Antiviral Chemotherapy: Successes and Challenges
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Transcript: English(auto-generated)
00:10
Thank you very much. It's a great pleasure to be here. This is my third visit to Lindau, and I hope not my last. I would like to take up some of the problems
00:22
which Dr. Weller has so beautifully described, particularly the viruses. We became involved in about 1970 with looking for antiviral agents that would be hopefully selective and not toxic. As you know, viruses grow in cells.
00:43
This creates the problem that if you have something which will inhibit the replication of the DNA or the RNA of the virus, that it is very likely to do the same thing to the cell. And we had some examples of that in the early 60s
01:01
with compounds which were indeed, had antiviral activity, but which were also very toxic and could therefore only be used externally on the skin or in the eye. Now, if I could have the first slide, please. What I'd like to talk to you about are particularly drugs
01:22
which can affect herpes simplex virus, types 1 and 2, which are the viruses responsible for herpes labialis or cold sores, encephalitis, which is a fatal brain disease, also various forms of genital herpes,
01:43
and varicella zoster, which as you've just heard, causes chickenpox in children but returns later in life in the form of shingles or zoster. Cytomegalovirus, which is a type of herpes virus which can be extremely serious, particularly in immunosuppressed patients
02:02
producing a pneumonia, a blinding retinitis, or colitis. And finally, the human immunodeficiency virus, which is the cause of AIDS. And the three compounds that I'd like to talk about, each has their own particular reason
02:21
for being selective on some of these viruses. And I'd like to compare them and show you some similarities and some differences. Could I have the next one, please? These three compounds are acyclovir, in which this sugar moiety that would ordinarily be present in a nucleoside
02:42
is partially removed. These two carbons in this hydroxyl group are removed. Ganciclovir, which is very closely related to acyclovir, which also has a guanine nucleus and now has an extra CH2OH group so that only one carbon is missing from the sugar.
03:01
And then finally, AZT, which also goes by the name azidobudene, which is the fine-mean moiety, the pyrimidine that's present in DNA, and it has an azido group on this three position. Could I have the next one, please?
03:22
Now, the thing that was unusual about acyclovir was the fact that it inhibited a variety of herpesviruses at very low concentrations without affecting the cells in which these viruses were grown. And this simply shows you a dose-response curve to acyclovir.
03:44
Notice that this is on a log scale, going all the way from 0.005 all the way to 100. And this is percent inhibition. And this is herpes simplex type 1, which is the cause of herpes labialis and encephalitis.
04:01
And you will see that the 50% inhibition point lies at about one-tenth micromolar. For herpes type 2, which causes genital herpes, it's at about one or two micromolar. And for varicella zoster virus, a little bit higher at about three or four micromolar.
04:21
But the cells in which these viruses are grown, such as the Vero cell, which is a monkey kidney cell, is not inhibited 50% till you get to 100 micromolar. Cytomegalovirus is the least sensitive and has inhibition at about 100 micromolar.
04:44
And WI38 is a human cell line in which one grows cytomegalovirus, for example, or varicella zoster virus, and that is not inhibited till you get out to 300. So this represents more than a thousand-fold difference between the sensitivity of the herpes simplex virus
05:04
and the cell in which it is grown. Well, this was very unusual and made us realize that we knew very little, really, about the differences between viruses and cells and began to look at what was the reason for this large differential.
05:22
Could I have the next one, please? Fortunately, by this time, high-pressure liquid chromatography was available. We were able to make radioactively labeled acyclovir to feed it to cells, extract the cells seven hours later, and then put the material on a high-pressure liquid chromatogram
05:42
to try and separate all the radioactive metabolites. And at the top, what you see is uninfected Vero cells that have been subjected to radioactive acyclovir, the radioactivity being shown in the red and the blue representing the ultraviolet absorption
06:03
of the various nucleotides. And what you see after seven hours is that the acyclovir remains unchanged. It's recovered just as when you put it in. On the bottom panel, we see herpes-infected cells subjected the same way,
06:21
but now you see that there are four radioactive compounds there represented by the red bars. We have the unchanged acyclovir, as you see at the beginning, and then you have radioactive materials in the monodie and triphosphate region of the chromatogram
06:43
near where adenine monophosphate, diphosphate, and triphosphate are. And we could show, indeed, that that's exactly what had happened, that the acyclovir had been converted to these three new compounds. And we then began to ask the question, how did this happen?
