When Angela Kashuba joined the faculty at the UNC Eshelman School of Pharmacy in 1997, she had some catch-up learning to do about her field of research—the pharmacology of HIV therapy. Ten years later, Kashuba’s research is helping to reshape policies and practices of HIV therapy and the prevention of the virus’s transmission.

Not bad for someone who says she saw all of three HIV-infected patients during her fellowship.

Academic research wasn’t the career Kashuba initially envisioned for herself. After earning her bachelor’s degree in pharmacy at the University of Toronto, she worked as a clinical pharmacist for several years before going to the State University of New York at Buffalo for her PharmD. She thought she would return to Toronto (her hometown) afterwards and work as a clinical coordinator in a hospital, but her research experience at SUNY Buffalo persuaded her to pursue academic research instead.

So she did a fellowship at the Clinical Pharmacology Research Center at Bassett Healthcare in Cooperstown, New York, and then came to UNC as an assistant professor in the School’s Division of Pharmacotherapy and Experimental Therapeutics.

“Where I did my fellowship, we didn’t have a lot of HIV work that was going on,” says Kashuba, now an associate professor. “During my fellowship, as I started looking for my own research niche, I realized that there were lots of pharmacology and interaction questions about HIV. As I talked to schools in medicine and infectious-disease physicians, the pharmacology challenges were really in the HIV area.

“When I came here, I had a huge learning curve to get up to speed with how HIV was being treated, what the nuances are for HIV therapy, what all the toxicities were, what the pharmacology questions were. But after that, it really expanded because there are so many pharmacology issues in HIV.”

Kashuba proved to be a fast learner, and her work is now helping to find solutions to some of those issues. Her HIV research focuses on several areas: drug interactions, the use of drugs to prevent transmission, and pharmacology issues in HIV-infected women. Since joining the School, Kashuba has developed an internationally recognized HIV clinical pharmacology program. The program focuses on understanding and predicting drug interactions, optimizing therapy in special populations, and most importantly, understanding the pharmacology of small molecules and their potential use in HIV prevention stretegies. She has been awarded more than $5 million in research grants and contracts. She has written more than seventy journal publications and eight book chapters and has also presented more than eighty peer-reviewed meeting abstracts.

In 2008 alone, Kashuba's group initiated four new research studies in healthy volunteer men and women to evaluate six new drugs and different approaches to prevent HIV transmission. She also initiated an animal model proof-of-concept study with the humanized mouse model for one of the promising compounds. Her group is also involved with ten other domestic and international pre-clinical, Phase I and II studies evaluating pharmacologic prevention approaches.

"No One Is in a Silo"

Collaboration is a vital part of Kashuba’s work, and she says the collaborative atmosphere at UNC is a big reason she came to the University.

“The nice thing about working in the HIV area at UNC is we do have a Center for AIDS Research, and we do have funded cores,” Kashuba says.

There are eighteen CFARs across the country, which are funded by the National Institutes of Health. The UNC CFAR is a consortium consisting of UNC, the Research Triangle Institute, and Family Health International. Kashuba is the director of the UNC CFAR’s Clinical Pharmacology/Analytical Chemistry Core. Because UNC is the only CFAR with a very robust pharmacology core, other CFAR members often come to UNC to run their samples. The core also works with investigators in Haiti, South America, China, Korea, Africa, and the United Kingdom.

“We have everything from the basic science all the way to behavioral and outcomes,” Kashuba says. “It’s a very collaborative atmosphere, which is one of the reasons I came here. After I interviewed both here [at the School of Pharmacy] and with the School of Medicine, I realized that there was multidisciplinary translational research occurring here, and that was not happening at any of the other universities that I had interviewed at. No one is in a silo; everyone is very collaborative and interested in making your research better.”

That atmosphere, Kashuba says, also helps ensure that the research has practical applications.

“There’re lots of people who are thinking about the big picture and how your research question can actually impact policy or impact treatment strategies,” she says. “So that’s one way we’re thinking about research questions: Not necessarily answering a particular question because it’s interesting, but how is it going to impact the way we administer therapy to patients, either here or abroad.

“Everything that we do usually stems from a clinical problem or a clinical question, and we take it back, try to work it out, and apply it again. Everything is very applied, so it’s usually bedside to bench to bedside again, as opposed to bench to bedside.”

Drug Interactions

Unlike treatment for many other diseases, HIV therapy involves a cocktail of antiretroviral drugs. How those drugs interact with each other and with medication that patients might take for other conditions is a key issue in drug development.

“Usually drug interaction is manageable in the general population because people are taking one or two drugs,” Kashuba says. “In HIV, just for the HIV therapy, people are taking at least three, sometimes up to five or eight drugs.”

Kashuba studies drug interaction through phenotyping, a technique that she has been honing since her fellowship. A known drug is given to a volunteer, and researchers measure how quickly a particular enzyme breaks down that drug, which establishes a baseline for comparison. Then for seven to fourteen days, the volunteer is given a drug that is under development. After that time, the volunteer is given the known drug again to measure how long it takes the enzyme to break down the known drug in the presence of the new drug. This will provide an idea about the drug-interaction potential of the drug under development.

