present in the other types. Just to give you an idea of how different they are, macrophages are monocytic cells that are isolated from donors and can remain in culture for maybe a month (longer maybe?) and HeLa cells are epithelial cells originally derived from a cervical cancer tumor in 1951 at Hopkins. They’ve been around for over 50 years and they grow like weeds. That’s my caveat.

What I noticed between the two studies is that there is virtually no overlap in the proteins found. Now, some of this is because of the assays used—the Brass study failed to find some of the proteins that we know are needed for HIV production, so we know that the screen didn’t identify all the proteins involved in HIV production. Also, Brass ruled out any knockdowns that severely limited cell viability or growth; some of those proteins might be found in the virus. I won’t rule out the human factor either, since the Brass article uses protein abbreviations and the Chertova article uses full names (my eyes are only so good, folks). Still, the amazing lack of concordance is really striking. Of all the proteins found in each of the studies (~200 each), I could only find one host protein that is both necessary for viral production and is also found in the viral particle: KIFC3 kinesin. I don’t know anything about this protein specifically, except that it is a motor and probably is involved in viral protein trafficking. It’s also interesting that the mass spec paper identified a bunch of Rab and VPS proteins that are incorporated in the virions and the RNAi paper found members of these classes that are needed for production—just not the same proteins. This could be due to trafficking differences between the cell types, but I found it odd that none of the proteins from these classes overlapped in the two studies. 

I am still not sure what this means. I have always felt that the host proteins found in the virion are probably important or necessary for viral production. However, after studying this in the lab for four years and having little-to-no success, maybe I need to reevaluate this stance. Certainly, the lack of correlation between these studies can be due to a number of factors. Also, the fact that host proteins in the virion may not be necessary for viral production may not mean they are not important in HIV disease. I would really like to see someone use these two assays in one paper to try and make some of these correlations. Even better, I wish someone would do an RNAi screen and produce virus, testing whether virus produced is less infectious. This might be a great way to produce a therapeutic vaccine. 

M. Linde

Day 67: Is it time to coformulate Viread with Viagra?

Ok, so I am sure the title of this entry will be the standard joke among HIV docs for a bit. Actually, it has probably been made far too many times already. The joke is based on recent data indicating that Viread (FTC/TDF) was shown to decrease the rate of vaginal infection among humanized mice. The mice essentially have human immune systems, which is why the can acquire HIV. Mice that received pre-exposure prophylaxis (PrEP) with Viread did not get infected when exposed vaginally, while 88% of the mice that did not receive PrEP acquired infection. The mice that received PrEP were given Viread 48 hrs and 24 hrs prior to HIV exposure and every 24 hrs for 5 days after exposure. The study was published by Denton and colleagues in PLoS Medicine this month. 

So, these data are kind of exciting. It’s humanized mice, so you can’t get too excited yet. I would assume a monkey study would be next and then a large-scale human trial. You have to take animal studies as they are—they don’t always translate to the same results in humans. But the data could be a boon for high-risk populations, especially women. One of issues with condoms is that it requires the cooperation of your partner. If there is an alternate method to block infection—one that does not require the partner’s cooperation—hopefully the transmission rate might be reduced. This could also be really helpful for serodiscordant couples who want to conceive. Granted, sperm-washing techniques (the process of eliminating virions from semen) are very successful, but this might be considerably less expensive and a lot easier. Viread PrEP could also be helpful for intravenous drug users, but it has yet to be established (as far as I know) if this method would protect against HIV acquired via needles.

Of course, there are also concerns. While I don’t think anyone would advocate using Viread PrEP instead of condoms (especially at this point), we have to establish whether Viread PrEP is as efficient in preventing infection as condom use. If PrEP with Viread is not as efficient as condom use for blocking infection, then you have to question the common utility of Viread PrEP. Additionally, condom use is one time and Viread PrEP might require a weeks worth of adherence, which might be difficult for some. Finally, Viread is not without side effects, although I doubt this would be a major concern considering Viread’s toxicity profile and the fact that the dosing would be intermittent instead of chronic. And cost is always an issue, especially in underdeveloped nations where HIV transmission is rampant. 

