Replies to post #224 on InterMune Inc. (ITMN)

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ITMN-5489 AASLD


Identification of Novel Non-Macrocyclic Inhibitors of HCV NS3/4A Serine Protease Activity
B. O. Buckman1; L. Pan1; L. Huang1; K. Kossen1; R. Rajagopalan1; S. Misialek1; S. K. Stevens1; H. Tan1; D. Ruhrmund1; V. Serebryany1; J. Matulik-Adamic1; A. Stoycheva1; S. Ammons1; D. K. Fuhrer1; L. M. Blatt1; L. Beigelman1; S. Seiwert1
1. Intermune, Inc, Brisbane, CA, USA.


Background: Agents that target the serine protease activity of NS3/4A have emerged as potentially significant components of therapies targeting the HCV virus. An agent’s dosing convenience, side effect profile, and efficacy all bear on its clinical utility. Here we describe a non-macrocyclic compound that has emerged from our ongoing drug discovery efforts involving macrocyclic and non-macrocyclic protease inhibitors. The performance characteristics of this compound, ITMN-5489, compare favorably with ITMN-191, the latter compound having recently demonstrated robust antiviral activity and promising safety in initial clinical studies.

Methods: Structure guided drug design was used in a campaign to refine potency, ADME properties and exposure in animals.

Results: Ongoing discovery efforts have identified a series of non-macrocylic compounds with favorable in-vitro and pharmacokinetic characteristics. Among these, ITMN-5489 displays an EC50 of ~1 nM against a genotype 1b replicon and 81 nM provides HCV replicon clearance in 14 day antiviral assays. The profile of inhibition against of a panel of 79 proteases and other cellular proteins suggests that off-target effects would compare favorably to several NS3/4A inhibitors currently in clinical development. ITMN-5489 exhibits significant stability in hepatocytes derived from rat, monkey and human, and plasma protein binding is uniform across these species. In primates, plasma exposure of ITMN-5489 compares favorably to that of ITMN-191. ITMN-5489 plasma concentrations 24 hours after dosing are higher than the 12 hr post dose concentration of ITMN-191 and overall AUC is 5 to 10 fold higher for ITMN-5489. Primate liver exposure of ITMN-5489 is > 7 fold higher than that of ITMN-191, and its liver to plasma ratio is approximately 60.

Conclusions: In short duration clinical studies, administration of ITMN-191 under both q12h and q8h schedules has shown very favorable virologic response and safety profiles. Here, we report a novel inhibitor of NS3/4A with in-vitro and pharmacokinetics characteristics, including exposure in primate that compare favorably to ITMN-191. ITMN-5489, like ITMN-191, shows a significantly higher concentration in liver as compared to plasma. Both plasma and liver exposure of ITMN-5489 are higher than those of ITMN-191, suggesting lower doses of this agent may achieve similar antiviral effect. The significant concentrations present 24 hours post dosing suggest once daily regimens may be possible with ITMN-5489.

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ITMN-191 Phase 1A


A Phase 1 Study of the Safety, Tolerability, and Pharmacokinetics (PK) of Single Ascending Oral Doses of the NS3/4A Protease Inhibitor ITMN 191 in Healthy Subjects
W. Z. Bradford1; C. Rubino2; S. Porter1; A. Forrest2; L. M. Blatt1; A. A. Patat3
1. Intermune, Inc, Brisbane, CA, USA.
2. ICPD/Ordway Research Institute, Inc., Albany, NY, USA.
3. Biotrial, Rennes, France.


