HIV/AIDS drugs could be repurposed to treat AMD, researchers suggest

Drugs that have been used for the past 30 years to treat HIV/AIDS, could be repurposed to treat the dry form of age-related macular degeneration (AMD), a new study suggests. Age-related macular degeneration is a progressive condition that is untreatable in up to 90 percent of patients and is a leading cause of blindness in the elderly worldwide. The two forms of age-related macular degeneration, wet and dry, are classified based on the presence or absence of blood vessels that have invaded the retina.

Age-related macular degeneration is a progressive condition that is untreatable in up to 90 percent of patients and is a leading cause of blindness in the elderly worldwide. The two forms of age-related macular degeneration, wet and dry, are classified based on the presence or absence of blood vessels that have invaded the retina. A detailed understanding of the molecular mechanisms underlying wet age-related macular degeneration has led to several robust FDA-approved therapies. In contrast, there are no approved treatments for dry age-related macular degeneration thus far.

Nucleoside reverse transcriptase inhibitors (NRTIs) are the most widely used class of anti-HIV drugs. NRTIs are thought to be therapeutic in HIV/AIDS patients because they target the enzyme reverse transcriptase, which is critical for replication of HIV. Previous work from the Ambati lab found that a type of toxic molecule called Alu RNA accumulate in the retina to cause dry age-related macular degeneration; interestingly, Alu RNA and HIV are similar in that they both require reverse transcriptase to fulfill their life cycle.

In their Science publication, Fowler et al. report that multiple FDA-approved NRTIs prevented retinal degeneration in a mouse model of dry age-related macular degeneration. Surprisingly, this effect of NRTIs in the eye was not due to the well-known function of these drugs to inhibit reverse transcriptase. Instead, NRTIs blocked an innate immune pathway called the "inflammasome," even in experimental systems in which the NRTIs were not capable of blocking reverse transcriptase. In their report, they also showed that NRTIs were effective in other disease models that share common signaling pathways with the dry age-related macular degeneration model, including the "wet" form of age-related macular degeneration -- a disease that when treated still does not lead to substantial vision improvement in two-thirds of patients -- and graft-versus-host disease which is the major obstacle preventing successful allogeneic hematopoietic stem cell transplantation.

"Repurposing of NRTIs could be advantageous, for one, because they are very inexpensive. Moreover, through decades of clinical experience, we know that some of the drugs we tested are incredibly safe. Since these NRTIs are already FDA-approved, they could be rapidly and inexpensively translated into therapies for a variety of untreatable or poorly treatable conditions," said Benjamin Fowler, the lead author and a postdoctoral fellow in the Ambati lab. Ambati added, "We are excited at the prospect of testing whether NRTIs could be effective in halting the progression of age-related macular degeneration in patients."

NRTIs were originally designed to treat cancer in the 1960s. They re-emerged in the late 1980s and became the first drugs the FDA-approved to treat HIV/AIDS.

Semen directly impairs effectiveness of microbicides that target HIV

Researchers have discovered why microbicides developed to prevent HIV succeed in the lab but fail in clinical trials: Semen. Semen enhances the infectiousness of HIV by causing the virus to cluster together, increasing its ability to attach to and infect cells. This effect is then sufficient to override the antiviral properties of the microbicides.

In the fight against HIV, microbicides -- chemical compounds that can be applied topically to the female genital tract to protect against sexually transmitted infections -- have been touted as an effective alternative to condoms. However, while these compounds are successful at preventing transmission of the virus in a petri dish, clinical trials using microbicides have largely failed. A new study from the Gladstone Institutes and the University of Ulm now reveals that this discrepancy may be due to the primary mode of transportation of the virus during sexual transmission, semen.

"We think this may be one of the factors explaining why so many drugs that efficiently blocked HIV infection in laboratory experiments did not work in a real world setting," explains co-first author Nadia Roan, PhD, a visiting scientist at Gladstone and an assistant professor-in-residence in the Department of Urology at the University of California, San Francisco. "We've shown previously that semen enhances HIV infection, but this is the first time we've shown that this activity markedly reduces the antiviral efficacy of microbicides."