07:01
What were the enzymes responsible? Could I have the next one, please? And what we found was that the thymidine kinase, which was coded for by the herpes virus, was the enzyme that was responsible for putting on this first phosphate that you see in red onto the side chain that would have been the sugar
07:24
if it had been a normal nucleoside. Once that first phosphate was on, the cell could recognize it as a nucleotide and put on the second and third phosphate. Now, the same thing was true for varicella zoster virus, which also had a specific thymidine kinase
07:44
which would convert this to a monophosphate. And, of course, the real antiviral compound is this triphosphate, not the original acyclovir itself. Unless it's converted to the triphosphate, it's inactive.
08:01
Could I have the next one, please? And what this triphosphate does, as you might expect, is that it inhibits the viral DNA polymerase. In other words, if it replaces the deoxyguanosine triphosphate and as you increase the amounts of drug, you get inhibition of the polymerization reaction
08:23
for making the tetranucleotide. And you'll notice two things, that as you increase the concentration, go from zero down to 0.25 micromolar, you get more and more inhibition of the viral polymerase.
08:40
But another thing is noticeable, and that is that it is not a completely competitive inhibition because if it were, you would have a series of straight lines, and instead of that, you have the lines tapering off so that in about 20 minutes at the higher concentration of the triphosphate,
09:01
the enzyme actually comes to a stop. It's no longer functional. Well, what was happening... Could I have the next one, please? First of all, we were getting more and more triphosphate formed in virus-infected cells, and this is simply to show you that as you add more drug to the medium,
09:22
this is 0.1, 1, 10, and 100 micromolar, for HSV-1-infected cells, you get more and more triphosphate. And as you can see, that also is a log scale so that if you have one micromolar in the medium, you get almost one micromolar acyclovir triphosphate.
09:43
And the similar thing is true with HSV-2, where one micromolar in the medium leads to about five-tenths micromolar triphosphate, and the blue lines represent the amount needed to inhibit the polymerase of that particular virus by 50%,
10:02
so that for HSV-1, the polymerase is exceedingly sensitive and has a KI below one-tenth micromolar. For HSV-2, even at one micromolar in the medium, we have enough triphosphate to inhibit that polymerase.
10:21
But if you now look at the uninfected Vero cell and expose it even to 100 micromolar in the medium, you still don't get enough triphosphate to inhibit the polymerase of the cell. So the selectivity here really lies in the fact that you get activation in virus-infected cells,
10:45
not in uninfected cells, and that the polymerase inhibition constants are quite different. Could I have the next one, please? Now, in addition to this inhibition, we have a situation where the viral polymerase can look upon acyclovir triphosphate
11:03
as though it were a real nucleoside triphosphate. It can incorporate one atom into the growing chain, and in this case, because the base is a guanine, it would couple a hydrogen bond with the cytosine of the template,
11:21
but because there's no 3-prime hydroxyl group, this terminates the chain. And so this termination of the chain results in tiny pieces of viral DNA which are inactive, but is also responsible for the inactivation of the enzyme because once this template is terminated,
11:43
it holds onto the enzyme, which is trying to put an additional nucleotide on there and doesn't let it go to another molecule. Could I have the next one, please? Now, the efficacy of acyclovir is rather a long story.
12:01
It took a number of years to find out all the advantages that we had with this drug, and one of the things that was very apparent early on was that it inhibited HSV-2 very well, the virus that causes genital herpes, and it was first tried in first episodes of genital herpes,
12:26
and it found that it reduced the healing time, it reduced the period of pain, it reduced the viral excretion, but since the real problem with genital herpes are the recurrences, we had to ask the question,
12:41
what would happen in patients who get frequent recurrences of genital herpes, and some people get as many as one a month or 12 a year for many years. So we took a group of about 200 patients in each of these two groups who had had genital herpes for years.