Background: ITMN-191 is a highly potent and selective inhibitor of the HCV NS3/4A serine protease that was specifically designed to achieve high liver concentrations with modest systemic exposure. Preclinical studies demonstrate a potency of 1.8 nM in an HCV genotype 1 replicon, and liver:plasma concentration ratios of 10:1 and 120:1 in rats and primates, respectively. The aim of the present study was to evaluate the safety, tolerability, and PK of single ascending doses of ITMN-191 in healthy adults.
Methods: In a double-blind, placebo-controlled study, healthy adult subjects were randomized to receive a single oral dose of ITMN-191 or matched placebo in a Phase 1 research facility. Study drug was administered orally in doses ranging from 2-1600 mg. In each of 4 cohorts, 8 and 2 subjects received ITMN-191 or placebo, respectively. In order to assess the effect of food on ITMN-191 PK, an additional 2 cohorts of 12 subjects each received ITMN-191 or placebo (10:2) in both the fed and fasted states, separated by a 5-day washout period.
Results: A total of 64 subjects were randomized and all completed the study. There were no serious adverse events (AE) or clinically significant laboratory or ECG abnormalities. AE’s were similar between groups, with the exception of a higher frequency of mild and transient diarrhea and abdominal pain in the ITMN-191 group at the highest tested dose (1600 mg). The PK of ITMN-191 was linear over a dose range of 100 – 800 mg; the relationship between dose and exposure was more than proportional at the 1600 mg dose. Co-administration with food increased the expected trough (based on q8h and q12h dosing intervals), and increased the AUC by 40-50%.
Conclusion: ITMN-191 was safe and well tolerated over the range of studied doses. AE’s were generally mild and transient, with no evidence of clinically significant laboratory abnormalities or ECG changes. These findings are consistent with the target PK profile that informed the molecular design of ITMN-191 and, given the predicted liver concentrations, suggest that doses in the lower range of those studied are likely to result in significant antiviral activity with modest systemic exposure.

 
 Mean (±SD) for selected ITMN-191 PK parameters 
 Single Dose(mg)         N       Cmax (ng/mL)    AUC0-∞ 
 (ng×h/mL) 
 2                       8       0.20 (0.05)     0.28 (0.06) 
 100                     8       25.4 (20.1)     24.8 (12.1) 
 200                     8       44.4 (37.2)     36.4 (14.8) 
 400(Fasted)             10      69.4 (30.2)     88.6 (41.9) 
 400(Fed)                10      75.2 (61.6)     122 (48.1) 
 800                     8       318 (286)       240 (136) 
 1600(Fasted)            10      622 (309)       693 (172) 
 1600(Fed)               10      528 (392)       1070 (380) 
 

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ITMN-191 Phase 1b

Treatment of Chronic Hepatitis C Virus (HCV) Genotype 1 Patients with the NS3/4A Protease Inhibitor ITMN-191 Leads to Rapid Reductions in Plasma HCV RNA: Results of a Phase 1b Multiple Ascending Dose (MAD) Study
N. Forestier1; D. G. Larrey2; D. Guyader3; P. Marcellin4; R. Rouzier5; A. A. Patat6; W. Z. Bradford7; S. Porter7; S. Zeuzem1
1. J.W. Goethe Universität, Frankfurt, Germany.
2. CHU , Montpellier, France.
3. Pontchaillou Hospital, Rennes, France.
4. Hôpital Beaujon, Clichy, France.
5. Centre CAP, Montpellier, France.
6. Biotrial, Rennes, France.
7. Intermune, Inc, Brisbane, CA, USA.


Background: ITMN-191 is an oral, highly potent, selective inhibitor of the HCV NS3/4A serine protease. Emerging evidence suggests combination regimens including direct inhibitors of viral enzymes will improve virologic response rates relative to the current standard of care. In order to identify doses for subsequent studies of ITMN-191-based combination regimens, we evaluated the safety, pharmacokinetics, and effects on plasma HCV RNA of multiple ascending doses of ITMN-191 in patients with chronic HCV infection.

Methods: In a double-blind, randomized, placebo-controlled study, 4 cohorts of treatment-naïve HCV genotype 1 patients and one cohort of non-responders (NR) were randomized to receive ITMN-191 or matched placebo for 14 days. Cohorts consisted of 8 and 2 patients receiving ITMN-191 and placebo, respectively. Patients were sequestered in a Phase 1 unit for 16 days and completed standardized clinical and laboratory evaluations.