The data gathered through phenotyping helps industry and the FDA decide which other drug-interaction studies to pursue. When the information is included in the drug’s package insert, it also helps physicians and pharmacists determine which concomitant medications can be given safely with the drug of interest.

Kashuba’s technique has caught the attention of the FDA. The agency issued a draft guidance document in September 2006 advising drug companies to use the method for testing drug interactions. The FDA used Kashuba’s research, among others, as the basis for the document.

“The drugs they recommend and the methods that they recommend are what I’ve been working on up to this point,” Kashuba says. “We have done a lot of work with industry, a lot of collaboration with industry, to use this technique to understand the drug-interaction potential for their drugs.

“We’re not the only ones who are working in this area, but we have got the techniques down very robustly and in a very sensitive manner. We’re one of the leaders in this particular area."

Using Drugs to Prevent HIV Transmission

The search for a vaccine against HIV has been ongoing for two decades but has yet to yield any viable candidates. The latest setback came in September 2007, when a vaccine being developed by Merck, which had advanced to the clinical trial stage, was declared a failure.

In the meantime, the HIV epidemic has continued to spread. By the end of 2007, an estimated 33.2 million people worldwide were living with HIV.

“If a vaccine is to be developed, it’s probably ten, fifteen years down the road,” Kashuba says. “Well, ten to fifteen years down the road, we’re going to have a whole lot more people infected with HIV.

“What we do have now are drugs, and we have generic drugs in developing countries that are cheap and available and becoming more available, so one of the research interests on campus is to use drugs to prevent the transmission of HIV.”

There are two ways to prevent transmission with drugs—giving drugs orally before and after high-risk behavior or applying drugs topically as a microbicide. Kashuba, who has a particular interest in women’s health, is studying both approaches. She is researching how quickly orally administered drugs get into the genital tract and how high the concentration is. Kashuba also is examining applying drugs topically to the genital tract to prevent the HIV virus from entering the body. That data will give groups such as the World Health Organization and the Centers for Disease Control and Prevention a pharmacologic basis for determining which antiretroviral drugs to move forward in clinical studies of HIV prevention.

“The reality is that we’re probably going to need a number of ways,” Kashuba says. “I don’t know if a pill is going to be more or less effective versus something applied topically. We need a menu of options women can choose from depending on what their situation is.”

Preventing Mother-to-Baby Transmission

Kashuba is collaborating with Charles van der Horst, a professor of medicine at UNC-Chapel Hill, on a study in Malawi that examines the use of drugs to prevent transmission of HIV from mother to infants.

To prevent mother-to-baby transmission, physicians use antiretroviral drugs to keep down the amount of the virus in women during pregnancy and do not allow HIV-infected mothers to breastfeed their infants. Kashuba says that approach has been very effective in North America but is not practical in developing countries, where access to antiretroviral therapy often is limited to patients with weak immune systems. Also, factors such as financial restraints, lack of clean water, and social stigma leave many mothers with no choice but to breastfeed their infants.

“It becomes very complicated in developing countries because there have been studies that have shown that even though babies become infected with HIV [through breastfeeding], if women don’t breastfeed, more babies die because of all the other benefits that breastfeeding gives to babies.”

The collaboration between Kashuba and van der Horst is looking into the possibility of giving HIV-infected mothers antiretroviral drugs only during the first six months of their babies’ lives, when the infants need to be breastfed. If that treatment effectively prevents transmission, it would allow women in developing countries who are not on long-term antiretroviral therapy to breastfeed without passing the virus to their babies. That research is being conducted as part of the Breastfeeding, Antiretrovirals, and Nutrition (BAN) study in the UNC-Malawi Project.

In July 2009, investigators working on the BAN study presented findings that showed giving daily antiretroviral syrup to breastfeeding infants or treating their HIV-infected mothers with highly active antiretroviral drugs is safe and effective in preventing mother-to-child transmission through breast milk. Infants in the study who received either of the interventions had significantly lower probability of HIV infection than those in the control group.

Treat the Patient, Not the Level

Kashuba is embarking on a new study that looks at the amount of antiretroviral drugs women receive during pregnancy. In the United States, physicians use potent antiretroviral therapy during the second and third trimesters to keep the viral load low in the mother so as to prevent transmission to the fetus. However, a study in recent years showed that the concentration of antiretroviral drugs in women’s blood dropped by about 50 percent in the third trimester. That study has led physicians to increase the dosage of drugs for pregnant women in the third trimester to compensate, a practice that concerns Kashuba.

“The problem is we weren’t seeing issues in women before we measured the drug concentrations,” Kashuba says. “So even though the drug concentrations were low, in these women we were seeing good responses to therapy. We weren’t seeing more virus in the blood. We weren’t seeing more babies being infected.