13:04
No, could we go back, please? That one, the bar graph. No, that one. Yes, thank you. What we were trying to do here is to determine
13:20
whether it would be possible to prevent recurrences of genital herpes, and we did this by taking the group and 200 in each group of people who had gotten 12 episodes a year for years. One of these groups was only treated when they got a recurrence of genital herpes,
13:40
and the other group was put on prophylaxis with two times a day of 400 milligrams of acyclovir. A year later, the group that only was treated when they got the recurrences was still getting 12 recurrences a year. The group which was taking the drug prophylactically
14:00
only had an average of about 1.7 recurrences a year. This group was then put on prophylaxis, and for the remaining five years, you'll see that both groups got fewer and fewer recurrences, down to the point here where, after five years, there was only about 0.7 recurrences a year,
14:23
meaning that about 70% of the patients had no recurrences in five years. At this point, we asked the patients if they would like to stop taking the drug, and the answer is no, thank you. However, we did induce some of them to stop because we were curious to know
14:41
whether they still had latent virus in their spinal ganglia, and the answer was yes, they did. The virus had not disappeared. What we were doing was simply preventing the virus from coming out again in the form of a recurrence, so that this drug has become a real prophylactic drug
15:02
for patients with genital herpes. Could I have the next one, please? Dr. Weller has already mentioned the seriousness of varicella zoster infections in the elderly and in the formation of shingles and of disseminated zoster, and this was a study comparing the effect of acyclovir
15:24
with treatment starting within 72 hours of the first outbreak on the duration of pain. Now, one of the most difficult things about shingles is that the pain persists for many months, and the older the patient is, the more this is true.
15:44
This is a group of patients under 50 years of age, and as you can see, the duration of pain, and we're looking here over almost a year, over 200 days, and you can see the proportion of patients who still have pain even out to that length of time.
16:03
Those who were treated with acyclovir, the duration of pain was shorter, and you could see that it was often as much as a month shorter. But this was even more true in the older patients, those over 50 years of age, who had persistent pain.
16:20
A much larger proportion of them, here you see 50%, still had pain out to 120 days, whereas with treatment we could reduce that by a month or more. And so this has again become the drug of choice for the treatment of zoster. Could I have the next one, please?
16:42
Now, the patients who suffered the most, I think, with herpes infections are those who are immunocompromised, those who have transplants, cancer, immunodeficiency diseases, and particularly AIDS patients. And what happens with those patients
17:01
when they get a herpes infection is that the virus continues to be shed for well over two weeks, duration of pain is about the same, and the healing time can be as much as a month. With treatment with intravenous acyclovir, and these are two different studies,
17:21
all in immunocompromised patients, one by Dr. Myers, the late Dr. Myers in Seattle, and Dr. Wade, and you can see there's a very rapid decrease in duration of viral shedding, a decrease in duration of pain, and a decrease in the healing time. And I think because pictures speak louder than words,
17:43
could I have the next one, please? We have a little boy here with an immunodeficiency disease who had a herpes infection of the mouth and it was esophagus, was unable to swallow, and was really going downhill very rapidly. In fact, it was so severe,
18:02
the doctors weren't quite sure whether this was due to herpes. But they nevertheless treated him with intravenous acyclovir – could I have the next one – for five days, and this is the same little boy a week later. So the drug has really justified all of our optimism for it
18:22
in its selectivity, very low incidence of side reactions. And could I have the next one, please? The one problem, as happens with most bacterial diseases as well as viral diseases, is is there going to be a problem of resistance?
18:44
Now, acyclovir has been on the market now for about 15 years, and so we've had a good opportunity to see how much of a problem is resistance. Well, as you can imagine, if the herpes thymidine kinase disappears from the virus,
19:02
then it will not activate acyclovir, and so you will get resistance. And so when resistance develops, it is almost always a reduction in the herpes thymidine kinase. Now, fortunately, this doesn't seem to happen in patients with a good immune response.
19:21
And the place we've seen this happening is mostly in very highly immunocompromised patients, and particularly in AIDS patients. Fortunately, there are some other antiviral drugs available for those patients that are not dependent on the herpes thymidine kinase, such as adenine arabinoside and phosphoformate.
19:45
The other possible ways of getting resistance for acyclovir would be if the viral DNA polymerase were altered so it was no longer susceptible to the triphosphate. That happens very rarely. Even in immunocompromised patients,
20:02
I think we have three examples of that in the literature over these past 15 years. And another possibility is that while the thymidine kinase may not disappear, it may be altered, so it doesn't accept acyclovir as a substrate.
20:20
And that also is extremely rare. There are only a handful of such examples in the literature. So on the whole, resistance has not been a problem, and we realize that from the patients who have genital herpes who have been on the drug for years and who don't develop resistant virus.