Results: Fifty patients were randomized and all completed the study. ITMN-191 was safe and well-tolerated. Adverse events were generally mild and transient and without association to treatment group or dose level. A single serious adverse event of benign paroxysmal positional vertigo was observed in a patient with a history of similar symptoms. The event was deemed unrelated to study drug and resolved rapidly without study drug discontinuation. ITMN-191 reduced HCV RNA in a dose dependent manner with both q8 and q12 hour schedules; reductions occurred rapidly and were typically sustained through day 14. Viral variants with reduced drug sensitivity were observed in the subset of patients that experienced virologic rebound but not in those who experienced a continual decline in HCV RNA.

Conclusion: ITMN-191 was safe and well tolerated. Treatment resulted in rapid and sustained reductions in HCV RNA, with a median reduction at day 14 of 3.8 log10 in patients receiving 200 mg q8h. Treatment response was lower in the NR cohort; this observation will inform regimen selection in future studies in this population. Based on these findings, a Phase 1b study to assess the safety and efficacy of ITMN-191 in combination with peginterferon α-2a plus ribavirin is currently underway.

 
 Change from baseline in Log10 (IU/mL) HCV RNA 
 Cohort 	        Dose            N               Median Change 
                                                 HCV RNA Log10 (IU/mL) 
                                                 Day 14 
 P (naïve)       Placebo         8               0.0 
 P (NR)          Placebo         2               0.0 
 1 (naïve)       100 mg q12h     8               -0.7 
 2 (naïve)       100 mg q8h      8               -1.7 
 3 (naïve)       200 mg q12h     5               -3.1 
 4 (naïve)       200 mg q8h      8               -3.8 
 5 (NR)          300 mg q12h     8               -2.5  
 

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Pharmacokinetic-Pharmacodynamic (PK-PD) Relationships for ITMN-191 in a Phase 1 Multiple Ascending Dose Trial in Patients with Genotype 1 Chronic Hepatitis C (CHC)Infection
C. Rubino1; W. Z. Bradford2; A. Forrest1; S. Porter2; L. M. Blatt2; S. Seiwert2; S. Zeuzem3
1. ICPD/Ordway Research Institute, Inc., Albany, NY, USA.
2. Intermune, Inc, Brisbane, CA, USA.
3. J.W. Goethe Universität, Frankfurt, Germany.


ITMN-191 is a highly potent, slowly dissociating NS3/4A protease inhibitor designed to achieve high liver concentrations with only modest plasma exposure. This study examines PK-PD relationships for ITMN-191 in CHC patients.
Methods: Four cohorts of 10 naïve patients were randomized (8:2) to receive oral ITMN-191 (100 mg q12h, 100 mg q8h, 200 mg q12h, or 200 mg q8h) or placebo for 14 days. PK parameters and PK-PD relationships were assessed using non-compartmental methods and non-linear models, respectively.
Results: ITMN-191 showed potent antiviral activity with relatively low plasma concentrations. At 200 mg q8h, the mean AUC0-24, Cmax, and Cmin values were 500 ng×h/mL, 91 ng/mL and 2.78 ng/mL, respectively, and the mean max. decline in HCV RNA was 3.9 log10.The dose-exposure relationship was not uniformly proportional.Predicted Cmin values for 2, 3, and 4 log10 HCV RNA declines were 0.07, 0.19, and 2.7 ng/mL, respectively. In an inhibitory Emax model, the strongest relationship was between Day 1 Cmin and max change in HCV RNA (figure); fitted Eo, Emax and EC50 values were -0.33 log10, 3.6 log10 and 0.08 ng/mL, respectively. Conclusion: ITMN-191 achieves significant antiviral effects with substantially lower plasma exposures than reported for other NS3 inhibitors. This is consistent with the high liver:plasma concentration ratios seen in animals and likely contributes to the favorable safety and antiviral activity in patients. These data, plus in vitro evidence of synergy with peginterferon, provide a strong rationale for the ongoing triple combination study to further evaluate the PK-PD and antiviral effect of an ITMN-191.


