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UK Biobank Makes Available 200,000 whole genomes Open Access

Reporter: Stephen J. Williams, Ph.D.

The following is a summary of an article by Jocelyn Kaiser, published in the November 26, 2021 issue of the journal Science.

To see the full article please go to https://www.science.org/content/article/200-000-whole-genomes-made-available-biomedical-studies-uk-effort

The UK Biobank (UKBB) this week unveiled to scientists the entire genomes of 200,000 people who are part of a long-term British health study.

The trove of genomes, each linked to anonymized medical information, will allow biomedical scientists to scour the full 3 billion base pairs of human DNA for insights into the interplay of genes and health that could not be gleaned from partial sequences or scans of genome markers. “It is thrilling to see the release of this long-awaited resource,” says Stephen Glatt, a psychiatric geneticist at the State University of New York Upstate Medical University.

Other biobanks have also begun to compile vast numbers of whole genomes, 100,000 or more in some cases (see table, below). But UKBB stands out because it offers easy access to the genomic information, according to some of the more than 20,000 researchers in 90 countries who have signed up to use the data. “In terms of availability and data quality, [UKBB] surpasses all others,” says physician and statistician Omar Yaxmehen Bello-Chavolla of the National Institute for Geriatrics in Mexico City.

Enabling your vision to improve public health

Data drives discovery. We have curated a uniquely powerful biomedical database that can be accessed globally for public health research. Explore data from half a million UK Biobank participants to enable new discoveries to improve public health.

Data Showcase

Future data releases

This UKBB biobank represents genomes collected from 500,000 middle-age and elderly participants for 2006 to 2010. The genomes are mostly of a European descent. Other large scale genome sequencing ventures like Iceland’s DECODE, which collected over 100,000 genomes, is now a subsidiary of Amgen, and mostly behind IP protection, not Open Access as this database represents.

UK Biobank is a large-scale biomedical database and research resource, containing in-depth genetic and health information from half a million UK participants. The database is regularly augmented with additional data and is globally accessible to approved researchers undertaking vital research into the most common and life-threatening diseases. It is a major contributor to the advancement of modern medicine and treatment and has enabled several scientific discoveries that improve human health.

A summary of some large scale genome sequencing projects are show in the table below:

BiobankCompleted Whole GenomesRelease Information
UK Biobank200,000300,000 more in early 2023
TransOmics for
Precision Medicien
161,000NIH requires project
specific request
Million Veterans
Program
125,000Non-Veterans Affairs
researchers get first access
100,000 Genomes
Project
120,000Researchers must join Genomics
England collaboration
All of Us90,000NIH expects to release 2022

Other Related Articles on Genome Biobank Projects in this Open Access Online Scientific Journal Include the Following:

Icelandic Population Genomic Study Results by deCODE Genetics come to Fruition: Curation of Current genomic studies

Exome Aggregation Consortium (ExAC), generated the largest catalogue so far of variation in human protein-coding regions: Sequence data of 60,000 people, NOW is a publicly accessible database

Systems Biology Analysis of Transcription Networks, Artificial Intelligence, and High-End Computing Coming to Fruition in Personalized Oncology

Diversity and Health Disparity Issues Need to be Addressed for GWAS and Precision Medicine Studies

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New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)

New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target

Curator: Stephen J. Williams, Ph.D.

UPDATED 10/2/2021

A Newly Identified Mutant RAS G12C  GTPase Activating Protein (GAP) may lead to discovery of new class of RAS inhibitors 

From the journal Science in Filling in the GAPs in understanding RAS: A newly identified regulator increases the efficacy of a new class of targeted anti-RAS drugs.

Source:

SCIENCE•8 Oct 2021•Vol 374Issue 6564•pp. 152-153•DOI: 10.1126/science.abl3639

    According to canonical thinking, mutationally activated RAS (constitutive activation) is insensitive to GTPase-activating proteins (GAPs).  GAPs accelerated the conversion of GTP bound to RAS to GDP, a critical step in the inactivation of RAS signaling cycle.  However a small molecule has just been FDA approved, sotorasib, in cancer cells by binding only the GDP bound KRAS G12C mutant. A few non small cell lung cancers (NSCLC) are resistant to another such inhibitor, adagrasib.  In the same Science issue, another paper by Li et. al. explains the potential that a GAP, RGS3, may play in this conundrum and demonstrates that certain other inhibitors of the RAS cycle, mainly the GEF (guanine exchange factor) SOS1, in combination with MEK inhibitors may circumvent this resistance.

Inhibitors of mutant KRAS
The KRASG12C (Gly12→Cys) mutant is refractory to canonical GAPs, p120 RASGAP and neurofibromin, but not RGS3, which promotes GTP hydrolysis. The resulting GDP-bound KRASG12C is an anticancer drug target. Combination with inhibitors of SOS1 or with inhibitors of downstream signaling may further improve efficacy.
GRAPHIC: C. BICKEL/SCIENCE
 
First a discussion of the RAS signalling cycle is shown below in a good review of RAS activation and signal termination.
 
PMCID: PMC3124093
NIHMSID: NIHMS294865
PMID: 21102635
Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?

Abstract

There is now considerable and increasing evidence for a causal role of aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases act as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. We assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.

Box 1

Ras superfamily of small GTPases

The human Ras superfamily comprised of over 150 members which is divided into five major branches on the basis of sequence and functional similarities. In addition to the three Ras isoforms, other members of the Ras family with important roles in cancer include Rheb and Ral proteins. The ~20 kDa core G domain (corresponding to Ras residues 4–166) is conserved among all Ras superfamily proteins and is involved in GTP binding and hydrolysis. This domain is comprised of five conserved guanine nucleotide consensus sequence elements (Ras residue numbering) involved in binding phosphate/Mg2+ (PM) or the guanine base (G). The switch I (Ras residues 30–38) and II (59–76) regions change in conformation during GDP-GTP cycling and contribute to preferential effector binding to the GTP-bound state and the core effector domain (E; Ras residues 32–40). Ras and Rho family proteins have additional C-terminal hypervariable (HV) sequences that commonly terminate with a CAAX motif that signals for farnesyl or geranylgeranyl isoprenoid addition to the cysteine residue, proteolytic removal of the AAX residues and carboxylmethylation of the prenylated cysteine. Some are modified additionally by a palmitate fatty acid to cysteine residues in the HV sequence that contributes to membrane association. Rab proteins also contain a C-terminal HV region that terminates with cysteine-containing motifs that are modified by addition of geranylgeranyl lipids, with some undergoing carboxylmethylation. Arf family proteins are characterized by an N-terminal extension involved in membrane interaction, with some cotranslationally modified by addition of a myristate fatty acid. Ran is not lipid modified but contains a C-terminal extension that is essential for function. Rho proteins are characterized by an up to 13 amino acid “Rho insert” sequence positioned between Ras residues 122 and 123 involved in effector regulation.

 

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The GDP-GTP cycle

Ras superfamily proteins possess intrinsic guanine nucleotide exchange and GTP hydrolysis activities. However, these activities are too low to allow efficient and rapid cycling between their active GTP-bound and inactive GDP-bound states. GEFs and GAPs accelerate and regulate these intrinsic activities. Members of the different branches of the superfamily are regulated by GEFs and GAPs with structurally distinct catalytic domains. Here we have utilized the Rho family as an example to illustrate the complexity of this process, where multiple GEFs and GAPs may regulate one specific GTPase. For the 20 human Rho GTPases there are 83 GEFs and 67 GAPs and a subset of Rho GTPases are not likely regulated by GEFs and GAPs (e.g., Rnd3/RhoE). Rho GTPases are activated by distinct RhoGEF families. Dbl family RhoGEFs (68) possesses a tandem Dbl homology (DH) catalytic and pleckstrin homology (PH) regulatory domain topology. DOCK family RhoGEFs (11) are characterized by two regions of high sequence conservation that are designated Dock-homology region regulatory DHR-1 and catalytic DHR-2 domains. Two other RhoGEFs have been described (SWAP70 and SLAT) contain a PH but no DH domain (2) and smgGDS (1) is an unusual GEF in that it functions as a GEF for some Rho as well as non-Rho family GTPases. At least 24 Dbl RhoGEFs have been reported to activate RhoA. Rho (and Rab) GTPases are also controlled by a third class of regulatory proteins, Rho dissociation inhibitors (RhoGDI) (of which there are 3) whose main function involves regulation of Rho GTPase membrane association by masking the isoprenoid group.

 

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So GEFs like SOS 1,2,3 are responsible for activation of the G Protien RAS upon Receptor Tyrosine Kinase (RTK) activation by a multitude of growth factors.  The GAPs are responsible for termination of the activated RAS cycle and now RAS, in its normal unmuted version, can now be activated again.  The G12C mutant keeps RAS in its activated state as GAPs cannot fascilitate the hydroylsis of GTP.  

Li et al. (in this issue) theorized that there must be cellular factors that stimulate formation of GDP-bound KRAS G12C, enabling its vulnerability to KRAS G12C inhibitors, and discovered a regulator of G protein signaling 3 (RGS3), which had GAP activity previously for heterotrimeric G proteins GαI and Gαq with unexpected GAP activity for the KRAS G12C mutant.   They also found the fully activated GTP bound KRAS G12C is dependent on RTK-mediated activation of SOS1.  This suggested that SOS1 inhibitors with MEK inhibitors could be effective against mutant KRAS.

The paper by Li is summarized below:

The G protein signaling regulator RGS3 enhances the GTPase activity of KRAS

SCIENCE•8 Oct 2021•Vol 374Issue 6564•pp. 197-201•DOI: 10.1126/science.abf1730

Abstract

Recently reported to be effective in patients with lung cancer, KRASG12C inhibitors bind to the inactive, or guanosine diphosphate (GDP)–bound, state of the oncoprotein and require guanosine triphosphate (GTP) hydrolysis for inhibition. However, KRAS mutations prevent the catalytic arginine of GTPase-activating proteins (GAPs) from enhancing an otherwise slow hydrolysis rate. If KRAS mutants are indeed insensitive to GAPs, it is unclear how KRASG12C hydrolyzes sufficient GTP to allow inactive state–selective inhibition. Here, we show that RGS3, a GAP previously known for regulating G protein–coupled receptors, can also enhance the GTPase activity of mutant and wild-type KRAS proteins. Our study reveals an unexpected mechanism that inactivates KRAS and explains the vulnerability to emerging clinically effective therapeutics.

