 |
|
|
| |
GSK CCR5 '140'
|
| |
Two abstracts were presented today at the Resistance Workshop. The first immediately reported below was an oral presentation & the second abstract was a poster.
--Oral presentation today by K Kitrinos GlaxoSmithKline) reported study data on one patient who received GSK CCR5 inhibitor. Mixed CXCR4/CCR5 tropic virus was seen transiently after initially being identified as CCR5 tropic at the beginning of the study. But patient still had .53 viral load reduction.
"Clonal analysis detects pre-existing R5X4-tropic virus in patient demonstrating population-level tropism shift on 873140 monotherapy"
K Kitrinos1, C LaBranche1, M Stanhope1, H Madsen1
and J Demarest1
1GlaxoSmithKline, Research Triangle Park, NC, USA
In a 10 day monotherapy study, 873140, a CCR5 antagonist, demonstrated potent antiviral activity (mean 1.66 log decrease in viral load at nadir, Lalezari et al., ICAAC 2004). Analysis of HIV-1 envelope (Env) tropism on one subject who
received 200 mg 873140 once daily detected R5X4- tropic (dual/mixed) virus at day 10, though only R5-tropic virus was detected at screening and days 1 and
5. Subsequent analysis revealed reversion to an R5-tropic only phenotype by day 24, with equivalent sensitivity to 873140 (fold IC50) as seen at day 1.
HIV Env tropism and Fold IC50 at the population level were determined by the ViroLogic PhenoSense HIV Entry assay. A clonal analysis of HIV env tropism, sequence and IC50 was performed at days 1, 10 and 24.
RESULTS
--30/31 subjects receiving 873140 remained R5-tropic at all other timepoints tested (days 5, 10, and 24)
--1 subject exhibited transient dual/mixed population tropism at day 10
--patient had -0.53 log reduction from baseline on day 11
--PK and receptor occupancy were comparable to others in the same dosing group
--Transient dual/mixed detection at day 10 is a mixture of R5 and R5/X4 viruses
--R5/X4 virus was present at days 1 and 24, but below the population assay threshold
--Both R5 and R5/X4 clones were sensitive to 873140 when tested on CCR5-only cells
--The V3 loop sequences of the R5 and R5/X4 Envs were distinct from each other
--Phylogenetic analyses and presence at day 1 suggest the presence of R5/X4 viruses at day 10 is the result of emergence, not switch
PROGRAM ABSTRACT SAID:
Of 48 clones evaluated for tropism at each timepoint, nearly half were non-functional. Of the functional clones, the dominant species were R5-tropic
at days 1 (22/23, 96%), 10 (16/22, 73%) and 24 (22/24, 92%). Interestingly, a low frequency of R5/X4 (dual)-tropic clones was detected at days 1 (1/23, 4%)
and 24 (2/24, 8%).
On day 10, 6/22 (27%) clones were R5X4 (dual)-tropic. No X4-tropic only clones
were detected at any timepoint. The dual-tropic clones had signature sequences localized to the V3 region that were similar to each other yet distinguished them from the R5-tropic clones. All clones were sensitive to 873140 in the PhenoSense assay (fold IC50 range 0.05-0.85), with the dual-tropic clones being slightly more sensitive than the R5-tropic clones.
The data indicate that the change in tropism at the population level observed on day 10 was the result of the emergence of pre-existing dual-tropic virus(-es) that were present but below the limits of detection on day 1. R5-tropic virus remained the dominant species at all timepoints and no X4-tropic only virus was detected. Population and clonal viruses were equally sensitive to 873140. Viruses present in a subject's quasispecies, which are below the limits of detection with currently available tropism assays, may become detectable following monotherapy with a
CCR5 antagonist.
POSTER AT WORKSHOP
"Studies with 873140, a Novel CCR5 Antagonist, Demonstrate Synergy with Enfuvirtide and Potent Inhibition of Enfuvirtide-Resistant R5-tropic HIV-1"
CC LaBranche1, D Davison2, RG Ferris1, GW Koszalka2, JF Demarest1, LR Boone1, ML Greenberg2
1GlaxoSmithKline, Research Triangle Park, NC; 2Trimeris, Inc., Durham, NC
...studies predict that 873140 will be effective against enfuvirtide-resistant R5-tropic HIV....
AUTHOR DISCUSSION
In this study, we have shown that 873140 potently inhibits infection of cMAGI cells by clinical HIV-1 isolates known to be resistant to enfuvirtide and that enfuvirtide resistance did not significantly alter the IC50 for 873140. This suggests a lack of cross-resistance between these two HIV entry inhibitors. Additionally, the results of drug combination studies suggest the potential for synergism between 873140 and enfuvirtide. This finding is consistent with previous studies demonstrating synergy between prototype CCR5 antagonists, TAK-779 and SchC, and enfuvirtide (Reeves et al, 2005; Tremblay et al, 2002). A mechanism has been proposed to explain the in vitro synergy observed between enfuvirtide and CCR5 antagonists (Doms et al. 2004). The sub-IC50 concentrations of the CCR5 antagonist present in the matrix drug combination in these studies may reduce the amount of CCR5 available to the virus, thus slowing the kinetics of infection and prolonging the availability of the enfuvirtide binding site. Perhaps more importantly, however, our data show that there
is no antagonism between 873140 and enfuvirtide, supporting use of these two entry inhibitors in combination in potent ART for treatment-experienced patients.
AUTHOR CONCLUSIONS:
--In vitro drug combination studies assessing the antiviral effects of 873140
and enfuvirtide demonstrate a lack of antagonism and, in fact, mild
synergism
--873140 is potently active against viruses that are resistant to enfuvirtide,
the currently approved entry inhibitor. Combination therapy is possible.