20:42
Now, I'd like to turn next to the second compound. Could I have the next slide, please? Namely, Gansyclovir, which, as you can see, is a very close relative of acyclovir, and which was actually made at the same time or in the same laboratory as acyclovir
21:01
and was tested on herpes viruses. Could I have the next one, please? And what we found was that the two were very similar. This represents the inhibitory dose of 50% inhibition for herpes type 1, herpes type 2, and varicella zoster, and the yellow being the acyclovir,
21:21
the orange being Gansyclovir. And you can see that they're very similar in their ability to inhibit these viruses. But where they differed was in cytomegalovirus. These are three different strains of cytomegalovirus, and acyclovir had variable activity on cytomegalovirus.
21:43
It could have an IC50 of about 20, but some strains it would be 100 or higher. Whereas Gansyclovir was quite active on cytomegalovirus, and so it has found its place as a treatment for cytomegalovirus.
22:01
And the question was, why is it so different with respect to CMV as compared with these other herpes viruses? Could I have the next one, please? And when we subjected the cytomegalovirus-infected cells to treatment with a Gansyclovir,
22:21
we found that it made a lot of triphosphate. This is over a period of seven days, the blue representing the amount of triphosphate formed from Gansyclovir, whereas those same infected cells made very little triphosphate from acyclovir. We realized then that we were dealing with a completely different enzyme
22:43
from the herpes TK, which could take either one of these drugs, and tried to find out what that enzyme was. Could I have the next one, please? One thing that became apparent was that when resistance developed to Gansyclovir,
23:01
so that the IC50 was high, those cells were making very little of the Gansyclovir triphosphate. In other words, if you wanted to get resistance, the resistance would be due to the fact that it could no longer make the triphosphate. Cells that were very sensitive with IC50s down in this region
23:22
were making a great deal of Gansyclovir triphosphate. So, we realized that the mechanism of resistance was getting rid of the ability to make the triphosphate. That gave us an opportunity to compare resistant virus with sensitive virus
23:40
to see where they were genetically different and see if we could identify what enzyme was responsible. Could I have the next one, please? And what indeed happened was that there was a piece of the genome of CMV, which, when it was removed, it was an open-reading frame called UL97.
24:04
Actually, the difference between the resistant strain and the sensitive strain, and the interesting thing about this UL97, which controlled the phosphorylation of Gansyclovir, was that it had many regions of homology with protein kinases,
24:21
which was rather unexpected, also with guanilacyclase and with aminoglycoside phosphotransferase. So, it was a very different virus from the one that caused the phosphorylation of acyclovir. And it diverged from protein kinases at certain motifs,
24:41
and we still haven't got a really pure form of that enzyme, although the laboratory is still working on that, realizing that that enzyme may be very useful for finding other drugs that would be phosphorylated by it. Could I have the next one, please? And to show that indeed this UL97 fragment
25:02
was responsible for the phosphorylation, it was transfected into E. coli, E. coli which could not originally phosphorylate Gansyclovir, but when transfected with UL97, it could indeed phosphorylate it. So, we knew we had the right piece of the CMV genome.
25:24
Could I have the next one, please? To give you some idea of the efficacy of Gansyclovir, which is used today mainly in AIDS patients for the treatment of CMV either of the retinitis, which is of a blinding variety, and this shows you the proportion of patients who relapse
25:44
or who do not relapse on treatment. Now, the problem here is that while they're on treatment, they don't relapse, but once they're off treatment, they relapse very quickly, usually within about 30 days. And one reason for taking patients off treatment
26:04
is that Gansyclovir happens to be quite toxic to bone marrow, unlike acyclovir, which is not. Gansyclovir seems to have an effect, and one has to very often take patients off drug to allow the bone marrow to recover.
26:22
And this is what happened here, but eventually resistance develops and even those on therapy begin to relapse. So, we still really need a better drug for cytomegalovirus. And I'd like to turn now to the third drug, AZT, for the treatment of AIDS.
26:43
Could I have the next one, please? Now, to remind you that AIDS is a relatively new disease, at least to our knowledge. In 1979, HTLV-1 was isolated. That is not the cause of AIDS. It's a different lymphotropic virus. It's a retrovirus, rather closely related to HIV.
27:05
And in 1981, the first cases of AIDS were reported in the United States. By 1983, that was associated, was known to be associated with the HIV, or sometime called HTLV-3, as the cause of the disease.