UPDATED 09/26/2021

The KRAS G12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients

Source: Hallin J, Engstrom LD, Hargis L, Calinisan A, Aranda R, Briere DM, Sudhakar N, Bowcut V, Baer BR, Ballard JA, Burkard MR, Fell JB, Fischer JP, Vigers GP, Xue Y, Gatto S, Fernandez-Banet J, Pavlicek A, Velastagui K, Chao RC, Barton J, Pierobon M, Baldelli E, Patricoin EF 3rd, Cassidy DP, Marx MA, Rybkin II, Johnson ML, Ou SI, Lito P, Papadopoulos KP, Jänne PA, Olson P, Christensen JG. The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov. 2020 Jan;10(1):54-71. doi: 10.1158/2159-8290.CD-19-1167. Epub 2019 Oct 28. PMID: 31658955; PMCID: PMC6954325.

Abstract

Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE: The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.

Introduction

KRAS is one of the most frequently mutated oncogenes in cancer; however, efforts to directly target KRAS have been largely unsuccessful due to its high affinity for GTP/GDP and the lack of a clear binding pocket (1–4). More recently, compounds were identified that covalently bind to KRASG12C at the cysteine 12 residue, lock the protein in its inactive GDP-bound conformation, inhibit KRAS-dependent signaling, and elicit antitumor responses in tumor models (5–7). Advances on early findings demonstrated that the binding pocket under the switch II region was exploitable for drug discovery, culminating in the identification of more potent KRASG12C inhibitors with improved physiochemical properties that are now entering clinical trials. The identification of KRASG12C inhibitors provides a renewed opportunity to develop a more comprehensive understanding of the role of KRAS as a driver oncogene and to explore the clinical utility of direct KRAS inhibition.

KRASG12C mutations are present in lung and colon adenocarcinomas as well as smaller fractions of other cancers. The genetic context of KRASG12C alteration can vary significantly among tumors and is predicted to affect response to KRAS inhibition. KRAS mutations are often enriched in tumors due to amplification of mutant or loss of wild-type allele (8, 9). In addition, KRAS mutations often co-occur with other key genetic alterations including TP53 and CDKN2A in multiple cancers, KEAP1 and/or STK11 in lung adenocarcinoma, or APC and PIK3CA in colon cancer (3, 8–12). Whether differences in KRAS-mutant allele fraction or co-occurrence with other mutations influence response to KRAS blockade is not yet well understood. In addition, due to the critical importance of the RAS pathway in normal cellular function, there is extensive pathway isoform redundancy and a comprehensive regulatory network in normal cells to ensure tight control of temporal pathway signaling. RAS pathway negative feedback signaling is mediated by ERK1/2 and receptor tyrosine kinases (RTK) as well as by ERK pathway target genes including dual-specificity phosphatases (DUSP) and Sprouty (SPRY) proteins (13–17). One important clinically relevant example is provided by the reactivation of ERK signaling observed following treatment of BRAFV600E-mutant cancers with selective BRAF inhibitors (18–20). The observed intertumoral heterogeneity and extensive feedback signaling network in KRAS-mutant cancers may necessitate strategies to more comprehensively block oncogenic signal transduction and deepen the antitumor response in concert with KRAS blockade (15, 21, 22).

Potential strategies to augment the response to KRASG12C inhibitor treatment are evident at multiple nodes of the signaling pathway regulatory machinery. RAS proteins are small GTPases that normally cycle between an active, GTP-bound state and an inactive, GDP-bound state. RAS proteins are loaded with GTP through guanine nucleotide exchange factors (e.g., SOS1) which are activated by upstream RTKs, triggering subsequent interaction with effector proteins that activate RAS-dependent signaling. RAS proteins hydrolyze GTP to GDP through their intrinsic GTPase activity, which is dramatically enhanced by GTPase-activating proteins (GAP). Mutations at codons 12 and 13 in RAS proteins impair GAP-stimulated GTP hydrolysis, leaving RAS predominantly in the GTP-bound, active state.

Potent covalent KRASG12C inhibitors described to date bind only GDP-bound KRAS (5–7). Although codon 12 and 13 mutations decrease the fraction of GDP-bound KRAS, recent biochemical analyses revealed that KRASG12C exhibits the highest intrinsic GTP hydrolysis rate and highest nucleotide exchange rate among KRAS mutants (23). Furthermore, the nucleotide-bound state of KRASG12C can be shifted toward the GDP-bound state by pharmacologically modulating upstream signaling with RTK inhibitors that increase the activity of KRASG12C inhibitors (7, 22, 24). Likewise, SHP2 is a phosphatase that positively transduces RTK signaling to KRAS. Accordingly, SHP2 inhibitors are active in cancers driven by KRAS mutations that are dependent on nucleotide cycling, including KRASG12C (25–27).

MRTX849 is among the first KRASG12C inhibitors to advance to clinical trials. The comprehensive and durable inhibition of KRASG12C by MRTX849 provides a unique opportunity to understand the extent to which KRAS functions as an oncogenic driver. In addition, the observation that the response to blockade of KRAS is markedly different in vitro and in vivo indicates that evaluation of the consequences of KRAS blockade in in vivo model systems is critical to understand the role of KRAS-driven tumor progression. The demonstration of partial responses in patients with lung and colon adenocarcinomas treated with MRTX849 in clinical trials indicates that results observed in tumor models extend to KRASG12C-positive human cancers. Our comprehensive molecular characterization of multiple tumor models at baseline and during response to KRAS inhibition has provided further insight toward the contextual role of KRAS mutation in the setting of genetic and tumoral heterogeneity. Finally, further interrogation of these genetic alterations and signaling pathways utilizing functional genomics strategies including CRISPR and combination approaches uncovered regulatory nodes that sensitize tumors to KRAS inhibition when cotargeted.

Introduction

KRAS is one of the most frequently mutated oncogenes in cancer; however, efforts to directly target KRAS have been largely unsuccessful due to its high affinity for GTP/GDP and the lack of a clear binding pocket (1–4). More recently, compounds were identified that covalently bind to KRASG12C at the cysteine 12 residue, lock the protein in its inactive GDP-bound conformation, inhibit KRAS-dependent signaling, and elicit antitumor responses in tumor models (5–7). Advances on early findings demonstrated that the binding pocket under the switch II region was exploitable for drug discovery, culminating in the identification of more potent KRASG12C inhibitors with improved physiochemical properties that are now entering clinical trials. The identification of KRASG12C inhibitors provides a renewed opportunity to develop a more comprehensive understanding of the role of KRAS as a driver oncogene and to explore the clinical utility of direct KRAS inhibition.

KRASG12C mutations are present in lung and colon adenocarcinomas as well as smaller fractions of other cancers. The genetic context of KRASG12C alteration can vary significantly among tumors and is predicted to affect response to KRAS inhibition. KRAS mutations are often enriched in tumors due to amplification of mutant or loss of wild-type allele (8, 9). In addition, KRAS mutations often co-occur with other key genetic alterations including TP53 and CDKN2A in multiple cancers, KEAP1 and/or STK11 in lung adenocarcinoma, or APC and PIK3CA in colon cancer (3, 8–12). Whether differences in KRAS-mutant allele fraction or co-occurrence with other mutations influence response to KRAS blockade is not yet well understood. In addition, due to the critical importance of the RAS pathway in normal cellular function, there is extensive pathway isoform redundancy and a comprehensive regulatory network in normal cells to ensure tight control of temporal pathway signaling. RAS pathway negative feedback signaling is mediated by ERK1/2 and receptor tyrosine kinases (RTK) as well as by ERK pathway target genes including dual-specificity phosphatases (DUSP) and Sprouty (SPRY) proteins (13–17). One important clinically relevant example is provided by the reactivation of ERK signaling observed following treatment of BRAFV600E-mutant cancers with selective BRAF inhibitors (18–20). The observed intertumoral heterogeneity and extensive feedback signaling network in KRAS-mutant cancers may necessitate strategies to more comprehensively block oncogenic signal transduction and deepen the antitumor response in concert with KRAS blockade (15, 21, 22).

Potential strategies to augment the response to KRASG12C inhibitor treatment are evident at multiple nodes of the signaling pathway regulatory machinery. RAS proteins are small GTPases that normally cycle between an active, GTP-bound state and an inactive, GDP-bound state. RAS proteins are loaded with GTP through guanine nucleotide exchange factors (e.g., SOS1) which are activated by upstream RTKs, triggering subsequent interaction with effector proteins that activate RAS-dependent signaling. RAS proteins hydrolyze GTP to GDP through their intrinsic GTPase activity, which is dramatically enhanced by GTPase-activating proteins (GAP). Mutations at codons 12 and 13 in RAS proteins impair GAP-stimulated GTP hydrolysis, leaving RAS predominantly in the GTP-bound, active state.

Potent covalent KRASG12C inhibitors described to date bind only GDP-bound KRAS (5–7). Although codon 12 and 13 mutations decrease the fraction of GDP-bound KRAS, recent biochemical analyses revealed that KRASG12C exhibits the highest intrinsic GTP hydrolysis rate and highest nucleotide exchange rate among KRAS mutants (23). Furthermore, the nucleotide-bound state of KRASG12C can be shifted toward the GDP-bound state by pharmacologically modulating upstream signaling with RTK inhibitors that increase the activity of KRASG12C inhibitors (7, 22, 24). Likewise, SHP2 is a phosphatase that positively transduces RTK signaling to KRAS. Accordingly, SHP2 inhibitors are active in cancers driven by KRAS mutations that are dependent on nucleotide cycling, including KRASG12C (25–27).

MRTX849 is among the first KRASG12C inhibitors to advance to clinical trials. The comprehensive and durable inhibition of KRASG12C by MRTX849 provides a unique opportunity to understand the extent to which KRAS functions as an oncogenic driver. In addition, the observation that the response to blockade of KRAS is markedly different in vitro and in vivo indicates that evaluation of the consequences of KRAS blockade in in vivo model systems is critical to understand the role of KRAS-driven tumor progression. The demonstration of partial responses in patients with lung and colon adenocarcinomas treated with MRTX849 in clinical trials indicates that results observed in tumor models extend to KRASG12C-positive human cancers. Our comprehensive molecular characterization of multiple tumor models at baseline and during response to KRAS inhibition has provided further insight toward the contextual role of KRAS mutation in the setting of genetic and tumoral heterogeneity. Finally, further interrogation of these genetic alterations and signaling pathways utilizing functional genomics strategies including CRISPR and combination approaches uncovered regulatory nodes that sensitize tumors to KRAS inhibition when cotargeted.

Results

MRTX849 Is a Potent and Selective Inhibitor of KRASG12C, KRAS-Dependent Signal Transduction, and Cell Viability In Vitro

A structure-based drug design approach, including optimization for favorable drug-like properties, led to the discovery of MRTX849 as a potent, covalent KRASG12C inhibitor (Fig. 1A; Supplementary Table S1). An LC/MS-based KRASG12C protein modification assay revealed that MRTX849 demonstrated much greater modification of KRASG12C when preloaded with GDP compared with GTP (Supplementary Table S2), supporting that MRTX849 binds to and stabilizes the inactive GDP-bound form of KRASG12C. Indeed, introducing a comutation that impairs the GTPase activity of KRASG12C (24) attenuated the inhibitory activity of MRTX1257, a close analogue of MRTX849 (Supplementary Fig. S1A). Secondary mutations that modulate the nucleotide exchange function of KRASG12C also affected inhibition by MRTX1257, supporting that the MRTX compound series is dependent on KRASG12C nucleotide cycling.

