April  2003  Volume 1   Number 1

Thoughtleaders
...a series of interviews by Medical Advocates for Social Justice with the people who are shaping our responses to infectious diseases and to the marginalized affected by these diseases.

 The Irresistible Dale Kempf Discusses Kaletra Resistance

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Thoughtleaders © 2003 by Medical Advocates for Social Justice

The Irresistible Dale Kempf Discusses Kaletra Resistance

John Hawes Interviews Abbott's Resistance Maven at the 10th Conference on Retroviruses and Opportunistic Infections

Dale Kempf, PhD, is a Senior Research Fellow and the Senior Project Leader for Antiviral Chemistry, Global Pharmaceutical Research and Development, at Abbott Laboratories in North Chicago. Dr. Kempf received his PhD from the University of Illinois and carried out post-doctoral research at Columbia University prior to joining Abbott in 1984. Since 1987, his research experience has ranged from drug discovery to the analysis of HIV protease inhibitor resistance and its application. Dr. Kempf has coauthored more than 85 scientific publications and holds more than 50 US patents. He has received national recognition as 1997 Inventor of the Year from the Intellectual Property Owners Association and the 1999 Discoverers’ Award from the Pharmaceutical Research and Manufacturers of America for his role in the discovery of ritonavir.
If you have heard Dale Kempf speak at any of the HIV-related conferences held over the past several years, you would not be surprised if someone told you he was a clinical virologist. However, he isn’t. Dale Kempf is a chemist and, as employed as such by Abbott Laboratories, was the “inventor” of the protease inhibitor ritonavir (Norvir)—the discovery that paved the way for the subsequent development of Kaletra (lopinavir/ritonavir), currently the most widely used drug in this class. At these conferences, Dr. Kempf often presents the results of ongoing studies involving Kaletra, especially the pharmacokinetic and resistance work. Medical Advocates for Social Justice sat down with Dr. Kempf during the 2003 Retrovirus Conference in Boston to discuss his presentation of the latest resistance findings at the conference and Kaletra resistance data in general.

MASJ: Before we talk about the resistance findings related to your poster presentation [Comparative Incidence and Temporal Accumulation of PI and NRTI Resistance in HIV-Infected Subjects Receiving Lopinavir/ritonavir or Nelfinavir as Initial Therapy*]  you were involved in the discovery and development of ritonavir and lopinavir, right?

Kempf: Yes, I was one of the inventors of ritonavir in 1991. In early 1995, a colleague of mine synthesized lopinavir, and I helped to shepherd it from the discovery phase into the development phase. This was simultaneous with the filing of the Norvir NDA by the clinical team in December 1995. About the same time we put lopinavir into development, the head of the clinical team, John Leonard, invited me to participate in the Norvir team’s meetings preparing for the FDA advisory panel, acting as a resistance expert. For two months, we had meetings every day—and a free lunch. I mostly went and sat while the team covered essentially every part of the development program. It was a tremendous learning experience for me, getting exposed to the development side. When Kaletra moved into development, I continued to participate in the development team so that over the last 5 or 6 years, I have played a dual role.

MASJ: Most people think of you as a clinical virologist.

Kempf: (laughing) I guess to a certain extent I am. These days I’m still involved with the clinical development side, but I also head up the antiviral chemistry group in Abbott's Discovery Division, working on compounds for HIV and hepatitis C.

MASJ: Was ritonavir developed through rational drug design?

Kempf: Yes, it was definitely rational drug design, but I would distinguish that from computer-assisted drug design, which takes rational design a step further. Although we had some three-dimensional structural information that did impact the design of ritonavir, it was not the iterative process that we would have liked to use and have now used for other projects where you make your compound and then you get a crystal structure and then you make the next one, and so forth. We did not have that capability in the early days with the protease enzyme.