ABSTRACT
Background: Development of novel antiretroviral agents is critical in the face of
continuing evolution of viruses resistant to currently available therapies. It is
essential to characterize the activity of new drugs against resistant viruses as well as their interaction with drugs in current antiviral regimens. In this study, 873140, a novel spirodiketopiperazine CCR5 antagonist currently in Phase 2/3 clinical trials, was tested in vitro for efficacy against viruses resistant to the only currently approved entry inhibitor, enfuvirtide. 873140 and enfuvirtide were also tested for their antiviral activity in combination.
Methods: HIV-1isolated from patient PBMCs were characterized for susceptibility
to enfuvirtide using MAGI and cMAGI target cells. Two R5-tropic, enfuvirtidesensitive and 4 R5-tropic, enfuvirtide-resistant isolates were subsequently tested for susceptibility to 873140 using the same format. In separate assays, the ability of the combination of 873140 and enfuvirtide to inhibit HIV-1 Ba-L was evaluated. HOS-CD4-CCR5-LTR-luc cells were pre-incubated with checkerboard titrations of enfuvirtide and 873140 for 1 hr at 37oC, then exposed to an inoculum of HIV-1 Ba-L sufficient to produce a signal-to-background of at least 50 fold in the absence of drug.
Results: Both enfuvirtide-sensitive and -resistant R5-tropic HIV-1 isolates were
highly susceptible to inhibition by 873140. The median IC50 was 0.28nM (range
0.17-0.38) for enfuvirtide-sensitive isolates and 0.26nM (range 0.12-0.32) for
enfuvirtide-resistant isolates. For the enfuvirtide/873140 combinations, the
average deviation from dose wise additivity was -0.07 +/- 0.02 (p=0.0009),
indicating a slight but statistically significant synergistic interaction between
these agents.
Conclusions: The data from these studies predict that 873140 will be effective
against enfuvirtide-resistant R5-tropic HIV-1. In addition, these in vitro experiments demonstrate synergism and, importantly, the absence of antagonism between 873140 and enfuvirtide, suggesting that these agents would be suitable for use in combination. This study suggests that entry inhibitors used in combination may provide new therapeutic approaches for the treatment of ART-experienced individuals.
Two virus isolates from the same patient, one enfuvirtide-sensitive and the other
enfuvirtide-resistant, were both potently inhibited by 873140 in the cMAGI cell
assay, with IC50 values of 0.38 and 0.32 nM, respectively. These data show that
resistance to a fusion inhibitor does not affect susceptibility to the 873140 CCR5
antagonist in this clinical virus isolate.
The ability of combinations of enfuvirtide and 873140 to inhibit the
replication of HIV-1/Ba-L in HOS.CD4.CCR5-Luc cells were analyzed based on the principles of dose-wise additivity. Fractional Inhibitory Concentrations (FIC) were determined at the 50% inhibitory level. The resulting isobologram indicates a synergistic interaction between enfuvirtide and 873140 with an average deviation from the line of additivity of -0.07 + 0.02 (p=0.0009).
INTRODUCTION
One entry inhibitor, the potent inhibitor of HIV fusion enfuvirtide, has been
approved for therapeutic use.
Another type of entry inhibitor, the CCR5 antagonist 873140, is currently in
Phase 2/3 clinical trials.
These two antiviral agents target distinct and sequential steps in the HIV entry cascade.
Combination therapy is required to achieve durable suppression of HIV
replication. The availability of a second entry inhibitor raises the possibility of
therapeutic strategies that combine entry inhibitors acting at different steps.
Virological resistance to enfuvirtide has been observed to develop in subjects
with incomplete suppression of virus replication.
To assess the impact of resistance to enfuvirtide on the antiviral activity of
873140, we have examined the in vitro activity of 873140 against clinical isolates
shown to be resistant to enfuvirtide. In addition, we have examined the in vitro
antiviral potency of 873140 and enfuvirtide when applied in combination.
METHODS
873140 Susceptibility Assay. HIV-1 isolated from patient PBMCs were characterized for susceptibility first to enfuvirtide and subsequently to 873140 using cMAGI target cells (CXCR4+ HeLa cells that stably express CD4, CCR5 and a HIV-1 LTR-driven ß-galactosidase reporter construct; Chackerian et al 1997) as previously described (Hu et al 2000) with the following exceptions. On the day of the assay, medium containing eleven serial 3-fold dilutions of 873140 was added to duplicate wells, with the highest final concentration of compound being 2µM (1%DMSO final). Control wells without virus and/or 873140 also contained 1% DMSO. As some viruses were T20-resistant, infection was limited to a single round through addition of the T1249 fusion inhibitor peptide (Kilby and Eron, 2003) 24h after virus inoculation. Prior to fixing and staining, the plates were visually scored for gross cytotoxicity, evidenced by confluence <75%. No wells within this experiment showed signs of gross cytotoxicity.
Drug Combination Assay. Drug combination studies were performed in
HOS-CD4-CCR5-LTR-luc cells, as previously described (Jenkinson et al., 2003).
GW873140 and test compounds were serially diluted in half-log increments at 4X
the final concentration and combined in a concentration matrix (full factorial
design). The range of concentrations was chosen based on single compound
titrations: 30 - 0.03 nM for 873140 and 1 - 0.001 µg/mL for enfuvirtide. HOSCD4-
CCR5-LTR-luc cells were pre-incubated with 1X combination matrix of
agents for 1 hr at 37°C prior to infection. After 4 days at 37°C, luciferase activity
was assayed using Steady-Glo substrate (Promega), and read in a Topcount
(Packard) luminometer at 1s/well.
| |
| |
| |
| |
|
|
|
|
|