Figure 1.

 

MRTX849 is a potent, covalent KRASG12C inhibitor in vitroA, Structure of MRTX849. B, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated from 1 hours to 72 hours with MRTX849 at 100 nmol/L. C, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated for 24 hours with MRTX849 over a 13-point dose response. D, Left y-axis shows active RAS ELISA assay to determine the reduction in RAS-GTP abundance following MRTX849 treatment in MIA PaCa-2 cells for 24 hours. The vehicle value was normalized to 1 by dividing all average values by the vehicle value. Right y-axis shows quantitation of KRAS band shift by MRTX849 treatment in MIA PaCa-2 cells for 24 hours as assessed by Western blot and densitometry. E, In-cell Western blot assay to evaluate modulation of pERK in MIA PaCa-2 cells grown in standard tissue-culture conditions treated with MRTX849 over a time course. F, CellTiter-Glo assay to evaluate cell viability performed on seven KRASG12C-mutant cell lines and three non–KRASG12C-mutant cell lines grown in 2-D tissue-culture conditions in a 3-day assay (left plot) or 3-D conditions using 96-well, ULA plates in a 12-day assay (right plot).

 

We next determined the cellular activity of MRTX849 utilizing the KRASG12C-mutant H358 lung and MIA PaCa-2 pancreatic cancer cell lines. In both models, MRTX849 demonstrated an upward electrophoretic mobility shift of KRASG12C protein band migration by immunoblot, indicative of covalent modification of KRASG12C. A maximal mobility shift was observed by 1 hour, was maintained through 72 hours (Fig 1B; Supplementary Fig. S1B), and was evident at concentrations as low as 2 nmol/L with near-maximal modification observed at 15.6 nmol/L (Fig. 1C; Supplementary Fig. S1C). Comparable inhibition of active RAS was observed as determined by a RAF RAS-binding domain capture ELISA assay (Fig. 1D; 1D). MRTX849 also inhibited KRAS-dependent signaling targets including ERK1/2 phosphorylation (pERK; Thr202/Tyr204 ERK1), S6 phosphorylation (pS6; RSK-dependent Ser235/236), and expression of the ERK-regulated DUSP6, each with IC50 values in the single-digit nanomolar range in both cell lines (Fig. 1B and C; Supplementary Fig. S1B and S1C). The evaluation of pERK over a time course of 48 hours indicated maximal inhibition was observed at 24 hours (Fig. 1E; Supplementary Fig. S1E). Treatment with the des-acrylamide version of MRTX849, which is unable to covalently bind to KRASG12C, did not demonstrate significant inhibition of ERK phosphorylation (Supplementary Fig. S1F). The H358 cell line was selected for determination of MRTX849 cysteine selectivity utilizing an LC/MS-based proteomics approach able to detect approximately 6,000 cysteine-containing peptides. After treatment for 3 hours, decreased KRASG12C Cys12-free peptide was detected with treated-to-control ratios of 0.029 and 0.008 determined at 1 and 10 μmol/L, respectively, indicating near-complete engagement of the intended target (Supplementary Table S3). In contrast, the only other peptides identified were from lysine-tRNA ligase (KARS) at Cys209 near the detection limit, indicating a high degree of selectivity toward KRASCYS12.

To evaluate the breadth of MRTX849 activity, its effect on cell viability was determined across a panel of 17 KRASG12C-mutant and 3 non–KRASG12C-mutant cancer cell lines using 2-D (3-day, adherent cells) and 3-D (12-day, spheroids) cell-growth conditions. MRTX849 potently inhibited cell growth in the vast majority of KRASG12C-mutant cell lines with IC50 values ranging between 10 and 973 nmol/L in the 2-D format and between 0.2 and 1,042 nmol/L in the 3-D format (Supplementary Table S4; Fig. 1F). In agreement with prior KRASG12C inhibitor studies (5), MRTX849 demonstrated improved potency in the 3-D assay format, as all but one KRASG12C-mutant cell line exhibited an IC50 value below 100 nmol/L. Although MRTX849 was broadly effective in inhibiting viability of KRASG12C-mutant cell lines, IC50 values varied across the cell panel by 100-fold, suggesting a differential degree of sensitivity to treatment. All three non–KRASG12C-mutant cell lines tested demonstrated IC50 values greater than 1 μmol/L in 2-D conditions and greater than 3 μmol/L in 3-D conditions, suggesting the effect of MRTX849 on cell viability was dependent on the presence of KRASG12C.

To determine whether the difference in sensitivity across the cell panel correlated with the ability of MRTX849 to bind to KRAS or inhibit KRAS-dependent signal transduction, seven KRASG12C-mutant cancer cell lines were selected from the panel for further evaluation. In each cell line, MRTX849 demonstrated a very similar concentration-dependent electrophoretic mobility shift (IC50) for KRASG12C protein migration, suggesting that the ability to bind to and modify KRASG12C does not readily account for differences in response in viability studies (Fig. 1B and C; Supplementary Figs. S1B and S1C and S2A and S2B). The effect of MRTX849 on selected phosphoproteins implicated in mediating KRAS-dependent signaling was also evaluated across the cell panel by immunoblot and/or reverse-phase protein array (RPPA) following treatment for 6 or 24 hours. Notably, the concentration–response relationship and maximal effect of MRTX849 on inhibition of ERK and S6S235/236 phosphorylation varied across the cell panel (Supplementary Fig. S2A and S2C; Supplementary Table S7). MRTX849 demonstrated only partial inhibition of pERK in KYSE-410 and SW1573 cells and a minimal effect on pS6S235/236 in SW1573, H2030, and KYSE-410 cells (Supplementary Fig. S2A and S2C). Each of these cell lines were among those that exhibited a submaximal response to MRTX849 in both 2-D and 3-D viability assays (Fig. 1F). Although KRAS is implicated in mediating signal transduction through the PI3K and mTOR pathways, there was minimal evidence of a significant and/or durable effect of MRTX849 on AKT (S473, T308) or 4E-BP1 (T37/T46, S65, T70) phosphorylation at any time point in any cell lines evaluated (Supplementary Fig. S2D). However, MRTX849 demonstrated concentration-dependent partial inhibition of the mTOR-dependent signaling targets p70 S6 kinase (T412) and/or pS6 (S240/44) in the H358, MIA PaCa-2,H2122, and H1373 cell lines, each of which exhibited a maximal response to treatment. Together, these data suggest that maximizing inhibition of KRAS-dependent ERK and S6 signaling may be required to elicit a robust response in tumor-cell viability assays.

MRTX849 Treatment In Vivo Leads to Dose-Dependent KRASG12C Modification, KRAS Pathway Inhibition, and Antitumor Efficacy

Studies were conducted to evaluate MRTX849 antitumor activity along with its pharmacokinetic and pharmacodynamic properties in vivo, both to understand the clinical utility of this agent and to provide insight toward response to treatment. MRTX849 demonstrated moderate plasma clearance and prolonged half-life following oral administration (Supplementary Table S1; Supplementary Fig. S3). To evaluate the pharmacodynamic response to MRTX849 and to correlate drug exposure with target inhibition, MRTX849 was administered via oral gavage over a range of dose levels to H358 xenograft–bearing mice, and plasma and tumors were collected at defined time points. The fraction of covalently modified KRASG12C protein was proportional to the plasma concentration of MRTX849 (Fig. 2A). When evaluated over time after a single oral dose at 30 mg/kg, the modified fraction of KRASG12C was 74% at 6 hours after dose and gradually decreased to 47% by 72 hours (Fig. 2B). This extended pharmacodynamic effect, despite declining levels of MRTX849 in plasma, was consistent with the irreversible inhibition of KRASG12C by MRTX849 and the relatively long half-life for the KRASG12C protein (∼24–48 hours; Supplementary Table S5). The modification of KRASG12C was maximized after repeated daily dosing for 3 days at 30 mg/kg (Fig. 2B), and higher dose levels did not demonstrate additional KRASG12C modification in multiple tumor models (data not shown). The maximum level of modification of approximately 80%, despite increasing dose and plasma levels of MRTX849, suggests that accurate measurement of complete inhibition of KRASG12C utilizing LC/MS may not be attainable, potentially due to a pool of active, noncycling, or unfolded KRASG12C protein in tumors. Together, these studies demonstrated a dose-dependent increase in covalent modification of KRASG12C by MRTX849 and that the majority of targetable KRAS was covalently modified by MRTX849 over a repeated administration schedule at dose levels at or exceeding 30 mg/kg.

Figure 2.

 

MRTX849 modifies KRASG12C and inhibits KRAS signaling and tumor growth in vivoA, MRTX849 was administered orally as a single dose to mice bearing established H358 xenografts (average tumor volume ∼350 mm3) at 10, 30, and 100 mg/kg. KRAS modification and MRTX849 plasma concentration data from n = 3 mice are shown as mean ± SD. KRASG12C modification was statistically significant versus vehicle control using the two-tailed Student t test. **, P < 0.01. B, MRTX849 was administered orally as a single dose or daily (QD) for 3 days to mice bearing established H358 xenografts (average tumor volume ∼350 mm3) at 30 mg/kg. Plasma was collected at 0.5, 2, 6, 24, 48, and 72 hours after administration of the last dose, and tumors were collected at 6, 24, 48, and 72 hours after dose. KRASG12C modification and MRTX849 plasma concentration data are shown from n = 3 mice as mean ± SD. Induction of modified KRASG12C protein at all time points was determined to be statistically significant versus vehicle control using two-way ANOVA. In addition, induction of modified KRASG12C protein at 72 hours in day 1 samples and 48 and 72 hours in day 3 samples was statistically significant versus the 6-hour time point. Brackets indicate P < 0.05 as compared with left-most sample. C, MRTX849 was administered as in A. Tumors were collected 6 hours after dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed by immunoblot and quantified by densitometric analysis. Relative fluorescence intensity of pERK1/2 and pS6 was normalized by dividing pERK1/2 and pS6 by total ERK1/2 and total S6, respectively. Vehicle-treated tumors were normalized to 1 by dividing all average values by the vehicle value. Average pERK1/2 and pS6 values were divided by the average value in vehicle-treated tumors. Data shown represent the average of 2 to 3 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity was determined to be statistically significant versus vehicle control using the two-tailed Student t test. Brackets indicate P < 0.05 compared with left-most sample. D, MRTX849 was administered as in B. Tumors were collected at 6, 24, 48, or 72 hours after administration of the last dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed as in C. Data shown represent the average of 3 to 4 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity on day 3 was determined to be statistically significant versus vehicle control using two-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. E, MRTX849 was administered via daily oral gavage at the doses indicated to mice bearing established MIA PaCa-2 xenografts. Dosing was initiated when tumors were approximately 350 to 400 mm3.MRTX849 was administered to mice daily until day 16. Data are shown as mean tumor volume ± SEM. Tumor volumes at day 16 were determined to be statistically significant versus vehicle control via two-tailed Student t test. **, P < 0.01; *, P < 0.05.