But it’s true that our drug design program was rationally based in that the concept behind the whole series of compounds that led to both ritonavir and lopinavir was that HIV protease exists as a symmetric homodimer. What I mean by that is that it has two identical parts that come together—not like a left and right hand but like two right hands—to make one active enzyme. Since the two pieces are identical, you can rotate the enzyme around halfway and end up with the same structure. So we designed our molecules to have that same symmetric property and that led us to an initial active series of compounds that were unique from inhibitors that others were studying. Both ritonavir and lopinavir maintain some of the remnants of that original design concept.

MASJ: Here you are now, years later, acting as a “clinical virologist” presenting information about resistance mutation data from the pivotal clinical study for Kaletra. Why is this data being presented at this year’s Retrovirus Conference?

Kempf: First of all, it’s important information for practicing physicians. Although I’m not a clinician, it is clear that there are treatment decisions that have to be made every day, and one of the most important decisions for a physician is, “If I decide to start therapy, what therapy do I use?” A lot of information is available to help clinicians, primarily from clinical trials. Physicians currently have some sense of what the risks and benefits are with any given drug combination in terms of long-term suppression of viral replication and the kind of side effects expected. What has been missing and continues to be missing are semi-quantitative data on one specific adverse effect of therapy, the development of resistance. The data presented here represent a first attempt to fill in some of this missing information.

Resistance can represent a risk of therapy in the same sense that side effects represent a risk of therapy. If the virus becomes resistant, then that has long-term consequences. But quantitating the risk of resistance has generally not been attempted with available data.

MASJ: The incidence of resistance from the pivotal trial database has been presented previously. Here you are presenting data on when the resistance mutations appear. What is the importance of the temporal aspects of this data?

Kempf: Well, we have this large clinical study—and much of this data has been presented before [41st ICAAC*  and 11th International HIV Drug Resistance Workshop*]—for which the clinical and resistance data has been systematically analyzed and compared between the two study regimens [3TC+d4T+Kaletra vs 3TC+d4T+nelfinavir]. So our particular approach here was to ask the question, “What is the probability of resistance development over a two-year period if therapy is initiated with one of these two regimens?” To answer this, the appropriate approach is a Kaplan-Meier analysis, which allows an assessment of that probability—in essence, an assessment of the risk of resistance over time. The Kaplan-Meier approach is a less biased way of looking at this information because it accounts for subjects dropping out. It also enables us to assess the risk of resistance in the entire population rather than only in those with detectable viral load, which is what we had done previously. The previous information is very valuable, but it’s like a snapshot; it doesn’t give you a sense of how fast resistance will emerge. Nor does it give you a quantitative sense of the risk of developing resistance after starting therapy with a particular regimen if one stays on that therapy for a given period of time. The hope was—and is—that quantifying resistance rates over time will enable physicians to begin to assess resistance development as a risk of initiating a particular therapy. And we think that’s important in making more educated treatment decisions.

MASJ: Why is it important for clinicians to know how fast resistance develops?

Kempf: The clinicians that I talk to would like to be able to develop a lifetime plan for successful treatment for each of their patients. Many considerations go into devising such a plan, but few things will compromise it like the development of resistance. If we can provide physicians with quantitative data that estimates the risk of resistance development over time, not only to individual drugs but also to individual drugs within different drug combinations, then that information will enable them to plan better for durable success. For example, we know 3TC resistance can be fairly rapid. But in our study, the estimated risk of 3TC resistance after two years was about four times as high in the nelfinavir-based combination regimen than in the Kaletra-based regimen. So the context in which a certain drug is used can make a big difference in determining how long that drug might be effective before resistance develops. These differences can be very important in planning for successful long-term therapy.

MASJ: This data shows remarkably different rates of resistance development between the Kaletra and nelfinavir regimens.

Kempf: That's correct. In our study, the overall probability of developing dual resistance to nelfinavir and 3TC was 20% over 96 weeks of nelfinavir therapy. In contrast, we observed no protease inhibitor resistance in Kaletra-treated patients. There was a large difference in the risk of 3TC resistance as well, even though the two nucleoside analogues used in the study were identical between treatment arms. Nelfinavir-treated patients had a 29% overall risk of 3TC resistance, whereas the risk in Kaletra-treated patients was only 7%.