27:23
And the antibody test for HIV then became available in 1984 so that one could test to see whether patients were indeed infected. Well, by 1985, we had almost 10,000 cases, 49% deaths.
27:43
The following year, we had over 12,000 cases. And as you've already heard, we're now well into the hundreds of thousands of cases and a prediction for millions of cases by the end of the century. And the percent of deaths was very high.
28:02
Our laboratory had been working on purine and pyrimidine nucleosides for many years, and one of the compounds that we decided to look at to see whether once this virus became identified, whether or not we had anything which would affect these retroviruses. And the compound that I showed you a little earlier,
28:22
AZT, which had a ZYTO group on the three position of the sugar in pyrimidine, was one of the compounds we looked at and found that it had activity against a few animal retroviruses, like the friend leukemia virus and the Maloney sarcoma virus,
28:41
and then in collaboration with Drs. Broder and Matsuya at the National Cancer Institute, asked them if they would look on it, look for activity in HIV-infected cells. Could I have the next one, please? And indeed what they found was that,
29:01
let me explain this bar graph, this represents percent of viable cells and, at this point, infected cells, infected with HSV-3. Infection causes the cells to be destroyed. It is cytotoxic to the cells if there's no infection, the cells continue to grow.
29:23
This is over a three-day period. Or if this virus is inactivated, it also does not affect the cells. Now, as you increase the amount of AZT in the medium, you can see that at this point, the inoculum, which is where the dotted line is,
29:42
the cells aren't destroyed, but neither do they grow. But when you get to one micromolar, you now protect the cells from destruction and you can go on up to five and ten and you are not affecting the uninfected cells so that you have here at least a tenfold difference
30:01
between the concentration which will affect the virus and begin to affect the cell. Could I have the next one, please? Now, we assumed that the activation of this AZT was similar to what we had seen with ACV,
30:21
with acyclovir and ganciclovir, but that turned out not to be the case. And so what we looked for was how much nucleotide is formed, how much of that AZT is phosphorylated in infected cells versus uninfected cells. And the answer was it didn't make any difference. What you see here is the uninfected cells in blue
30:43
and the HIV-infected cells in orange and the amount of monophosphate or diphosphate or triphosphate is very similar for the two. So, there was no selectivity in this case with regard to activation of AZT.
31:01
So, what was the reason for its activity on the virus and not on the cell? Could I have the next one, please? And the difference lay in the sensitivity of the polymerases. This is the concentration of azitothymidine triphosphate necessary to inhibit the reverse transcriptase,
31:24
which is the polymerase of the virus, where the virus is converting RNA to DNA and is sensitive to at about 50% here, you can see, less than one micromolar, whereas the DNA polymerase of the cell
31:43
is sensitive to hundreds of micromolar. In other words, there's about a 200-fold difference between the concentration that's needed to inhibit the reverse transcriptase and the cellular DNA polymerase. Now, this is a calfimous template
32:02
and even if you use reverse transcriptase with the calfimous template, it is still very much more sensitive than the cellular one. Could I have the next one, please? And this is simply shown in another way here with different amounts of AZT in the medium, showing you how much triphosphate is made,
32:23
and as you noticed before, the amounts of triphosphate formed are really relatively small in the case of AZT, but they're certainly high enough to inhibit the reverse transcriptase, because this represents, for example, at one micromolar, you get one micromolar triphosphate,
32:43
but all you need for the reverse transcriptase is one hundredth of a micromolar, whereas the cellular polymerase, you need over 200, and even for the cellular beta DNA polymerase, about 70. So, the selectivity really lies
33:01
in the sensitivity of the polymerases. Could I have the next one, please? And before I show you some of the clinical data with AZT, I simply want to compare for you these three drugs with regard to activation. This requires activation by the herpes thymidine kinase
33:22
or the zoster thymidine kinase. Gan cyclovir requires activation. It can be activated by HSV and VZV thymidine kinase, but in particular, it's activated by the cytomegalovirus-induced enzyme, and AZT is not selectively activated.
33:43
With regard to their activity on the polymerases, the viral DNA polymerase is more sensitive than the cellular and is also inactivated because of that incorporation. Gan cyclovir, the viral DNA polymerase, is also more sensitive than the cellular one.