 

To evaluate the effect of MRTX849 on KRAS-dependent signal transduction in vivo, a single dose of MRTX849 at 10, 30, or 100 mg/kg was administered to H358 tumor–bearing mice. Dose-dependent inhibition of ERK1/2 and pS6S235/36 phosphorylation was observed at 6 hours after dose based on immunoblot and densitometric analysis (Fig. 2C). MRTX849 also demonstrated marked inhibition of ERK1/2 and S6S235/36 phosphorylation after one or three daily doses at 6 or 24 hours, and levels gradually recovered by 72 hours after the final dose (Fig. 2D). pERK1/2 and pS6S235/36 were further evaluated in formalin-fixed, paraffin-embedded sections from vehicle-treated and MRTX849-treated xenografts in four tumor models utilizing IHC methods coupled with image analysis algorithms. These studies demonstrated increased pERK1/2 and pS6 in nontumor/stromal cells following MRTX849 administration, indicating that immunoblotting studies with bulk tumor lysate likely underrepresent the degree of pathway inhibition in tumor cells, whereas IHC-based evaluation may more accurately reflect both the degree and spatial impact of pathway inhibition. Maximal inhibition was observed for both ERK and S6S235/36 phosphorylation after a single dose at the 6-hour time point, with a rebound in signaling evident 24 hours after single dose in each model (Supplementary Fig. S4). Marked inhibition of ERK phosphorylation was observed at 6 hours after administration, with 89%, 94%, and 94% inhibition observed compared with vehicle controls in MIA PaCa-2, H1373, and H2122 tumors, respectively (H358 pERK not quantifiable). This indicates that dose levels at or exceeding 30 mg/kg dose maximized inhibition of ERK phosphorylation in multiple models (Supplementary Fig. S4A and S4B). Inhibition of S6 phosphorylation at 6 hours was more variable, with percent inhibition values of 76%, 50%, 86%, and 56% observed in MIA PaCa-2, H1373, H358, and H2122 tumors, respectively (Supplementary Fig. S4B). Together, these data indicate that consistent acute (6 hours) inhibition of KRAS-dependent ERK phosphorylation was maximized in all evaluated models, whereas inhibition of S6S235/36 was more variable, presumably due to varying degrees of KRAS-independent activation of this pathway in different tumor models.

MIA PaCa-2 and H358 were selected as MRTX849-responsive tumor models, thereby enabling a high-resolution understanding of dose–response relationships. Significant, dose-dependent, antitumor activity was observed at the 3, 10, 30, and 100 mg/kg dose levels in the MIA PaCa-2 model (Fig. 2E). Evidence of rapid tumor regression was observed at the earliest post-treatment tumor measurement, and animals in the 30 and 100 mg/kg cohorts exhibited evidence of a complete response at study day 15. Dosing was stopped at study day 16, and all 4 mice in the 100 mg/kg cohort and 2 of 7 mice in the 30 mg/kg cohort remained tumor-free through study day 70 (Supplementary Fig. S5A). In a second MIA PaCa-2 study, dose-dependent antitumor efficacy was observed at the 5, 10, and 20 mg/kg dose levels, and 2 of 5 mice at the 20 mg/kg dose level exhibited complete tumor regression (Supplementary Fig. S5B). Significant dose-dependent antitumor efficacy was also observed in the H358 model, including 61% and 79% tumor regression at the 30 and 100 mg/kg dose levels, respectively, at day 22 (Supplementary Fig. S5C). MRTX849 was well tolerated, and no effect on body weight was observed at all dose levels evaluated (Supplementary Fig. S5D). These studies indicated that MRTX849 demonstrated dose-dependent antitumor efficacy over a well-tolerated dose range and that the maximally efficacious dose of MRTX849 is between 30 and 100 mg/kg/day.

MRTX849 Demonstrates Broad-Spectrum Tumor Regression in KRASG12C Cell Line and Patient-Derived Xenograft Models

To evaluate the breadth of antitumor activity across genetically and histologically heterogeneous KRASG12C-mutant cancer models, MRTX849 was evaluated at a fixed dose of 100 mg/kg/day (a dose projected to demonstrate near-maximal target inhibition in most models) in a panel of human KRASG12C-mutant cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) models. MRTX849 demonstrated tumor regression exceeding 30% volume reduction from baseline in 17 of 26 models (65%) at approximately 3 weeks of treatment (Fig. 3A; Supplementary Table S6). By comparison, MRTX849 did not exhibit significant antitumor activity at 100 mg/kg in three non–KRASG12C-mutant models (Fig. 3A; Supplementary Table S6). Together, these results indicate that KRASG12C-mutant tumors are broadly dependent upon mutant KRAS for tumor-cell growth and survival and that MRTX849 elicits antitumor activity through a KRASG12C-dependent mechanism.

Figure 3.

 

Antitumor activity of MRTX849 in KRASG12C-mutant and non–KRASG12C-mutant human tumor xenograft models. A, MRTX849 was administered via oral gavage at 100 mg/kg every day to mice bearing the CDX or PDX model indicated. Dosing was initiated when tumors were, on average, approximately 250 to 400 mm3. MRTX849 was formulated as a free base and resuspended as a solution in 10% Captisol and 50 mmol/L citrate buffer, pH 5.0. The percent change from baseline control was calculated at days 19 to 22 for most models. Statistical significance was determined for each model and is shown in Supplementary Table S6. Status of mutations and alterations in key genes is shown below each model. MAF (%), percent KRASG12C-mutant allele fraction by RNA-seq; CNV, copy-number variation; * denotes very high CDK4 expression by RNA-seq and possible amplification. HER family status was determined by averaging EGFRERBB2, and ERBB3 RNA-seq expression for CDX (CCLE) or PDX (Crown huBase) models. Positive HER family calls denote greater than the median expression of the models tested. CDX and PDX model HER family calls were determined independently. B, Tumor-growth inhibition plots from representative xenograft models that were categorized as sensitive, partially sensitive, or treatment refractory.

 

Although MRTX849 exhibited marked antitumor responses in the majority of models tested, a response pattern ranging from delayed tumor growth to complete regression was observed across the xenograft panel. The response to treatment was categorized as sensitive, partially sensitive, or treatment refractory (Fig. 3B). Rank order and Pearson statistical analyses were performed to evaluate the correlation between in vitro potency (IC50 in 2-D or 3-D viability assays) and antitumor response in vivo (% regression or progression on day 22), and a significant correlation between response in cell lines compared with tumor models was not observed (Supplementary Fig. S6A and S6B). Thus, we focused on a comprehensive analysis of correlates with MRTX849 tumor response in vivo, including tumor histology, co-occurring genetic alterations, as well as baseline or drug-induced changes in expression of KRAS-related genes [RNA sequencing (RNA-seq)] and/or protein signaling networks (RPPA in 18 models, ref. 28; Supplementary Fig. S7). No individual genetic alteration, including but not limited to KRAS-mutant allele frequency, TP53, STK11, or CDKN2A, predicted the antitumor activity of MRTX849. Interestingly, baseline gene and/or protein expression of selected members of the HER family of RTKs and of regulators of early cell-cycle transition did exhibit a trend with the degree of antitumor response, suggesting these pathways may influence the response to KRAS inhibitors (Supplementary Fig. S7A). Together, these data indicate that there are no individual binary biomarkers that clearly predict therapeutic response and that the molecular complexity and heterogeneity present in distinct KRAS-mutated tumors may contribute to the response to target blockade.

MRTX849 Antitumor Activity Translates to RECIST Responses in Patients with Cancer

A 45-year-old female former smoker diagnosed with stage IV lung adenocarcinoma and refractory to multiple lines of therapy including carboplatin/pemetrexed/pembrolizumab, docetaxel, and investigational treatment with binimetinib and palbociclib was enrolled onto the MRTX849-001 phase Ib clinical trial with two bilateral lung lesions and mediastinal lymph node as target lesions. Targeted next-generation sequencing (NGS) demonstrated a KRASG12C mutation (c.34G>T). In addition, loss-of-function KEAP1 (K97M) and STK11 (E223*) mutations were detected and are predicted to be deleterious to their respective proteins. The patient was administered MRTX849 (600 mg twice a day) and had marked clinical improvement within 2 weeks, including complete resolution of baseline cough and oxygen dependency. A RECIST-defined partial response of 33% reduction of target lesions was observed at cycle 3 day 1 (45 days), and the patient continues on study (Fig. 4A).

Figure 4.

 

Activity of MRTX849 in patients with lung and colon cancers. A, Pretreatment and 6-week scans of a heavily pretreated patient with a KRASG12C mutation–positive lung adenocarcinoma indicating 33% reduction of target lesions. Patient continues on study. The top plots show a coronal view, and bottom plots an axial view of CT chest images prior to MRTX849 treatment (left) and after two cycles of MRTX849 treatment (right). B, Baseline, 6-week (Cycle 2), and 12-week (Cycle 4) scans of a patient with a KRASG12C mutation–positive colon adenocarcinoma. Partial response (PR) was confirmed at Cycle 4, and patient continues on study. Four lesions (TL1–4) are shown with axial views of CT images prior to MRTX849 treatment (top), after two cycles of MRTX849 treatment (center), and after four cycles of MRTX849 treatment (bottom).

 

A 47-year-old female never-smoker with metastatic adenocarcinoma of the left colon who exhibited progressive disease after receiving multiple lines of systemic therapy, including FOLFOX plus bevacizumab, single-agent capecitabine, FOLFIRI plus bevacizumab, and an investigational antibody–drug conjugate, was enrolled into the MRTX849-001 phase Ib clinical trial. This patient had extensive metastases involving the liver, peritoneum, ovaries, and lymph nodes. Targeted NGS identified a KRASG12C mutation. The patient was administered MRTX849 (600 mg twice a day) and demonstrated marked clinical improvement within 3 weeks and a visible decrease in size of her umbilical Sister Mary Joseph nodule. Her carcinoembryonic antigen levels decreased from 77 ng/mLat baseline to 11 ng/mL at cycle 2 day 1 and 3 ng/mL by cycle 3 day 1 (normal range, 0–5 ng/mL). A RECIST-defined partial response with a 37% reduction of target lesions and complete response of a nontarget lesion was observed at cycle 3 day 1(day 42). Confirmatory CT scans were conducted at cycle 5, day 1 (day 84) and indicated a confirmed RECIST partial response with further reduction of target lesions at −47% from baseline (Fig. 4B). The patient remains on treatment through Cycle 6.