Observed Data and Fitted PK-PD relationship for ITMN-191

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Combination of the NS3/4A Protease Inhibitor ITMN-191 (R7227) with the Active Moiety of the NS5B Inhibitors R1626 or R7128 Enhances Replicon Clearance and Reduces the Emergence of Drug Resistant Variants
H. Tan1; S. Rajyaguru2; T. Wu2; M. McCown2; S. Ali2; W. Jiang2; M. J. Otto3; P. A. Furman3; I. Najera2; K. Klumpp2; J. Symons2; N. Cammack2; L. M. Blatt1; S. Seiwert1
1. Intermune, Inc, Brisbane, CA, USA.
2. Roche Palo Alto LLC, Palo Alto, CA, USA.
3. Pharmasset Inc., Princeton, NJ, USA.


Background: ITMN-191 is an inhibitor of HCV NS3/4A protease activity, and R1626 and R7128 are nucleoside inhibitors of the polymerase activity of HCV NS5B. All three compounds promote multi-log10 reductions in circulating HCV RNA in chronic HCV patients when administered for short durations as monotherapy. Here, to support future clinical studies that would combine ITMN-191 with R1626 or R7128, we investigated the combined antiviral effect of these compounds.

Methods: In the HCV clearance assay, cells harboring an HCV genotype 1b replicon were treated for 2 weeks with ITMN-191, the active moiety of R1626 (R1479), the active moiety of R7128 (PSI 6130), or a combination of inhibitors in the absence of G418 selection for replicon retention. Cells were counted and aliquots harvested for RT-PCR-based quantification of replicon RNA levels under G418 selection over 4 subsequent weeks. In the colony formation assay, cells were treated with either one or two compounds at 1X, 10X, or 15X their respective EC50. After 3 weeks in culture with G418, cells were fixed and stained with crystal violet or total cellular RNA extracted. For drug-drug interaction studies, HCV replicon cells were treated for 3 days with a pair of inhibitors in checkerboard dilution and percent reductions of reporter gene activity obtained.
Results: In the HCV clearance assay, 18 nM ITMN-191 and low μM concentrations of the active moiety of R1626 and R7128 (18 μM & 27 μM, respectively) eliminated HCV replicon in the absence of G418 selective pressure for replicon retention. Addition of the lowest tested concentration of ITMN-191 (6 nM) to the lowest tested concentration of the active moiety of R1626 or R7128 (0.3 μM & 0.45 μM, respectively) resulted in replicon clearance, demonstrating significant combined antiviral effect. In the colony formation assay in the presence of G418 selective pressure for replicon retention, NS5B inhibitors at 10X or 15X EC50 supported replicon clearance and did not result in drug resistant colonies. Similar treatment with ITMN-191 selected resistant colonies, but these were suppressed by an NS5B inhibitor at 1X EC50. Drug-drug interaction studies over a 3 day incubation period demonstrated additive to slightly synergistic interactions between the two inhibitor classes.

Conclusions: The combination of ITMN-191 with the active moeity of either R1626 or R7128 results in enhanced antiviral activity and suppression of ITMN-191 resistant variants. These findings suggest that the combination of ITMN-191 with R1626 or R7128 may confer significantly greater antiviral activity than has been observed with these agents in previous monotherapy trials.

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A high throughput system for phenotypic analysis of NS3 sequences
J. Hong1; X. Qin1; S. Lim1; L. M. Blatt1; S. Seiwert1
1. Intermune, Inc, Brisbane, CA, USA.


Background: Systems to evaluate the phenotypic characteristics of NS3 sequences have many uses including the evaluation of drug sensitivity of patient-isolated NS3 following drug treatment. ITMN-191 (R7227) is a potent, slowly dissociating macrocyclic inhibitor of NS3/4A protease that has displayed robust antiviral activity in initial clinical studies. To support these studies, as well as larger future studies, we have developed a novel, scalable system to characterize drug sensitivity of NS3 sequences isolated from HCV patients.

Methods: NS3 protease domain is amplified using nested PCR. Amplicons allow determination of a consensus or population sequence and are cloned into a “phenotyping” plasmid which inserts NS3 sequence upstream of an NS4A sequence, an endoplasmic reticulum (ER) tethering sequence and a secreted luciferase (sLuc) sequence, each of which are separated by a NS3 cleavage site. Transfection into 293F suspension cells enables homogenous, solution based detection of NS3 activity: NS3 activity liberates sLuc from its ER tether allowing its secretion from cells. Dose response curves can be determined since cells treated with greater amounts of NS3 inhibitor release less sLuc into media. Automation of the above process allows determination of ~100 EC50 values per day.