Our data indicate that the pharmacological properties of Kaletra are superior to nelfinavir. That's reflected not only in Kaletra's superior clinical response but also in the large difference in the risk of resistance development.

MASJ: Do you have any ideas for the complete lack of protease resistance development to Kaletra in this study?

Kempf: We have developed a pharmacological model that may help to explain the rarity of resistance to lopinavir that we've observed in naive subjects. This model also predicts why nelfinavir should show relatively much more resistance. The model is based on what we call the inhibitory quotient or IQ, which we have defined for protease inhibitors as the ratio of the trough drug concentration to the IC50. The IC50 of the drug is the concentration needed to inhibit 50% of viral replication. We have been careful to use a standardized definition of IQ, with trough levels determined in patients and IC50 values determined under conditions as close to in vivo conditions as possible.

Before discussing this model, one needs to understand that in order for resistance to develop, you need to satisfy two criteria: you have to have drug present and you have to have viral replication. If you don't have both simultaneously, you will not get resistance because virus has to be replicating with some drug pressure in order for resistant mutants to arise. The question we can then ask is, "What will happen to viral replication when there are different amounts of drug present?"

We also need to understand the concept of protease inhibitor boosting. Kaletra is a “boosted” protease inhibitor, meaning it combines the active drug lopinavir with a low dose of ritonavir, which is used to boost the levels of lopinavir in the patient’s blood. If you look at a plot of lopinavir plasma levels over time (see figure 1), the concentration of lopinavir is well above the IC50 for wild-type virus during the 12-hour dosing interval when given as Kaletra. The IC50 value for mutant virus is higher, and defines the upper limit of a “zone of highest selective pressure.” If drug concentrations fall to levels within this zone, viral replication will lead to the rise of viral mutants through selective drug pressure. Drug concentrations below this zone will allow preferential replication of the wild-type virus, and concentrations above will suppress all replication. It is the drug concentrations within this zone that will suppress wild-type replication but will allow viral mutants to escape.

The longer drug concentrations remain in this zone, the more you will favor the replication of the mutant virus because you are inhibiting the majority of wild-type virus but the minority of the mutant virus. The width of this zone varies by drug, and for drugs such as 3TC and efavirenz, the zone is large. Resistance comes very easily to those drugs because the drug concentrations remain in this zone for relatively longer periods of time.

We believe that with Kaletra, drug levels don't reside in the zone of selective pressure for very long, and consequently there is little resistance development. With other drugs, such as nelfinavir, that reside longer in this zone, more resistance develops. Let's look at our graph and assume that one or two or more doses of Kaletra are missed, and then ask what happens to the pharmacokinetics of lopinavir?



Click here to enlarge figure 2

When lopinavir is given with ritonavir as Kaletra, the drug concentration initially decays in a somewhat gradual log-linear slope over the dosing interval (see figure 2). If it continued to decline at the same rate, you would eventually get into the zone of highest selective pressure and stay there for a substantial amount of time. But ritonavir’s concentration is decaying also, and at some point the amount of ritonavir is no longer high enough to effectively inhibit the metabolism of lopinavir. We know from our single-dose studies that the drop in lopinavir drug levels following its peak concentration is very steep if ritonavir is not also present. So the data suggest that in nonadherent patients, by the time lopinavir levels decay to the zone of highest selective pressure, the inhibitory effect of ritonavir is mostly gone, and lopinavir passes through the zone relatively quickly. This reduces the amount of overall selective pressure, and therefore resistance is rare.

MASJ: If I understand you, it seems better if the drug levels drop quicker?