“In pharmacy school, you’re always taught to treat the patient, not the level. What people are doing now is empirically increasing the doses of these drugs in women, potentially exposing them to more toxicity—which they don’t need in the third trimester of pregnancy—without any good benefit.”

When someone takes a drug, part of it binds to proteins in the blood, while the rest remains free floating. It is the free-floating portion—called free drug—that fights the virus. Kashuba says her study will investigate the concentration of free drug in the blood during the third trimester. If the free-drug concentration remains the same despite a decrease in the total drug concentration, then that means the immunity against HIV remains the same and no increase in dosage would be necessary.

“Because women have all these physiologic changes that occur in them [during pregnancy], it may be that the amount of free drug, the active form of the drug, actually isn’t changing in the third trimester,” Kashuba says. “And it may be that we don’t need to mess around with the doses in women and exposing them to toxicity.”

HIV in Older Patients

The age of the HIV-infected population is becoming older, which is both good news and bad.

On one hand, it reflects the success of antiretroviral therapy, as people with HIV now have lifespan similar to that of people without HIV. The bad news, however, is that more older people are becoming infected with HIV.

Kashuba says that due in part to drugs such as Viagra and Cialis, people are becoming more sexually active later in life, and that leads to higher risks of infection and new pharmacology questions.

To answer those questions, Kashuba is collaborating on a study with Kristine Patterson, an assistant professor in the School of Medicine, to study the pharmacology of HIV therapy in the older population, an area that has not been heavily researched before.

“People are sexually active later in life, and there is a recent survey that just came out that showed that older people are less likely to protect themselves from transmitted diseases and less likely to get tested for HIV because they don’t think they are at risk,” Kashuba says. “So we’re seeing more older people being diagnosed with HIV for the first time. What we don’t know is how these drugs are handled in an older population. We don’t know what the toxicities are in an older population. We don’t know whether the transmission rates are increased in older populations. So there’re a bunch of kinetic-dynamic issues that need to be addressed.”

Working with industry

Kashuba is conducting studies with the four newest antiretroviral drugs to receive FDA approval—darunavir (2006), maraviroc (2007), raltegravir (2007), and etravirine (2008).

Raltegravir and maraviroc represent two new classes of drugs to treat HIV infection. Raltegravir is an integrase inhibitor made by Merck. It targets integrase, an HIV enzyme that integrates the viral genetic material into human chromosomes. Without integrase, the virus cannot incorporate its DNA into the host cell’s DNA and use the cell to replicate itself.

Maraviroc, a compound produced by Pfizer, is a chemokine receptor antagonist, a class of drugs that binds to immune system cells and prevents the HIV virus from entering the cells. Kashuba says chemokine receptor antagonists are special because all but one of the existing antiretroviral drugs operate inside the cell—after the virus has invaded the cell. Chemokine receptor antagonists, on the other hand, work outside the cell, keeping the virus out altogether, which is the ideal treatment. It is also administered orally, making it a more practical prophylaxis than the other existing extracellular antiretroviral drug, which has to be injected.

Kashuba’s study on maraviroc found that it has a higher drug penetration in the genital tract than all the other antiretroviral drugs that have been investigated. The study also showed that most of the drug was in a protein unbound state, which means most of the drug found at this site is available to do its work.  The drug also showed a high concentration in the tissues—the site of the initial infection.

Furthermore, the study found that after a dose, the genital tract concentrations of maraviroc remained high for more than three days.

“These data suggest that this drug may be used only around the time of risky behavior rather than every day,” Kashuba says. “Current studies are designed for people to take a drug or two every day, which is not practical in the long term.”

Kashuba’s study was the first to measure the amount of any drug in a protein unbound state in genital secretions, the first to measure tissue concentrations of any orally administered antiretroviral drug, and the first to investigate whether the pharmacokinetics of antiretroviral drugs would favor episodic dosing (used only around the time of risky behavior rather than daily).

“We recently presented these novel data as a platform presentation at the CROI 2008 meeting on the oral formulation of maraviroc,” Kashuba says. “It created a lot of excitement, and was very well received.”

Kashuba also generated a great deal of buzz at the Microbicides 2008 meeting with her data on the topical formulation of the antiretroviral drug tenofovir.

“No one has ever looked to see how much drug stays in the female genital tract after topical dosing with a gel, or how much drug gets into tissues after the gel is applied,” Kashuba says. “We were the first to design this investigation and found very high concentrations of the drug in vaginal tissues that stay put for at least twenty-four hours after dosing. These data make the topical gel look even more promising than the oral pill.”

The data from Kashuba’s investigation lend support for a current study in South Africa that recently started a trial in which tenofovir gel is applied only around the time of possible HIV exposure.

“There was much controversy in the scientific community when this study was started, because everyone thought that the gel needed to be applied daily, regardless of whether risky behavior would be occurring on that day,” Kashuba says. “Based on our data, it appears that this study can soundly move forward.”