Temporal Effects of MRTX849 on KRAS-Dependent Signaling and Feedback Pathways and Relationship to Antitumor Activity Following Repeat Dosing in Xenograft Models

A comprehensive analysis was conducted to evaluate MRTX849-induced temporal molecular changes to further interrogate mechanisms of drug response across sensitive and partially sensitive models. To evaluate temporal changes in global gene expression, xenograft-bearing mice were administered vehicle or 100 mg/kg MRTX849, and RNA-seq was performed on tumors at 6 and 24 hours after treatment. Gene expression was evaluated at day 1 and day 5 for the sensitive models MIA PaCa-2 and H1373 to ensure sufficient tissue availability from regressing tumors, or at day 7 in the partially sensitive models H358, H2122, and H2030 to coordinate with tumor stasis plateau. The top differentially expressed gene set enrichment analysis (GSEA) hallmark gene sets, regardless of tumor response, in all five models were several KRAS-annotated gene sets confirming MRTX849 selectively inhibits multiple genes directly related to KRAS signaling. MYC, mTOR, cell cycle, and apoptosis/BCL2 pathway gene sets were also strongly differentially expressed, confirming MRTX849 broadly affected multiple well-established, KRAS-regulated pathways, several of which have proved difficult to directly inhibit with previous targeted therapies (Fig. 5A and B; Supplementary Fig. S8A—S8D). The marked impact of MRTX849 on a large number of genes that regulate cell cycle and apoptosis provides further insight into molecular mechanisms which mediate its antitumor activity.

Figure 5.

 

MRTX849 treatment in vivo regulates KRAS-dependent oncogenic signaling and feedback-inhibitory pathways. A, Volcano plots displaying differentially expressed genes in xenograft tumors 24 hours after oral administration of vehicle or 100 mg/kg MRTX849 in a representative MRTX849-sensitive (H1373) and MRTX849-partially sensitive (H358) model. Significance denoted in the legend (Padj < 0.01). B, GSEA heat maps depicting hallmark signature pathways differentially regulated in at least one model 24 hours following oral administration of a single 100 mg/kg MRTX849 dose compared with vehicle. Normalized enrichment score shown in all models 6 or 24 hours after a single dose (QD × 1) or 5 (QD × 5) or 7 (QD × 7) days dosing. C, Genes that feedback-inhibit MAP kinase signaling are downregulated following MRTX849 treatment in all five cell line xenografts assessed by RNA-seq. TPM, Transcripts Per Kilobase Million.

 

Targeted RNA-seq analysis was performed on genes implicated in the temporal regulation of external signaling inputs and feedback pathways which collectively temper signaling flux through the RAS–RAF–MEK–ERK MAP kinase (MAPK) pathway including DUSPSPRY, and PHLDA family genes (13, 18). These MAPK pathway–negative regulators were each ranked among the most strongly decreased genes following MRTX849 treatment, providing evidence that ERK-dependent transcriptional output is blocked and that pathways involved in reactivation of RTK- and ERK-dependent signaling were activated (Fig. 5C; Supplementary Fig. S4A).

On the basis of the observation of dynamic changes in transcriptional programs linked to KRAS pathway reactivation, IHC plus quantitative imaging of tumor cell–specific pERK and pS6 was evaluated over a range of time points. In the sensitive MIA PaCa-2 and H1373 tumor models, treatment with MRTX849 (100 mg/kg) demonstrated ≥90% inhibition of ERK phosphorylation at 6 and 24 hours on both days 1 and 5 (Supplementary Fig. S4). In contrast, in the partially sensitive H358 and H2122 models, robust inhibition of ERK phosphorylation was observed at 6 hours after a single dose; however, marked recovery of ERK phosphorylation was observed at 24 hours after single dose and at both 6 and 24 hours following 7 days of repeat-dose administration. Because DUSPSPRY, and ETV family transcripts remain downregulated through 5 to 7 days in all models, it is evident that other independent factors contribute to temporal reactivation of ERK (Fig. 5C). Similar to what was observed with single-dose administration, the effect of MRTX849 on pS6 was variable over time and did not track with the antitumor activity of MRTX849. Together, these results suggest that the extent and duration of inhibition of pERK may track with the magnitude of antitumor efficacy of KRASG12C inhibitors and that further evaluation of the role of S6 is required to understand if it plays a role in drug sensitivity.

The effect of MRTX849 on cell proliferation and apoptosis was characterized by IHC analysis of Ki-67 or cleaved caspase-3 after a single dose or repeat administration. The fraction of Ki-67–positive cells was significantly reduced in tumors after repeat administration in all four models tested, further supporting a broadly operative antiproliferative mechanism, independent of the magnitude of MRTX849 antitumor response (Supplementary Fig. S4). Induction of apoptosis as determined by cleaved caspase-3 immunostaining was also evident on day 1 of treatment (6 and/or 24 hours after treatment) in the sensitive H358, MIA PaCa-2, and H1373 models (79%–100% maximal regression) but not in the partially sensitive H2122 model (Supplementary Fig. S4). An expanded RPPA-based pathway analysis of several models also indicated a correlation between antitumor activity of MRTX849 and decreased survivin (statistically significant at days 5/7 in 7 models evaluated; Supplementary Fig. S7B) and a trend toward increased cleaved caspase-3 induction (day 1, P = 0.08, 16 models), supporting the induction of apoptosis as a key mediator of a cytoreductive antitumor response (Supplementary Fig. S7C). Interestingly, the magnitude of reduction of MYC and cyclin B1 protein levels at days 5/7 also closely correlated with MRTX849 antitumor activity, consistent with their roles as critical regulators of KRAS-mediated cell growth and survival pathways (Supplementary Fig. S7B). Collectively, these data support that durable inhibition of ERK activity and maximal inhibition of ERK-regulated outputs including MYC and E2F-mediated transcription are associated with induction of apoptosis and maximal response to MRTX849 treatment.

CRISPR/Cas9 Screen Identifies Vulnerabilities and Modifiers of Response to MRTX849 in KRASG12C-Mutant Cancer Cell Lines In Vitro and In Vivo

The correlative analysis of genomic or proteomic markers with response to MRTX849 in the defined panel of models provided only limited insight toward mechanism of therapeutic response or resistance. Therefore, we directly interrogated the role of selected genes in mediating therapeutic response utilizing a focused CRISPR/Cas9 knockout screen targeting approximately 400 genes including many genes involved in KRAS signaling. This was conducted in H358 and H2122 cells in vitro and in H2122 xenografts in vivo in the presence and absence of MRTX849 treatment (Supplementary Fig. S9A–S9F). In MRTX849-anchored screens in vitro, single guide RNAs (sgRNA) that target RAS signaling pathway genes including MYC, SHP2 (H2122), mTOR pathway (MTOR and RPS6), and cell-cycle genes (CDK1, CDK2, CDK4/6, and RB1) were identified to affect cell fitness. sgRNAs that target KEAP1 and CBL were enriched in the H2122 model, demonstrating cell-specific genetic routes toward improved fitness through loss of classic tumor-suppressor genes, including in the context of MRTX849 treatment. KRAS sgRNA dropout was less pronounced in the MRTX849-treated cells compared with DMSO control–treated cells, as would be expected with redundant depletion of the drug target (Supplementary Fig. S9C and S9D). To evaluate whether a distinct KRAS dependence or modulation of MRTX849 therapeutic response was observed in vitro versus in vivo, xenograft-bearing mice bearing H2122 cells (∼250 mm3) transduced with the sgRNA library were orally administered vehicle or MRTX849 for 2 weeks (Supplementary Fig. S9A, S9E, and S9F). In MRTX849-treated xenografts, sgRNAs targeting cell cycle, SHP2, MYC, and mTOR pathway genes remained among the top depleted sgRNAs, demonstrating that inhibition of these targets in vivo, in the context of KRAS inhibition, leads to further tumor-growth inhibition over and above the effects of KRAS inhibition alone (Supplementary Fig. S9E and S9F). sgRNAs targeting the tumor suppressor KEAP1 were enriched in MRTX849-treated xenografts, suggesting loss of KEAP1 may represent a mechanism of intrinsic or acquired resistance. Interestingly, NRAS was one of the top enriched genes in the vehicle-treated xenografts, suggesting NRAS functions as a tumor suppressor in this context; however, enrichment was not as pronounced in the MRTX849-treated xenografts, suggesting NRAS may compensate for KRAS in the context of KRAS inhibition (Supplementary Fig. S9F). Collectively, these data demonstrate the importance of selected proteins that regulate RTK- and RAS-dependent signaling and cell-cycle transition in mediating the oncogenic effects of mutant KRAS, and also provide a catalog of potentially druggable vulnerabilities that complement KRAS blockade.

Cancer Therapeutic Combination Screen to Identify Rational and Clinically Tractable Strategies to Address Feedback and Resistance Pathways

To further interrogate pathways that mediate the antitumor response to MRTX849 and to identify combinations capable of enhancing response to MRTX849, a combination screen was conducted in vitro using a focused library of small-molecule inhibitors across a panel of cell lines (Supplementary Fig. S10A and S10B; Supplementary Table S8). Approximately 70 compounds targeting relevant pathways (RTKs, MAPK/ERK, PI3K, mTOR, cell cycle) were tested in a 3- or 7-day viability assay, and synergistic combinations were identified and ranked. Multiple hits from this screen were then identified for additional evaluation in combination studies with MRTX849, including the HER family inhibitor afatinib, the CDK4/6 inhibitor palbociclib, the SHP2 inhibitor RMC-4550, and mTOR pathway inhibitors.

Combination Strategies That Target Upstream Signaling Pathways Implicated in Extrinsic Regulation of KRAS Nucleotide Cycling and Feedback/Bypass Pathways

MRTX849 in combination with HER family inhibitors synergistically inhibited tumor-cell viability in the majority of cell lines evaluated and were the top hit in the combination screen in vitro (Supplementary Fig. S10). Cell lines with the highest (top 50th percentile) average composite baseline RNA expression values of selected HER family members exhibited the highest synergy scores to these combinations (Supplementary Fig. S11A). Afatinib was selected as a prototype HER family inhibitor based on its broad in vitro combination activity. Combination studies were conducted with MRTX849 and afatinib in five tumor models that were partially sensitive or treatment refractory to single-agent MRTX849. The MRTX849 and afatinib combination demonstrated significantly greater antitumor efficacy compared with either single agent in all five models evaluated, including multiple models exhibiting complete or near-complete responses to the combination (Fig. 6A; Supplementary Fig. S11B).

Figure 6.

