Brendan Larder reported the data from this study. He is with Virco, the European based company that offers the Antivirogram Phenotypic resistance test and the VircoGen Genotypic resistance test, which uses ABI genotypic sequencing. Both tests are only available through LabCorp, which is based in the US. In brief summary, Larder reported that 96/107 highly PI cross-resistant clinical isolates were fully sensitive (< 4-fold resistance or a mean of 2-fold resistance) to tipranavir when tested in vitro. A clinical isolate is a blood sample taken from a person. Eight of the 107 isolates had intermediate resistance (4-10 fold resistance), and only 3 clinical isolates had resistance (>10-fold). This makes tipranavir a potentially good candidate to help people with PI resistance and in need of an effective salvage therapy combination. Larder reported some isolates with PI resistance had hypersensitivity to tipranavir, but I’m unsure if this will translate into actual increased activity in humans. Larder characterized the resistance mutation profiles they observed in this preliminary study and they are reviewed below. A number researchers at the Resistance Workshop expressed they felt this data is promising. I think this data is promising in offering potential help to individuals with PI resistant virus. But the data is in vitro, and preliminary until studies are conducted showing how effective TPV can be for individuals with varying degrees or PI resistance.

TPV is a potent non-peptidic protease inhibitor shown to be active in phase II studies. Previous reports indicated TPV has antiviral activity against HIV resistant to other Pis. Brendan Larder and Upjohn suggested that this could be due to the way tipranavir was designed and the flexibility that it shows in the protease active site. Larder showed a slide picture of a superposition of crystal structures of PNU-140690 (tipranavir, TPV) with crystal a wild-type protease enzyme and a mutant enzyme with V82F/84. He suggested that TPV can bind in different ways to the enzyme depending on whether the enzyme is wild-type or mutant. TPV twists a bit or changes its conformation. A few researchers I spoke with at the meeting did not agree TPV has this characteristic of flexibility. Frankly, in the end what matters in this case and what usually matters is the effect clinically--whether TPV is effective for humans with PI resistant virus.

The objective of this study is to evaluate the activity of TPV against diverse PI resistant clinical isolates, and to identify patterns of PI resistance mutations associated with TPV sensitivity and resistance. They selected previously characterized PI resistant clinical

isolates from Virco’s repository. Larder said they represented a broad spectrum of PI susceptibility; 107 isolates were highly cross-resistant to 3 or 4 PIs; 28 isolates had resistance to a single PI (RTV, NFV, SQV). Larder said they couldn’t find isolates with resistance to IDV that didn’t have cross-resistance to other PIs. He said all recombinant viruses were sequenced to confirm their mutation pattern before phenotypic analysis. Virco uses ABI genotypic sequencing.

The isolates were rated to be sensitive, resistant or to have intermediate sensitivity to TPV. Resistant is defined here as an increase in IC50>10-fold; intermediate resistance is between 4-10 fold increase in IC50, and sensitive is <4-fold. Larder reported that 96 of the 107 multiply PI resistant isolates were sensitive to TPV. Eight isolates showed intermediate resistance or reduced susceptibility, and 3 showed a greater than 10-fold reduced susceptibility.

They wanted to characterize the relationship between the TPV sensitivity or resistance and the mutational patterns observed. In other words, which mutational patterns resulted in sensitivity or resistance to TPV. The mean phenotypic resistance to other PIs for the group of 107 isolates ranged from about 40 to 90 fold: 40-fold to IDV, 90-fold to RTV, 40-fold to NFV and about 45-fold to SQV, but only 2-fold resistant to TPV. The frequency of primary PI genotypic mutations in this group were as follows: 82A (70%), 48V (20%), 90M (70%), 84V (45%). There were no 88 or 30 mutations seen in this group. The frequency of other mutations seen were—10 (75%), 54 (35%), 71 (60%), 77 (40%).

For the ritonavir-only resistant isolates (n=13) they had about 35-fold reduced sensitivty to ritonavir, 1-fold TPV resistance (that is, no TPV resistance), and susceptibility to other PIs (NFV, SQV, IDV). The genotypic mutations observed the most were 90 (40%), 82A (70%), 84V (25%), 54 (80%), 71 (60%), 10 (50%), 46 (20%). There were no 48s.

The mutation patterns seen with NFV resistant isolates (n=10) were quite different. Again, these isolates were only resistant to NFV, and not resistant to other PIs including TPV. There was a dominance of 30 (100%), 88 (80%), and no other primary mutations were seen. But mutations at 71 (60%), 36 (40%), 10 (40%) were seen.

For the saquinavir resistant isolates there were as to be expected a dominance of 48 (100%) and 90 (80%) mutations. Secondary mutations observed were 10 (40%), 36 (40%), 54 (20%), 71 (40%), 77 (40%). Larder reported that surprisingly they observed a 2.5 fold "hypersensitive" response to TPV from the SQV-resistant isolates.

Regarding this observation of hypersensitivity they found 21 isolates in the group of 107 that were hypersensitive (mean 3.3-fold hypersenstitive) to TPV, and 11 isolates that were TPV resistant (mean 13-fold). They defined TPV resistance by a >4-fold increase in IC50, and hypersensitivity was defined as a >2.5-fold increase in sensitivity. The 21 isolates showing on average a 3.3-fold hypersensitivity had mean resistance to the other 4 PIs ranging from 30 to 60 fold. Of the other 11 isolates the mean resistance to the 4 other PIs ranged from 60 to 100 fold and on average they were 13 fold TPV resistant.

With the hypersensitive viruses (n=21) which were again all >2.5 fold hypersensitive, Larder reported seeing a lot of 48 mutations (55%), 82A (75%), and 90M (60%), and secondary mutations: 10 (90%), 46 (45%), 54 (50%), 71 (60%), 77 (50%). But there were no 84V mutations reported seen in this group.

For the TPV resistant isolates (n=11), Larder reported no 48 or 82A mutations were seen. But 82T (50%) and a lot of 84V (80%), and again with 90M (60%) were seen. In general these viruses had a lot of mutations; a lot of secondary mutations were observed: 10 (100%), 20 (50%), 24 (20%), 36 (80%), 46 (40%), no 48, no 50, 54 (55%), 71 (100%), 73 (30%), no 82A, no 88.

Larder summarized that all 28 isolates resistant to only 1 PI (either NFV, RTV, or SQV) were fully sensitive to TPV, or were hypersensitive. Combinations of mutations that included 48V/82A were associated with TPV hypersensitivity. But when the 90M was added to these two mutations this was associated with sensitivity. Combinations of mutations that included 82T/84V or 48V/90M were associated with TPV resistance. The 82T/84V may have the stronger association. But many samples with 84V/90M plus secondary mutations remained TPV sensitive. This probably is due to background mutations and Virco is conducting additional research to explore this.

Upjohn is starting clinical studies to explore PNU140690 in a variety of situations including in individuals with varying degrees of PI resistance.