Kempf: It's best if drug levels stay well above the IC50 at all times. As long as drug levels are high and well above the zone, then you would like them to drop very slowly. However, if adherence is poor, and drug levels drop into the zone of selective pressure, you don't want them to stay there long. In other words, you would like your drug to have a long half-life as long as there is no viral replication, but a short one once replication begins, so that selective pressure is low. The best analogy I can think of is a light switch. Kaletra is very powerful when it is "turned on". But when doses are missed, it turns itself off rapidly. The mutant viruses grow best in the twilight, which is a short period of time with Kaletra. That's what we think may be unique about Kaletra, although we don't know yet how it specifically compares with other boosted protease inhibitors.

MASJ: This seems like it stands the "forgiveness factor" concept on its head.

Kempf: In fact, it provides an excellent example of how different drugs can have different "forgiveness factors." Kaletra might be an excellent choice for patients who are at risk for low adherence, because if medications are not taken consistently, the overall time in the "zone of highest selective pressure" is less than with some other regimens. To fully understand Kaletra's pharmacologic barrier to resistance, you need to understand pharmacokinetics during times of poor adherence. We don't understand all the details of that yet, but we know that the emergence of resistance has to be related in some quantitative way to the overall time that drug levels induce selective pressure on the virus.

MASJ: Will we see this data presented in the near future?

Kempf: It's hard to prospectively gather this data in patients because we are looking at the effects of missed doses over a long period of time. The pharmacokinetic data we have right now is single-dose data, but we're collecting more data from studies in healthy volunteers where starting and stopping the drug is acceptable. If we can quantitate the terminal phase of the lopinavir plasma decay curve at steady state, we will be better able to estimate the time that lopinavir spends in the zone. Some studies have been done, but I haven't seen the data yet.

MASJ: What else does this model explain?

Kempf: If you construct the same model for nelfinavir, it shows that a missed dose of nelfinavir allows drug concentrations to quickly enter the zone and remain for relatively long periods of time before they drop below the zone. So the overall selective pressure is higher.

Perhaps, more interestingly, this model also appears to apply to the activity of Kaletra in protease inhibitor-experienced patients. In contrast to naïve patients, protease inhibitor-experienced patients who do not adequately suppress virus on Kaletra therapy develop resistance relatively easily. There are probably two reasons for this. One is that several mutations are already in place from prior protease inhibitor therapy. In this case, the model predicts a broader zone because a new mutation is likely to produce a greater amount of resistance compared to that same mutation in the context of the wild type virus. Second, the zone will be higher; so if adherence is imperfect, lopinavir's metabolism will still be adequately inhibited by ritonavir when it enters the zone, and it will pass through more slowly. So the model predicts that protease inhibitor-experienced patients treated with Kaletra would have a higher rate of resistance compared with naïve patients, and in fact that is what we see.

MASJ: You also looked at secondary protease mutations in the study you presented here.

Kempf: There are two reasons we included secondary mutation analyses. One is that we wanted to present all the resistance data we have collected to date. Two is that although no primary mutations to protease developed with Kaletra, there were secondary mutations that could arise due to drug pressure.

Resistance to protease inhibitors has traditionally been broken down into primary and secondary mutations. There is a bit of fuzziness to their definitions, but most primary mutations are those at amino acid positions that make up the walls of the active site cavity where the drugs bind. By changing the shape of the active site, the affinity of the drug molecule for the enzyme becomes lower, and there is some degree of drug resistance.

Secondary mutations are not found in the active site; in fact, some are found on the surface far away from the active site. Nobody really understands on an atomic structural basis how they also contribute to resistance, but it is probably a combination of inducing subtle changes in the affinity of drug binding as well as increasing viral fitness to overcome the sacrifice the virus makes to introduce a primary mutation. Some secondary mutations also exist as polymorphisms, which are mutations that arise from natural variation, not due to drug pressure. In our analysis, we found seven Kaletra-treated patients for whom a new secondary mutation was present at rebound that wasn't present at baseline. Five patients developed a new mutation at position 36, one patient at position 10, and one patient at 71. Now, a couple of important points here: First, mutations at positions 10, 36, and 71 are all common polymorphisms, so we can't readily distinguish whether this was simply natural variation or due to drug pressure from Kaletra- but I wouldn't be surprised if it was due to drug pressure. Nonetheless, phenotypic characterization was obtained on all of those viruses, and there is no difference in the susceptibility of those viruses and that of wild-type virus, so the presence of these secondary mutations doesn't contribute anything significant to susceptibility. Yes, they might be there as a result of selective pressure, but you can't qualify it as resistance because the virus is still phenotypically sensitive.