 

HER family and SHP2 inhibitor combinations further inhibit KRAS signaling and exhibit increased antitumor responses. A, MRTX849 at 100 mg/kg, afatinib at 12.5 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or KYSE-410 cell line xenografts (n = 5). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.01. B, Quantification of KRAS mobility shift and pERK in H2122 cells treated for 24 hours with MRTX849 (0.1–73 nmol/L), afatinib (200 nmol/L), or the combination assessed by Western blot densitometry. C, MRTX849 at 100 mg/kg, afatinib at 12.5 mg/kg, or the combination was administered once or daily for 7 days via oral gavage to mice bearing H2122 cell line xenografts (n = 3/group). Tumors were harvested at 6 and 24 hours following the final dose. Tumor sections were stained for pERK and pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK or pS6 staining intensity was determined to be statistically significant relative to vehicle or either single agent using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. D, Quantitation of KRAS band shift and pERK after 24-hour treatment with MRTX849 (0.1–73 nmol/L), RMC-4550 (1 μmol/L), or the combination in H358 cells assessed by Western blot densitometry. E, MRTX849 at 100 mg/kg, RMC-4550 at 30 mg/kg, or the combination was administered daily via oral gavage to mice bearing the KYSE-410 or H358 cell line xenografts (n = 5/group). Combination treatment led to a statistically significant reduction in tumor growth compared with either single agent on the last day of dosing. *, Padj < 0.05. F, MRTX849 at 100 mg/kg, RMC-4550 at 30 mg/kg, or the combination was administered via oral gavage to mice bearing KYSE-410 cell line xenografts (n = 3/group), and tumors were harvested at 6 and 24 hours post-dose. Tumor sections were stained with pERK or pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK staining intensity was determined to be statistically significant relative to RMC-4550 alone using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample.

 

To evaluate whether afatinib affected covalent modification of KRASG12C by MRTX849, partially sensitive H2122 cells were treated with increasing concentrations of MRTX849 alone or in the presence of afatinib (200 nmol/L, IC90), and the mobility shift in KRAS protein was densitometrically determined from immunoblots. A clear shift in the concentration response to MRTX849 was apparent in the presence of afatinib, indicating that the combination increased the fraction of modified KRASG12C consistent with the putative role of HER family receptors in extrinsic regulation of KRASG12C GTP loading (Fig. 6B). The concentration–response relationship for inhibition of ERK phosphorylation was also clearly shifted in the presence of afatinib. To further evaluate the effect of the combination on KRAS-dependent signaling, four cell lines (H2030, H2122, H358, and KYSE-410) were treated with a range of MRTX849 concentrations in the presence or absence of afatinib for 6 or 24 hours, and key signaling molecules were evaluated by RPPA. Afatinib demonstrated clear inhibition of EGFR (pY1068) and HER2 (pY1248) activity and partial inhibition of ERK, AKT (S473), and p70S6K phosphorylation at both time points (Supplementary Fig. S11C). The effect of afatinib on S6 (S235/236, S240/244) and p90 RSK (S380) phosphorylation was variable and exhibited only minimal inhibition in most of the cell lines evaluated. The combination of afatinib and MRTX849 demonstrated markedly enhanced concentration-dependent inhibition and/or a greater magnitude of effect on ERK, RSK, p70 S6K, and S6 (S235/236) phosphorylation compared with MRTX849 alone at both 6 and 24 hours. Of note, neither afatinib nor MRTX849 alone inhibited S6 phosphorylation at the S240/244 site regulated by mTOR/S6K, whereas the combination demonstrated marked inhibition at 24 hours.

In vivo, the combination also exhibited a trend toward increased pERK and pS6 (S235/236) inhibition in the partially sensitive H2122 model in combination groups as determined by quantitation of immunostaining after 1- or 7-day administration (Fig. 6C). Similar results were observed in the MRTX849-refractory KYSE-410 model, and the combination also increased the number of apoptotic cells in this model (Supplementary Fig. S12A–S12C). Collectively, these data indicate that upstream baseline HER family activation may limit the ability of MRTX849 to achieve robust inhibition of the ERK and mTOR–S6 signaling pathways. Accordingly, the combination of afatinib and MRTX849 can limit feedback reactivation of ERK and demonstrate complementary inhibition of AKT–mTOR–S6 signaling, resulting in significantly improved antitumor activity.

SHP2 inhibition has been shown to inhibit the growth of cells that harbor KRASG12C mutations, and this effect is likely mediated, in part, by decreasing KRAS GTP loading (25–27). To evaluate whether SHP2 inhibition enhanced covalent modification of KRASG12C by MRTX849, H358 and H2122 cells were incubated with increasing MRTX849 concentrations with or without the SHP2 inhibitor RMC-4550. In both cell lines, cotreatment with RMC-4550 (1 μmol/L, IC90) demonstrated a MRTX849 concentration-dependent increase in KRASG12C protein modification and a concomitant decrease in ERK phosphorylation compared with MRTX849 alone (Fig. 6D; Supplementary Fig. S13A). RPPA analysis of KRAS-dependent signaling was conducted at 6 or 24 hours after treatment in three cell lines (H358, H2030, H2122) over a range of MRTX849 concentrations in the presence or absence of RMC-4550. RMC-4550 demonstrated robust inhibition of ERK phosphorylation and partial inhibition of p90 RSK (S380) and p70 S6K (T412) at both time points (Supplementary Fig. S13B). The combination of RMC-4550 and MRTX849 demonstrated incrementally increased concentration-dependent inhibition of ERK and RSK phosphorylation in all cell lines at both 6 and 24 hours and markedly improved inhibition of S6 (S235/236) phosphorylation compared with MRTX849 alone in H2122 and H358 cells at 24 hours. In addition, the combination demonstrated near-complete inactivation of KRAS in MRTX849-refractory KYSE-410 xenografts as determined using an active RAS ELISA assay, and this was significant compared with single agents (Supplementary Fig. S13C). On the basis of these findings, combination studies were conducted with MRTX849 and RMC-4550 in six KRASG12C-mutated tumor models in vivo, and the combination demonstrated significantly greater antitumor efficacy compared with either single agent in 4 of 6 models evaluated (Fig. 6E; Supplementary Fig. S13D). Consistent with the in vitro data, the combination also demonstrated a significant decrease in ERK phosphorylation compared with either single agent in the KYSE-410 model as determined by quantitation of tumor-cell immunostaining on day 1 at 6 and 24 hours and day 7 at 6 hours after dose (Fig. 6F). Together, these data indicate that EGFR family and SHP2 blockade can augment the antitumor activity of KRASG12C inhibitors through enhancing covalent target modification and establishing a more comprehensive blockade of KRAS-dependent signaling.

Combinations That Inhibit Bypass Pathways Downstream of KRAS and Exhibit Increased Antitumor Activity in Xenograft Models

KRAS is implicated in regulation of the oncogenic S6 protein translation pathway through both ERK-dependent activation of RSK, which phosphorylates S6 at Ser235/236, and cross-talk with the PI3K and mTOR pathway that additionally phosphorylates S6 at Ser240/244 (29). However, the S6 pathway can also be activated independently of mutated KRAS in tumor cells through hyperactivated RTK signaling, PI3K activation, or STK11 mutations, each of which converge on mTOR-mediated activation of S6. In the in vitro combination screen, mTOR inhibitors demonstrated synergy in a subset of evaluated cell lines (Supplementary Fig. S14A). To further evaluate the effect of the combination on KRAS and mTOR pathway–dependent signaling, four cell lines were treated with MRTX849 in the presence or absence of the selective ATP-competitive mTOR inhibitor vistusertib (1 μmol/L), for 6 or 24 hours, and several signaling molecules were evaluated by RPPA. Vistusertib demonstrated clear and robust inhibition of several components of the PI3K–mTOR signaling pathway including AKT (S473), p70 S6K (T412), S6 (pS235/236, S240/244), and 4E-BP1 (S65, T70) phosphorylation in each cell line at both time points consistent with its mechanism of action (Supplementary Fig. S14B). MRTX849 alone did not affect 4E-BP1 or S6 (S240/244) activity, and it exhibited a variable and cell line–dependent effect on p70 S6K and S6 (pS235/236) phosphorylation in these cell lines. Vistusertib also demonstrated marked induction of ERK phosphorylation, often several-fold over vehicle control, at both time points in all four cell lines, consistent with prior reports (30). The combination of vistusertib and MRTX849 demonstrated a comparable level of inhibition of ERK phosphorylation compared with single-agent MRTX849, indicating that the activation of ERK signaling by vistusertib was impeded by the combination of the two agents. In addition, MRTX849 combined with vistusertib further inhibited p70 S6K and AKT S473 phosphorylation compared with either single agent. Near-complete inhibition of S6 (S235/236, S240/244) phosphorylation at limit of detection was observed for the combination in each cell line at evaluated time points.

Consequently, a cohort of tumor models was identified, and the combination of MRTX849 with the selective mTOR inhibitor vistusertib demonstrated marked tumor regression and significantly improved antitumor activity compared with either single agent in all six models evaluated (Fig. 7A; Supplementary Fig. S14C). MRTX849 in combination with a second, differentiated mTOR inhibitor, everolimus, which inhibits TORC1 but not TORC2, in the H2030 xenograft model also demonstrated a striking combination effect (Supplementary Fig. S14D). In the KRASG12C, STK11-mutant H2030 model, MRTX849 demonstrated marked inhibition of ERK phosphorylation through 24 hours, but exhibited only partial inhibition of pS6235/36 at 6 hours after dose, on days 1 and 7 (Fig. 7B and C). Vistusertib demonstrated marked inhibition of pS6235/36 at 6 hours after treatment with evidence of recovery by 24 hours. The combination of vistusertib and MRTX849 did not have a further effect on ERK phosphorylation but demonstrated a significant reduction in pS6235/36 on day 1 at 24 hours compared with vistusertib alone and a trend toward reduced pS6235/36 on both day 1 and day 7 at 6 hours compared with either single agent (Fig. 7B; Supplementary Fig. S14E). Together, these data indicate that MRTX849 and mTOR inhibitor combination demonstrates complementary inhibition of the ERK and mTOR–S6 signaling pathways, resulting in broad antitumor activity in KRASG12C-mutant tumor models.

Figure 7.