Semen markedly enhances the infectiousness of HIV through the presence of protein aggregates called amyloid fibrils. HIV binds to these fibrils, causing the virus to cluster together and increasing its ability to attach to and infect cells in the host -- in this case the sexual partner of the infected individual. This effect is then sufficient to increase the infectiousness of the HIV virus, thereby diminishing the antiviral properties of the microbicides.

In the study researchers tested the effectiveness of several different types of microbicides targeting the HIV virus on cells that had been exposed to HIV alone compared with cells that were treated with both HIV and semen. Across the board, they saw that not only did the cells with semen have rates of HIV infection approximately ten-fold higher than normal, these microbicides were up to twenty times less effective at blocking the virus in these cells than in those not exposed to semen.

Senior author Jan Munch, PhD, from the University of Ulm says, "Our findings suggest that targeting amyloids in semen is an alternative strategy to improve drug efficacy. The next step is to create a compound or cocktail of drugs that targets both the HIV virus and these amyloid fragments and to test its effectiveness. Also, given that semen is the main means of transmission of HIV, future testing of microbicides in the lab should be performed in the presence of semen to better predict antiretroviral efficacy in real life."

To test that it was the HIV-enhancing ability of semen that was having this effect on the microbicides and not some other substance, the researchers repeated the experiments using semen from men whose semen does not enhance HIV infection due to a disorder called ejaculatory duct obstruction. In the presence of these samples, there was no decrease in effectiveness of the anti-viral microbicides, confirming the importance of the HIV-promoting effects of semen in counteracting the effectiveness of these drugs.

Most microbicides work by targeting the virus itself, attempting to break it down or blocking its ability to infect a cell. However, the heightened infectiousness of HIV in the presence of semen appears to over-power any anti-viral effects the microbicides possess. The one exception to this finding is a different type of microbicide that acts on the host cells' receptors, stopping the virus from latching on from within. In the current study, this microbicide, called Maraviroc, was equally effective in preventing infection both with and without the presence of semen.

"There are important potential clinical implications for this study," says Warner Greene, MD, PhD, director of the Gladstone Institute of Virology and Immunology and a senior author on the paper. "Microbicides were originally developed as a way to empower and protect women in sub-Saharan Africa who often don't have a way to negotiate safe sex or condom use. However, the first generation of microbicides were largely ineffective or worse, some even leading to increased transmission of the virus. This study sheds light on why these microbicides did not work, and it provides us with a way to fix this problem by creating a new compound drug combining antivirals and amyloid inhibitors."

Altered milk protein can deliver aids drug to infants

A novel method of altering a protein in milk to bind with an antiretroviral drug promises to greatly improve treatment for infants and young children suffering from HIV/AIDS, according to a researcher.

That's critical because an estimated 3.4 million children are living with HIV/AIDS, the World Health Organization reports, and nine out of 10 of them live in resource-limited countries in sub-Saharan Africa, where effective antiretroviral treatments still are not widely accessible or available. International medical experts believe less than a third of affected children worldwide receive an antiretroviral drug.

Complicating treatment is that most antiretroviral drugs are not well tolerated by very young children. One of the most commonly prescribed antiretroviral drugs for treating and preventing HIV infection, Ritonavir, has undesirable side effects and important oral-delivery problems. Its physicochemical properties challenge its administration to infants, explained Federico Harte, associate professor of food science.

"Ritonavir has a high hydrophobicity and low solubility in water, which lead to a low dissolution rate in the gastrointestinal fluid and, hence, to insufficient bioavailability. The liquid formulation used to treat infants over one month of age contains 43 percent ethanol and has an awful flavor that has been described as bitter-metallic, medicinal, astringent, sour and burning," he said.

"Moreover, when coming into contact with the stomach mucosa, Ritonavir causes nausea, vomiting and diarrhea. Therefore, we need to develop alternative pediatric formulations of Ritonavir and overcome its poor water solubility to improve its oral administration to infants and children."

To solve that problem, Harte looked to a group of proteins in cow's milk celled caseins. Casein proteins form spherical aggregates called casein micelles, which are responsible, incidentally, for the white color of milk. The casein micelles in mammals' milk are natural delivery systems for amino acids and calcium from mother to young, and Harte reasoned, might deliver Ritonavir molecules as well.