In contrast, we found that secondary mutations much more commonly emerged in the nelfinavir- treated patients who experienced rebound-about 50% of these patients. Some mutations were found at polymorphic positions 10, 36, and 71, but there also were some at positions 46, 54, and 88, which are not common polymorphisms, indicating significant drug selection. So these secondary mutation analyses are another way of looking at the overall selection pressure of a drug regimen.

MASJ: You have done a lot of resistance studies to date. How does this study fit into your overall resistance program?

Kempf: It fits into our overall program by continuing to fully characterize Kaletra resistance. The traditional way of characterizing resistance is to start out with wild-type virus, put drug pressure on it to get mutant virus, and then you describe what the phenotype and genotype of those mutant viruses are. We have done that in the test tube, but we are still waiting for this to happen in antiretroviral-naïve patients. So in the meantime, we used a different tactic to characterize resistance in patients by looking at mutant viruses generated in patients who had failed on other protease inhibitors. Then, we asked the following questions: "How susceptible are these viruses to lopinavir?" And, "if they have reduced susceptibility, what mutations in these viruses contribute to that reduced susceptibility?" That is, we correlated the phenotype and genotype with respect to lopinavir resistance. Using statistical methods, we came up with a set of 11 mutations, which we called the "lopinavir mutation score." 1 We were then able to characterize the response to Kaletra in protease inhibitor-experienced patients using the mutation score. We found that patients with fewer mutations did the best, but that Kaletra was powerful enough to retain at least some activity against viruses that have up to seven mutations. We have also taken those protease inhibitor- experienced patients who didn't completely respond to Kaletra and characterized the additional resistance that emerged. So we have looked at the effect of preexisting mutations on response to therapy, and we also have looked at the effect of preexisting mutations on selection of additional resistance.

This present study in naïve patients fits in with the broader scope of our program in that we would like to characterize not only what happens to make the virus resistant but also under what conditions can the virus become resistant, and how often does it become resistant. Those are the key questions we are asking with this study. So we haven't yet been able to characterize a "resistance pattern" for Kaletra in naïve patients, but we have good information on how often resistance is going to happen and we know it is going to be rare.

MASJ: Any other future plans?

Kempf: Yes. Regarding Kaletra, we are in the middle of analyzing more data from protease inhibitor-experienced patients, trying to assess what makes these mutant viruses either likely or not so likely to select additional resistance by Kaletra and under what conditions. These data appear to be consistent with our pharmacological model as well and are part of our continuing program focusing on clinical outcomes from a resistance standpoint. That kind of educational information will hopefully contribute to improved therapeutic strategies and better quality of life for those living with HIV. In addition, we continue our discovery efforts into finding new protease inhibitors for HIV and novel drug therapies for hepatitis C. We believe that the expertise we've developed so far will help us to develop new and innovative drugs in both of these areas.

1 These 11 mutations are detailed in the full-text paper. Reduced Susceptibility to the Protease Inhibitor Lopinavir Among Viral Isolates from Protease Inhibitor Experienced Patients. Kempf DJ, J Isaacson JD, King MS, et al.  J Virology, 2001, 75, 7462-7469

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John Hawes is a freelance medical writer with an extensive working background in medical education, publishing, and special projects.  His experience includes biopharmaceutical products related to infectious disease, including HIV infection, and oncology. John can be contacted via e-mail at  .jhawes7@comcast.net.
 
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