All in all, the study is good news. We desperately need more ways to prevent infection. A pill is a good start as it would help circumvent some of the social and political problems faced with condoms. We still need a barrier microbicide, but the data for PReP are encouraging. So, assuming this does work, does Viread go over the counter?

M. Linde

Day 60: HAART and cardiovascular events

For a number of years now there has been some concern over the potential for increased risk of cardiovascular events among patients on HAART. Bozzette and colleagues weigh in on the debate with a very large study from the VA system. They compiled relative risk rates from over 41,000 patients between January 1993 and December 2003, comprising over 168,000 person-years of follow up. The date range is such that it covers pre-HAART, early-HAART, and modern-HAART.

The article, published in JAIDS, shows that, not surprisingly, the risk of death declined between 1995 and 2003. If they hadn’t seen that, you would have to ask some serious questions. Of note, risk of serious cardiovascular event was about the same for any death, dropping over time as mortality did. Inpatient stays for myocardial infarction did not increase significantly over the study and there was no significant increase in risk of cardiovascular event for patients on HAART at 2, 4, or 6 years of HAART use.

The authors note that their cohort is mostly older men who are not as advanced in disease progression. However, the large scale nature of the study is reassuring that these adverse events should not affect HAART use on a population level. Certainly, individual patients may have other cardiovascular factors that play into treatment choice.

M. Linde

Day 58: SOCS1 and gag trafficking

A Japanese group recently reported some interesting findings on a new player involved in HIV trafficking. The report comes in the Proceedings of the National Academy of Science (PNAS), which usually means the article is pretty good, although because of the PNAS submissions process, you have to be careful. Occasionally articles get through that belong in a lower impact journal. This article, by Ryu et al. turns out to be well done. The authors implicate the protein Suppressor of Cytokine Signaling 1 (SOCS1) in gag protein trafficking. They start by doing gene arrays in the T-cell line MOLT-4. Now, cell lines and gene arrays can be deceiving because cell lines are so genetically messed-up to begin with. Ryu and colleagues found that SOCS1 is upregulated in these cells and, importantly, they confirm these findings using RT-PCR and western blots in primary PBMCs from two different donors. 

The authors then go on to show that over-expressing SOCS1 in T cells increases release of viral particles. These viral particles look normal (at least with the one electron micrograph shown). Further, SOCS1 has a dose-dependent increase on intracellular gag cleavage products without decreasing the amount of uncleaved gag (p55) and also increases gag localization at the plasma membrane of the cell. Now, this means that SOCS1 is either increasing the transcription of gag or it is decreasing the degradation. Using a reporter construct with the HIV LTR, the authors show that SOCS1 over-expression does not increase gag transcription. Later, they show evidence suggesting that SOCS1 expression may decrease degradation. 

Using various biochemical assays, Ryu and colleagues show that SOCS1 binds to the matrix (p24) and nucleocapsid (NC) portions of gag and that the p17 binding is the important interaction for increasing HIV release. SOCS1 also has a domain—an SH2 domain—that is necessary for binding. This SH2 domain is important for the function of SOCS1, which is involved in protein ubiquitination. 

The findings are confirmed by SOCS1 knock-down using siRNAs targeting SOCS1. Knocking down the cellular protein causes an increase in gag perinuclear localization. When knocking down SOCS1, gag colocalizes with lysosomal markers, but not late endosome markers. The data suggest that in the absence of SOCS1, gag is potentially shifted to lysosomal degradation. Blocking lysosomal degradation and silencing SOCS1 causes an increase in gag compared with just SOCS1 silencing. Notably, the authors comment on another paper which found that silencing SOCS1 in dendritic cells increases env-specific CD8 responses, CD4 response, and antibody production. My first thought is that in the absence of SOCS1, gag (and possibly env—based on the env CD8 response increase) is not getting to the plasma membrane and is entering into the antigen presentation pathway. 