Results: Repeated analysis of drug sensitivity of a single NS3 genotype 1b sequence over three days provided consistent EC50 values (EC50 = 1.5 ± 1.0 nM, n = 28, CV ~66%, average R2 of 0.93 for dose response curves), indicating the assay has a high degree of precision. Analysis of 72, 48, and 24 quasi-species derived from three patients harboring genotype 1b HCV provided average EC50 = 1.5 nM, 0.6 nM, and 0.9 nM, respectively, for a close analog of ITMN-191. Importantly, mixing of quasi species prior to mammalian cell transfection in a population-based assay yielded drug sensitivities that were within 2-fold of the average drug sensitivities when individual sequences were characterized in a clonal approach, indicating population-based phenotyping is viable. Characterization of NS3 sequence variants identified in the course of in vitro selection experiments demonstrated the phenotyping assay recapitulates the rank order potency observed when these substitutions are introduced into HCV replicons.

Conclusions: The methods reported here to analyze NS3 sequence and drug sensitivity provide a robust and scalable solution for the characterization of NS3 sequences derived from clinical samples. This system accurately reflects drug sensitivities observed in the HCV replicon system and will be deployed to analyze NS3 sequences obtained from ongoing clinical studies of ITMN-191.

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Identification of novel HCV NS3 helicase inhibitors through the application of optimized unwinding and ATPase assays in high throughput screens (HTS)
K. Kossen1; J. Kraemer2; D. Winkler2; T. Hesterkamp2; S. Misialek1; R. Rajagopalan1; B. O. Buckman1; L. M. Blatt1; S. Seiwert1; L. Beigelman1
1. Intermune, Inc, Brisbane, CA, USA.
2. Evotec AG, Hamburg, Germany.


Background: The RNA helicase activity of HCV NS3 is essential for viral replication and thus represents an attractive target for new antiviral compounds. As the ATPase and unwinding activities of the NS3 motor protein are coupled, we performed HTS assays against both enzymatic activities to maximize the chances of identifying tractable chemical matter.

Methods: The ATPase activity of NS3 is robust whereas duplex unwinding requires more specialized conditions. Therefore, assay optimization focused on improving the unwinding activity by varying the duplex substrate and assay buffer. The optimized conditions were incorporated into fluorescence polarization (FP)-based assays for both ATPase and unwinding, miniaturized to an assay volume of ~ 1μL, and employed in parallel HTS assays of ~300,000 compounds each. The ATPase assay uses a format originally developed for the characterization of kinase inhibitors. The unwinding assay uses a novel design in which opposite strands of a duplex DNA substrate incorporate either a fluorophore or biotin, respectively. NS3 helicase promotes the dissociation of these two strands. Detection is accomplished by addition of streptavidin which forms a high FP complex with substrate but not product.

Results: The performance of the unwinding assay is significantly influenced by both the choice of duplex substrate and the buffer conditions. Optimization of duplex length and fluorescent labels identified an optimized substrate that afforded an approximately 2-fold increase in dynamic range. Optimization of reaction buffer resulted in further enhancement of unwinding activity. Interestingly, under the optimized assay conditions NS3 shows significant unwinding activity when duplex substrate is in excess over enzyme. The final unwinding assay is linear over ~1 hr with a dynamic range of 120 mP. The conditions of the unwinding assay required minimal modifications when transferred to the ATPase assay which yielded a dynamic range of 80 mP. Application of these conditions to HTS provided excellent assay performance with average Z’ values of 0.76 and 0.74 for the unwinding and ATPase end-points, respectively. Following resynthesis and hit verification, a total of 12 structural families of active leads were identified.

Conclusions: NS3 helicase is considered a difficult target for small molecule drug discovery and to date no inhibitors have entered clinical trials. Here, the application of optimized assay conditions for NS3 catalyzed unwinding and ATPase activities in parallel HTS screens resulted in identification of 12 chemically tractable lead families. Optimization of these leads is currently ongoing.