"I have been working with bovine casein micelles for a few years now, and we have investigated the structure and functionality of these proteins," he said. "What we found is these micelles are able to carry molecules that have very little solubility in water, that have low molecular weight and that are very hydrophobic -- such as Ritonavir."

Significantly, Harte discovered in his research that subjecting milk to ultrahigh-pressure homogenization enhances the binding properties of the casein micelles. In previous studies, he learned that casein micelles could be bound to triclosan -- an antimicrobial used in deodorants -- and vitamin D, which is added regularly to skim milk.

Normal milk is homogenized at 10 to 15 megapascals, he pointed out. Milk in this research was homogenized at between 400 and 500 megapascals, disassociating the casein micelles and improving the protein's binding qualities to attach to drug molecules.

"As a result of this enhanced binding of molecules, we believe a milk powder containing Ritonavir can be used as baby formula, providing a transport system for a drug that is not very soluble in water. Right now we are running tests, and we are in the final stages of an experiment in which we gave three different formulations to piglets," Harte said.

"We are taking blood serum samples every three hours to study the kinetics of the drug in the piglets," he said. "The hope is that -- and we don't have the data yet -- we find that the Ritonavir is being adequately delivered by the protein in milk. So if that works, I think we are pretty close to having a formulation that can be used with hydrophobic drugs."

Harte noted that with his proposal for research funding he included a letter from a pediatrician at St. Jude Children's Research Hospital describing the challenge of orally administering Ritonavir to infants and young children.

"She has been treating patients with AIDS, and the awful flavor and ethanol content of Ritonavir were big issues for her," he said. "I am hopeful that this may lead to an application that works against AIDS. We have not done any clinical trials yet, so we will need to get data from those trials to say for sure."

First Immature form of HIV seen at high resolution surprises researchers

The first structure of the immature form of HIV at a high enough resolution has been obtained by researchers, allowing them to pinpoint exactly where each building block sits in the virus. The study reveals that the building blocks of the immature form of HIV are arranged in a surprising way.

Sientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany and collaborators from Heidelberg University, in the joint Molecular Medicine Partnership Unit, have obtained the first structure of the immature form of HIV at a high enough resolution to pinpoint exactly where each building block sits in the virus. The study reveals that the building blocks of the immature form of HIV are arranged in a surprising way.

"The structure is definitely different from what we'd expected," says John Briggs from EMBL, who led the work. "We assumed that retroviruses like HIV and Mason-Pfizer Monkey Virus would have similar structures, because they use such similar building blocks, but it turns out that their immature forms are surprisingly different from each other. At this point, we don't really know why."

Briggs and colleagues used cryo-electron microscopy to study the protein lattice that surrounds the virus' genetic material. After infecting one of the cells in our immune system, HIV replicates, producing more copies of itself, each of which has to be assembled from a medley of viral and cellular components into an immature virus. This is the form that leaves the cell. The protein building blocks that make up the virus are then rearranged into the virus' mature form, which can infect other cells.

The first cryo-electron microscopy images of immature HIV, obtained at EMBL in the 1990s, surprised researchers by showing that the virus did not have a regular symmetrical structure, as had been assumed. That meant it was going to be difficult to get a detailed picture of the structure of its protein lattice. Two decades on, by optimising both how data is collected at the microscope and how it is analysed, Florian Schur, a PhD student in Briggs' lab, has now achieved an unprecedentedly detailed structure.

With this structure in hand, scientists have a basis to probe further. They can use it to decide where to focus efforts for achieving the even greater detail needed to explore potential drug targets, for instance. It will also enable researchers to understand how mutations might influence how the virus assembles. And the techniques themselves can be applied to a variety of questions.

"This approach offers so many possibilities," says Schur. "You can look at other viruses, of course, but also at complexes and proteins inside cells, with a whole new level of detail."

In future, the EMBL scientists will use the approach to look at other viruses and at the vesicles that transport material inside cells. They also aim to push the techniques even further, to allow them to see other parts of the viral proteins that are currently beyond their reach, but which they suspect play an important role in HIV maturation.

"In the long term, we'd also like to investigate how drugs which are known to inhibit virus assembly and maturation actually work," Briggs concludes.