Gag ubiquitination is known to be important for gag trafficking and SOCS1 is known to be a player in ubiquitination. From this report, it appears that SOCS1 is potentially decreasing gag degradation and increasing the trafficking to the plasma membrane, possibly through SOCS1 modulation of gag ubiquitination. The fact that silencing SOCS1 increases CD8 responses against HIV makes this protein an interesting therapeutic candidate in two fronts: blocking it will decrease viral release and may also increase the cell mediated response, hopefully helping people control existing infection.

M. Linde

Day 55: Maintaning resistance

One of the fascinating aspects of HIV (and really all virology and microbiology) is the relationship between viral evolution and the selective pressures on the virus. HIV, like living creatures, adapts to its environment. If conditions are very harsh or not harsh at all, the viral population tends to be very uniform. In a very harsh environment, only viral particles with a very specific make-up may be able to spread; in the absence of these pressures, the viral particles that can replicate the fastest end up dominating the population. Somewhere in the middle, the viral population becomes diverse, with a number of different quasispecies or genetic variants. This is a key concept for antiviral therapy. Antiviral therapy needs to be stringent enough to place extremely harsh conditions on the virus. That’s why anti-HIV therapy needs to include three active drugs. 

Now, if the selective pressure on the virus during antiviral therapy lessens, which can happen for several reasons (not the least of which is non-adherence to a regimen), the virus can replicate and a viral variant will emerge that can replicate in the less harsh environment. In common terms, this is called resistance development. For useful drugs, there are usually only a few viral variants that can survive in these conditions. These viral variants may have to sacrifice certain advantages they would normally have to replicate under a moderate selective pressure. Often, they don’t replicate as well as normal virus (called wild-type) does in the absence of selective pressure. So, you get a scenario where it is believed that a resistant virus replicates alright when the drug is around, but not quite as well as a wild-type virus does in the absence of drug. 

This may have clinical importance. If the rate a viral variant replicates (called the replicative capacity) is slower, does that mean that a person carrying this variant as the predominant quasispecies will have a slower disease progression? Well, several studies have suggested that this is the case, but unfortunately it is difficult to determine how fast a viral variant replicates in the body. Outside of the body, yes, you can determine this rate, but no one knows if what happens outside the body is true in the body. Now, if the answer to the above question is “yes”, it would argue that for certain antiretrovirals, the development of resistance should not necessarily mean that drug is no longer a useful component of ant-HIV therapy. The idea is to maintain selective pressure on the virus and keep the predominant variant a person carries less fit than the wild-type virus, hopefully delaying disease progression. 

Recently in the Journal of Medical Virology, Gianotti and colleagues looked at the replicative capacity of HIV from people on lamivudine (3TC) monotherapy who have 3TC-associated resistance mutations (the methionine-to-valine switch at position 184 in reverse transcriptase, noted as M184V). Now, since you can’t measure the replicative capacity in the body, the authors looked at ex vivo samples from patients with the M184V mutation, comparing variants at 24 and 48 weeks from patients on 3TC monotherapy to patients who had stopped all therapy. What the authors found was that virus from patients who maintained their resistance mutations had reduced replicative capacity compared with those who “lost” these mutations (note: you don’t really lose these mutations, they just become a minority population while another variant takes over the majority role). Furthermore, those patients who lost the mutations had greater reductions in their CD4/CD8 T cell ratio. 

The study is important because it helps tie the reduced fitness hypothesis to what is observed in the clinic. This strategy of maintaining drugs to keep the viral replicative capacity down may help extend the options for patients who have been through several treatment options or those who have difficulty with adherence (provided they already have the M184V mutation). Of course drug companies also love this stuff because it suggests that some patients should stay on their meds, even after the development of resistance. There are necessary questions to answer; such as, is it really worth staying on these drugs for the reduced replicative capacity? Antiviral therapy is not without side effects and cost. However, now that there has been at least a preliminary link between reduced viral fitness and an immunologic parameter, the answer appears to be headed in the affirmative. Whether this holds for drugs other than 3TC remains to be determined. 

M. Linde