 

CDK4/6 and mTOR combinations suppress independently hyperactivated downstream pathways and exhibit increased antitumor responses. A, MRTX849 at 100 mg/kg, vistusertib at 15 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or H2030 cell line xenografts (n = 5/group). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.05. B, MRTX849 at 100 mg/kg, vistusertib at 15 mg/kg, or the combination was administered once or daily for 7 days via oral gavage to mice bearing H2030 cell line xenografts (n = 3/group). Tumors were harvested at 6 and 24 hours following the final dose. Tumor sections were stained with pERK and pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK or pS6 staining intensity was determined to be statistically significant relative to vehicle or either single agent using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. C, Protein Western blot analysis of KRAS pathway targets in H2030 xenografts treated with MRTX849 (100 mg/kg), vistusertib (15 mg/kg), or the combination, 6 or 24 hours after a single dose. D, Protein Western blot analysis of KRAS pathway and cell-cycle targets in H2122 cells treated for 24 hours with MRTX849, palbociclib, or the combination. E, Normalized RNA-seq gene-expression data on E2F targets in H2122 xenografts treated with MRTX849, palbociclib, or the combination, 6 and 24 hours after a single daily dose or seven daily doses. F, MRTX849 at 100 mg/kg, palbociclib at 130 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or SW1573 cell line xenografts (n = 5). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.05.

 

Signaling through KRAS is known to mediate cell proliferation, at least in part, through the regulation of the cyclin D family and triggering RB/E2F-dependent entry of cells into cell cycle. Loss-of-function mutations and homozygous deletions in the cell-cycle tumor suppressor CDKN2A (p16) are coincident in a subset of KRAS-mutant non–small cell lung cancer (NSCLC) and hyperactivate CDK4/6-dependent RB phosphorylation and cell-cycle transition. In the CDKN2A-null H2122 and SW1573 cell lines in vitro, MRTX849 demonstrated concentration-dependent partial inhibition of RB phosphorylation (pRB pS807/811) and concurrent increase in p27 in H2122 cells, but not SW1573 cells, at 24 hours (Fig. 7D; Supplementary Fig. S15A). MRTX849 in combination with the CDK4/6 inhibitor palbociclib (1 μmol/L) demonstrated near-complete inhibition of pRB in both H2122 and SW1573 cells and further induced p27 in H2122 cells. Interestingly, pS6 (S235/236) was also much more effectively suppressed by the combination in both H2122 and SW1573 cells, which is consistent with a recent report (31). RNA expression of target genes and RPPA analysis of target protein signaling events were also used as a readout of cell-cycle inhibition in the H2122 tumor model in vivo, and the combination of MRTX849 and palbociclib significantly inhibited E2F1 and selected E2F family target genes, induced p27 protein expression to a greater degree compared with either single agent, and further reduced the number of Ki-67–positive cells after 7 days of administration (Fig. 7E; Supplementary Fig. S15B and S15C). In addition, the combination demonstrated a significant decrease in pRB (S780) compared with either single agent after 7 days of administration in SW1573 tumors in vivo (Supplementary Fig. S15D). This combination also induced tumor regression in five tumor xenograft models that was significant compared with either single-agent control (Fig. 7F; Supplementary Fig. S15E). Although not significant, a trend was noted in which models with CDKN2A homozygous deletion exhibited an increased antitumor response to the combination of MRTX849 and CDK4/6 inhibition compared with models lacking evidence of genetic dysregulation of key cell-cycle genes (Supplementary Fig. S15F and S15G).

Discussion

The identification of MRTX849 as a highly selective KRASG12C inhibitor capable of near-complete inhibition of KRAS in vivo provides a renewed opportunity to better understand the role of this mutation as an oncogenic driver in various cancers and to guide rational clinical trial design. The lack of a significant correlation between sensitivity to MRTX849 antitumor activity in in vitro versus in vivo model systems made it necessary to further study KRAS oncogene dependence in tumor models in vivo, a more clinically relevant setting. The demonstration that MRTX849 exhibited significant antitumor efficacy in all evaluated KRASG12C-mutated cancer models and demonstrated marked regression in the majority (65%) confirms that this mutation is a broadly operative oncogenic driver and that MRTX849 represents a compelling therapeutic opportunity. This evidence of activity extended to patients, as demonstrated by RECIST partial responses in 2 patients enrolled in a phase I clinical trial of MRTX849. Collectively however, these data also illustrate that the degree of dependence of cancer cells on the presence of a KRASG12C mutation for growth and survival can vary across tumors and that co-occurring genetic alterations observed in KRAS-mutated cancers may influence response to direct targeted therapy. The further observation that KRAS mutations occur across different cancers and that no single co-occurring genetic alteration predicted response to treatment illustrates the genetic heterogeneity of KRAS-driven cancers. Findings in the present studies are consistent with other functional genomics or therapeutic strategies to block KRAS function across panels of cell lines or models which demonstrated a highly significant response of KRAS-mutant cells to target knockdown, a heterogeneous magnitude of response, and no clear co-occurring aberrations that predict resistance to target blockade (5, 32, 33). Interestingly, despite the implication that certain mutations that co-occur with KRAS including TP53, STK11, and KEAP1 may limit therapeutic response in KRASG12C-positive lung cancers, none of these mutations correlated with response or resistance in the cell-line panel. In addition, the partial response we reported in the patient with lung adenocarcinoma was observed in a patient harboring deleterious comutations in both STK11 and KEAP1. Together, these data further illustrate the heterogeneity and complexity of KRAS-mutated cancers and suggest that no binary co-occurring genetic event may be predictive of therapeutic response.

Temporal and dose–response analysis indicated maximal modification of KRASG12C and durable inhibition of KRAS-dependent signaling was important in maximizing therapeutic response. The recovery of ERK signaling and the inability to inhibit mTOR–S6 signaling despite continued treatment were each associated with transient or submaximal response to MRTX849. ERK1/2 is implicated in direct phosphorylation and negative feedback regulation of EGFR (T669), FGFR1 (S777), and SOS1, and each of these targets may facilitate KRASG12C-independent resetting of ERK signaling flux (34–36). The rapid and remarkable suppression of ERK pathway–regulated transcripts such as DUSP and SPRY/SPRED family members by MRTX849 in all models evaluated is consistent with that observed for RAF inhibitors and is implicated in reactivation of ERK and RTK signaling (18, 19). The dual-specificity phosphatases DUSP4 and 6 were strongly suppressed by MRTX849 and are implicated in dephosphorylating and inactivating ERK1/2 (14, 18, 37), whereas SPRY family members are implicated in the negative regulation of RTKs and adaptor proteins (e.g., GRB2), and may participate in modifying RAS family nucleotide exchange and effector binding (e.g., RAF1; ref. 38). Although suppression of DUSP and SPRY/SPRED was broadly observed in all models, the magnitude of signaling reactivation and response to MRTX849 varied across models. This suggests some tumor models harbor additional factors that bypass KRAS dependence or affect RAS pathway signaling flux, such as expression or activation of selected RTKs (e.g., ERBB2 amplification in the KYSE-410 model) or STK11 loss-of-function mutations, and may be primed for feedback reactivation of RAS-dependent signaling and/or limit the degree of signaling inhibition by MRTX849. This phenomenon was observed for BRAFV600E-mutant colon cancer (but not melanoma) which exhibits high baseline EGFR expression, is primed for rapid feedback activation of this RTK, and is resistant to single-agent inhibition but highly responsive to cotargeting BRAF (and/or MEK) and EGFR (20). In addition, blockade of BRAF or MEK1/2 resulted in feedback-mediated activation of the PI3K–mTOR signaling pathway in concert with the coactivation of upstream RTKs (e.g., EGFR), resulting in bypass of ERK pathway dependence and therapeutic resistance (17, 20, 39). The observations that baseline expression of HER family RTKs trended with MRTX849 antitumor activity and that CRISPR-based drug-anchored screens implicated EGFR, SHP2, and mTOR–S6 pathways as cotargetable vulnerabilities both support the hypothesis that these targets act as conditional response modifiers.

Activation of RTK signaling in the context of KRASG12C-mutant cancer was predicted to limit MRTX849 therapeutic response both by enhancing extrinsic regulation of GTPase activity and initiating KRAS-independent ERK and mTOR–S6 pathway activation. Therefore, HER family and SHP2 inhibition were employed as strategies to either block the critical RTK family in KRAS-mutant cells or block collective RTK signaling downstream, respectively. As MRTX849 binds only GDP-KRASG12C, both HER family and SHP2 inhibition each enhanced KRASG12C modification by MRTX849 and significantly improved antitumor activity. This observation is consistent with the putative role of activated RTKs in the engagement of SHP2 to mediate SOS1-dependent RAS GTP loading and to diminish RAS GAP activity, each of which converge on enhanced RAS activation state (40). The afatinib combination demonstrated a clear and marked inhibition of both the ERK–RSK and AKT–mTOR–S6 signaling pathways, whereas the SHP2 inhibitor combination demonstrated a clear impact on ERK–RSK signaling and a relatively less prominent impact on mTOR–S6 signaling. Although afatinib may more effectively address mTOR–S6 bypass signaling, SHP2 inhibition should be an effective combinatorial strategy to combat other RTKs outside of the HER family, such as FGFRs or MET, that could affect KRAS dependence. To further address bypass signaling mediated by RTK activation or STK11 mutations, each of which activate the mTOR–S6 signaling pathway independently of KRAS, mTOR inhibition in combination with MRTX849 was also evaluated. MRTX849 in combination with vistusertib, in fact, demonstrated significantly improved antitumor activity in vivo compared with either single agent in all six tumor models evaluated, regardless of STK11 mutational status. Consistent with the mechanism of action of vistusertib, comprehensive inhibition of AKT–mTOR–S6 signaling was observed for vistusertib alone and near-complete inhibition of pS6S235–36 and pS6240–44 was observed in combination. In addition, the marked feedback reactivation of ERK by vistusertib was relieved by the combination. The induction of ERK activity has been observed in tumor cells following mTORC1 inhibition by rapalogs or ATP-competitive inhibitors and has been implicated in limiting antitumor activity of this class of agents (30, 41, 42), supporting the suppression of ERK signaling by MRTX849 as a key mechanism of response to the combination. Notably, all three combination strategies converge on more comprehensive inhibition of KRAS-dependent signaling, converging on ERK and S6 activity. In addition, although the inhibition of the AKT–mTOR–S6 pathway did not correlate with model response to MRTX849 (potentially due to tumor heterogeneity), the observations that both MTOR and RPS6 drop out in drug-anchored CRISPR screens and that effective combination strategies more comprehensively block this pathway illustrate its likely importance in maximizing therapeutic response in KRAS-mutated cancers.

Cell-cycle dysregulation due to genetic alterations in cell-cycle regulators identified additional factors that could modify the therapeutic response to MRTX849. In addition, CDKN2A, RB1, CDK4, and CDK6 were all identified as gene targets that affected cell fitness in CRISPR screens. Genetic alterations including homozygous deletion of CDKN2A or amplification of CDK4 or CCND1 comprise up to 20% of KRAS-mutated NSCLC (43). Combination studies with MRTX849 and palbociclib in vivo demonstrated more comprehensive inhibition of RB and E2F family target genes and increased antitumor activity compared with either single agent in NSCLC models. In addition, these studies indicated that the combination resulted in more effective inhibition of S6 (S235/236) phosphorylation, establishing a previously unappreciated connection between cell-cycle blockade and protein translation pathways. Notably, this combination was especially effective in CDKN2A-deleted models, suggesting that this combination strategy may be primarily beneficial in a molecularly defined subset of patients characterized by decoupling of cell-cycle regulation from KRAS.