34:01
On the other hand, here there's a very large difference between the effect on reverse transcriptase and the cellular one. In the case of a cyclovir, you have DNA chain termination. In the case of Gan cyclovir, you do not because that has an extra three hydroxyl group and it can actually be incorporated into the DNA.
34:25
In the case of zyto thymidine, you also have chain termination because the azido group is not available to extend the chain. Now, a little bit about the activity of AZT. Could I have the next one, please?
34:41
The first trial with AZT was done back in 1986. At that time, we already knew that AIDS was a disease that was fatal, and 180 AIDS patients and 121 so-called AIDS-related complex
35:02
that's not frank AIDS but well on the way toward AIDS were put into two groups. It was a double-blinded study. The group was treated with AZT, which has the generic name as zydovudine, and the other group with placebo. And this was put in the hands of a group
35:22
who were not at borough's welcome but rather a completely separate group who would look at the data every two months to see if they could see any difference between groups A and B. We wanted to have the thing completely blinded from the scientists who were actually starting this study.
35:43
And within four months, it became apparent that there was a difference. And when it was unblinded, it turned out that the 19 deaths had all been on placebo and there had been one death in the patients on zydovudine. And it was at this point that the double-blind study was terminated
36:02
and all patients were then offered zydovudine. And it was really on the basis of this study as well as the subsequent studies that followed that the FDA was convinced that this was a drug that was very important for patients with AIDS.
36:21
Could I have the next one, please? And in fact, in the group of patients who had not been in this study and who were studied over this two-and-a-half-year period, this represents the survival. And you can see by the end of 840 days, we have about only 20 percent survival as opposed to two groups that were being treated with AZT.
36:43
And although this is not what one would like something better than this, nevertheless, we've reduced the mortality down to about 45 percent at the end of two-and-a-half years. Well, what is happening now? Could I have the next one, please?
37:01
As we expected and as has certainly took place fairly rapidly, the development of resistance to AZT. And what was interesting was that the rate at which resistance developed depended to some extent on how sick the patients were when they started the drug. If they were patients who already had AIDS,
37:23
resistance developed, and this shows you the IC50 starting down here at about one-tenth micromolar and rising here to about six micromolar. Within a year, many of these patients had resistant virus. If the patients were symptomatic but not yet have frank AIDS,
37:45
the resistance developed much more slowly, and over a period of three years, you have an increase here of resistance. But if the patients were asymptomatic and were treated for three years, those viruses were still not resistant.
38:00
So, resistance isn't an absolute thing. It depends a lot on the state of the individual because of these patients are immunocompromised to a large extent and have very low CD4 counts. Well, what does one do about resistance and what is the nature of this resistance? Could I have the next one, please?
38:22
The interesting thing about the resistance was that if a patient was treated, for example, for a year with AZT and had now developed resistant virus, so that the IC50, instead of being a tenth, was a ten, that patient was now put on another reverse transcriptase inhibitor,
38:44
like DDI, which is dideoxyinosine, to which the virus was sensitive, even though it was a reverse transcriptase inhibitor. As time went on, the patient or the virus became resistant to DDI, but at the same time, the sensitivity to AZT returned
39:05
so that the development of resistance was not an irreversible thing. It was very strange and we wondered what had happened to this mutated virus that had become resistant when it was treated with a different reverse transcriptase inhibitor
39:22
to which it was now becoming resistant. And when one actually looked at the nature of the mutations, the mutations were all in the reverse transcriptase, in other words, the polymerase. Could I have the next one, please? And the interesting thing was that if you look at a mutation
39:44
from leucine to valine at position 74 of the polymerase, that does not affect the sensitivity of AZT, but it does make it much less sensitive to DDI or DDC. On the other hand, if you have a mutation at 154
40:03
from threonine to tyrosine, you now have about a hundred-fold increase in the IC50 for AZT and at the same time the sensitivity to DDI and DDC go down. Well, what happens if you have both mutations?
40:23
The interesting thing was that these mutations do not disappear, but when both mutations are present, essentially the second one overcomes or changes the resistance of the first one so that you can still have tyrosine to threonine,
40:43
but when you add a second mutation, you get sensitivity to AZT again. And I think this really led us to conclude that what we really needed to treat AIDS were combinations of drugs. I think we had learned that lesson in cancer years ago
41:02
that no single drug was going to do the job and that if resistance was the problem, then the way to overcome or to prevent resistance was to subject the virus to several different drugs acting at different places and see whether or not we could then prevent resistance from developing.
41:23
Could I have the next one, please? About a month ago, a paper appeared in Science by Mellors et al. in Pittsburgh in which they did a retrospective study of AIDS patients that had been in their clinic for a period of 10 years and asked the question,
41:41
could they predict from the viral DNA, the load of viral DNA in the plasma, how long these patients would live? How long would it be before they developed AIDS? And a very striking, this is in a group of about 200 patients that had been followed for a period of 10 years,
42:01
and what they found was that if the viral load, when the patients were first admitted or within the first six months, was less than 5,000 per milliliter, that's 5,000 molecules of viral RNA, those patients lived a very much longer time
42:20
than the patients who had a very high load of virus, over 37,000 and sometimes as much as 100,000 molecules per milliliter in which case the prognosis was very poor, and you can see that half of them had gone by four years. And this is a very important finding
42:41
because it now enables us to check the viral titers in patients who are being treated with drugs or with combinations of drugs and to have some assurance that that is a meaningful value to know, first of all, to how long it will go before they develop AIDS
43:01
and possibly how long they will live so that one doesn't want to have to test a drug for 10 years to find out how effective it is. What one would like to know is how rapidly can that drug decrease the viral titer with the hope that that will then lead to longevity.
43:20
Could I have the next one, please? And such combination studies have begun. This is a study with AZT and another reverse transcript titer, and it's lasting here for a whole year. The combination of two drugs has now gone several years,
43:42
and everyone is convinced that it prevents the formation of resistance, as you can see here, but now we're talking in possibility. Could I have the next one, please? This is a case of patients who had already had AZT for a long time.
44:00
In other words, they had already developed some resistance, and as you can see, if you continue to treat them with AZT, the change in the viral titer actually slightly increased, whereas with a combination, even in these patients that were already resistant to AZT, the combination of AZT with 3TC caused a very profound effect
44:24
on the viral titer, and over the course of, I believe this is 24 weeks, you can see again that this has leveled off and not returned to the original. And because we now have a little more confidence in what the amount of virus means in prognosis,
44:44
could I have the next one, please? This is simply a study done in vitro in our laboratories trying a triple combination, and these triple combinations are mainly two reverse transcriptase inhibitors plus a protease inhibitor,
45:01
because the protease inhibitors which have come out recently acted a completely different part of the life cycle of HIV. They act on the protein coat that is necessary to complete the virus so that it becomes infective to another cell. And by combining, and this simply shows you,
45:23
these red lines are the dose response curves to AZT alone, and then what happens when you add to that AZT two drugs, another reverse transcriptase inhibitor, in this case DDC, plus a protease inhibitor,
45:41
and you can see that what you do to the viral titer here actually represented by the amount of cytopathic effect that you have on the cells and culture down to where you can now get the same effect with one-tenth micromolar AZT. This represents the actual blood levels,
46:02
the nadir and the maximum for AZT, so that you're well below that. And as you can see with these triple combinations, the effectiveness is very much greater than for AZT alone. These triple combinations are currently in clinical trial, and I think the early studies, the early effects,
46:23
can be already seen in the fact that the viral titers have fallen to such an extent in patients that sometimes they're not even measurable. We can only hope for the future that with this triple combination, we will not develop the kind of resistance we've seen with single drugs.
46:41
And if I could have the last slide, please. I simply want to indicate the importance of resistance studies for what they tell us about the activity of the compounds and how we can get around them. And in the case of HIV, we've learned a lot from the variety of polymerase mutants that are present,
47:02
the fact that you can get cross-resistance between two drugs, such as DDI and DDC. On the other hand, you can also get collateral sensitivity so that if something becomes resistant to DDI, it can become AZT more sensitive.
47:20
And the effect of multiple mutations is always not what we expect, that one mutation can actually counteract the other rather than add to it. And then, of course, we've also learned something from resistance studies on the nature of activating enzymes as in the case of ganciclovir with cytomegalovirus.
47:42
I think that's all for the slides. And what I simply want to share with you is a feeling of optimism that we now have that we can do something more for AIDS patients than we've been able to do in the past by understanding something about the mechanism by which these drugs work,
48:03
but also understanding that the only way we're going to get away from resistance is going to be with combination chemotherapy. Thank you very much.