Collectively, models exhibiting a cytoreductive response to single-agent MRTX849 demonstrated a more comprehensive and durable inhibition of KRAS-dependent signaling and induction of an apoptotic response. These data suggest that maintaining durable inhibition of KRAS-dependent signaling below a defined threshold is required to elicit tumor regression. The elucidation of mechanisms that limit the therapeutic response to single-agent KRAS inhibition has provided insight toward strategies to enhance therapeutic activity in KRAS-mutant tumors. Of the 35% of models (9/26) that did not exhibit durable regression with single-agent MRTX849 treatment, five models (KYSE410, SW1573, H2122, H2030, and LU6405) were selected for rational combination studies, and at least one combination demonstrated significant improvement in antitumor efficacy and elicited a >50% tumor regression in all five models evaluated. These results suggest that essentially all KRASG12C-mutated cancers can derive clinical benefit from direct KRAS inhibitor–directed therapy either alone or in combination. Furthermore, rational pathway-centric combination regimens directed at hallmark signaling nodes may be directed to genetically defined patient subsets. For example, KRAS-mutated NSCLC exhibits mutually exclusive, co-occurring genetic alterations in STK11 and CDKN2A (43). The present data suggest that KRASG12C/STK11-mutated NSCLC could be readily addressed by combining a KRASG12C inhibitor with an RTK or mTOR inhibitor, whereas KRASG12C/CDKN2A-mutated NSCLC could be more effectively addressed by combination with a CDK4/6 inhibitor. Collectively, the present studies support the broad utility of covalent KRASG12C inhibitors in treating KRASG12C-mutated cancers and provide defining strategies to identify patients likely to benefit from single-agent therapy or rationally directed combinations.

  

UPDATED 02/07/2021

The November 1st issue of Science highlights a series of findings which give cancer researchers some hope in finally winning a thirty year war with the discovery of drugs that target KRAS, one of the most commonly mutated oncogenes  (25% of cancers), and thought to be a major driver of tumorigenesis. Once considered an undruggable target, mainly because of the smooth surface with no obvious pockets to fit a drug in, as well as the plethora of failed attempts to develop such an inhibitor, new findings with recently developed candidates, highlighted in this article and other curated within, are finally giving hope to researchers and oncologists who have been hoping for a clinically successful inhibitor of this once considered elusive target.

For a great review on development of G12C KRas inhibitors please see Dr. Hobb’s and Channing Der’s review in Cell Selective Targeting of the KRAS G12C Mutant: Kicking KRAS When It’s Down

Figure 1Mechanism of Action of ARS853 showing that the inhibitors may not need bind to the active conformation of KRAS for efficacy

Abstract: Two recent studies evaluated a small molecule that specifically binds to and inactivates the KRAS G12C mutant. The new findings argue that the perception that mutant KRAS is persistently frozen in its active GTP-bound form may not be accurate.

Although the development of the KRASG12C-specific inhibitor, compound 12 (Ostrem et al., 2013), was groundbreaking, subsequent studies found that the potency of compound 12 in cellular assays was limited (Lito et al., 2016, Patricelli et al., 2016). A search for more-effective analogs led to the development of ARS853 (Patricelli et al., 2016), which exhibited a 600-fold increase of its reaction rate in vitro over compound 12 and cellular activities in the low micromolar range.

A Summary and more in-depth curation of the Science article is given below:

After decades, progress against an ‘undruggable’ cancer target

Summary

Cancer researchers are making progress toward a goal that has eluded them for more than 30 years: shrinking tumors by shutting off a protein called KRAS that drives growth in many cancer types. A new type of drug aimed at KRAS made tumors disappear in mice and shrank tumors in lung cancer patients, two companies report in papers published this week. It’s not yet clear whether the drugs will extend patients’ lives, but the results are generating a wave of excitement. And one company, Amgen, reports an unexpected bonus: Its drug also appears to stimulate the immune system to attack tumors, suggesting it could be even more powerful if paired with widely available immunotherapy treatments.

Jocelyn Kaiser. After decades, progress against an ‘undruggable’ cancer target. Science  01 Nov 2019: Vol. 366, Issue 6465, pp. 561 DOI: 10.1126/science.366.6465.561

The article highlights the development of three inhibitors: by Wellspring Biosciences, Amgen, and Mirati Therapeutics.

Wellspring BioSciences

In 2013, Dr. Kevan Shokat’s lab at UCSF discovered a small molecule that could fit in the groove of the KRAS mutant G12C.  The G12C as well as the G12D is a common mutation found in KRAS in cancers. KRAS p.G12C mutations predominate in NSCLC comprising 11%–16% of lung adenocarcinomas (45%–50% of mutant KRAS is p.G12C) (Campbell et al., 2016; Jordan et al., 2017), as well as 1%–4% of pancreatic and colorectal adenocarcinomas, respectively (Bailey et al., 2016; Giannakis et al., 2016).  This inhibitor was effective in shrinking, in mouse studies conducted by Wellspring Biosciences,  implanted tumors containing this mutant KRAS.

See Wellspring’s news releases below:

March, 2016 – Publication – Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State

 

February, 2016 – Publication – Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism

 

Amgen

Amgen press release on AMG510 Clinical Trial at ASCO 2019

THOUSAND OAKS, Calif., June 3, 2019 /PRNewswire/ — Amgen (NASDAQ: AMGN) today announced the first clinical results from a Phase 1 study evaluating investigational AMG 510, the first KRASG12C inhibitor to reach the clinical stage. In the trial, there were no dose-limiting toxicities at tested dose levels. AMG 510 showed anti-tumor activity when administered as a monotherapy in patients with locally-advanced or metastatic KRASG12C mutant solid tumors. These data are being presented during an oral session at the 55th Annual Meeting of the American Society of Clinical Oncology (ASCO) in Chicago.

“KRAS has been a target of active exploration in cancer research since it was identified as one of the first oncogenes more than 30 years ago, but it remained undruggable due to a lack of traditional small molecule binding pockets on the protein. AMG 510 seeks to crack the KRAS code by exploiting a previously hidden groove on the protein surface,” said David M. Reese, M.D., executive vice president of Research and Development at Amgen. “By irreversibly binding to cysteine 12 on the mutated KRAS protein, AMG 510 is designed to lock it into an inactive state. With high selectivity for KRASG12C, we believe investigational AMG 510 has high potential as both a monotherapy and in combination with other targeted and immune therapies.”

The Phase 1, first-in-human, open-label multicenter study enrolled 35 patients with various tumor types (14 non-small cell lung cancer [NSCLC], 19 colorectal cancer [CRC] and two other). Eligible patients were heavily pretreated with at least two or more prior lines of treatment, consistent with their tumor type and stage of disease. 

Canon, J., Rex, K., Saiki, A.Y. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223 (2019) doi:10.1038/s41586-019-1694-1

Besides blocking tumor growth, AMG510 appears to stimulate T cells to attack the tumor, thus potentially supplying a two pronged attack to the tumor, inhibiting oncogenic RAS and stimulating anti-tumor immunity.

Mirati Therapeutics

Mirati’s G12C KRAS inhibitor (MRTX849) is being investigated in a variety of solid malignancies containing the KRAS mutation.

For recent publication on results in lung cancer see Patricelli M.P., et al. Cancer Discov. 2016; (Published online January 6, 2016)

For more information on Mirati’s KRAS G12C inhibitor see https://www.mirati.com/pipeline/kras-g12c/

KRAS G12C Inhibitor (MRTX849)

Study 849-001 – Phase 1b/2 of single agent MRTX849 for solid tumors with KRAS G12C mutation

Phase 1b/2 clinical trial of single agent MRTX849 in patients with advanced solid tumors that have a KRAS G12C mutation.

See details for this study at clinicaltrials.gov

UPDATED 02/07/2021

Amgen scientists’ rapid work to challenge the undruggable KRAS G12C mutation in cancer

Inside a 40-year quest to challenge the KRAS G12C mutation in cancer
By Amgen Oncology
Amgen’s sotorasib, an investigational lung cancer treatment, has been submitted to the FDA for review

 

 

Nearly four decades have passed since researchers first identified the RAS gene family, which includes HRASNRAS and KRASRAS is the most frequently mutated family of oncogenes – or potentially cancerous genes – in human cancers.1,2 While research  efforts have been able to identify and develop treatments for other driver gene mutations that contribute to cancer growth, success with treating KRAS, the most frequently mutated variant of the RAS family, has remained elusive.2 But now there is hope.

Amgen, a leading biotechnology company, has taken on one of the toughest challenges of the last 40 years in cancer research.3 Chemical biologist Kevan Shokat’s lab at the University of California, San Francisco, identified a small molecule that could slip into a groove on a KRAS mutation called G12C in 2013.4 Building on their own research strategies and this new insight, scientists at Amgen used structural biology and medicinal chemistry to identify an adjacent groove, and by November 2017, made the initial decision to advance the molecule that would become investigational sotorasib.5

KRAS G12C is the most common KRAS mutation in NSCLC.6,7 In the U.S., about 13% of patients with NSCLC harbor the KRAS G12C mutation.8 There is a high unmet need and poor outcomes in the second-line treatment of KRAS G12C-driven non-small cell lung cancer (NSCLC) and, currently, there are no KRASG12C targeted therapies approved.

According to Amgen’s head of research and development David Reese, “the company’s scientists had an idea some time ago that the future of oncology would be led by the marriage of immuno-oncology and precision therapy. We wanted to go after high value targets, and RAS proteins are one of them.”

Because of this effort to rapidly accelerate the speed of innovation, investigational sotorasib entered clinical trials in humans less than 12 months.

 

 

At the same time that scientists discovered investigational sotorasib, the team was undertaking a project to map out every step it takes to progress a potential new treatment from an idea in a lab to being made available for patients. The goal was to shrink timelines and eliminate gaps to develop drugs more rapidly in order to reach patients with serious illnesses like NSCLC as quickly as possible.

Because of this effort to rapidly accelerate the speed of innovation, sotorasib entered clinical trials in humans less than 12 months.5 Sotorasib was the first investigational KRASG12C inhibitor to enter the clinic, and is now being studied in the broadest clinical program exploring 10 combinations with global investigational sites spanning five continents.9 In a little more than two years, the sotorasib clinical program has established a clinical data set of more than 700 patients studied across 13 tumor types.9

The investigational treatment was recently submitted to the FDA for review and was granted Breakthrough Therapy designation, a distinction designed to expedite the development and review of drugs.5 It was also accepted into the FDA’s Real-Time Oncology Review pilot program, which aims to explore a more efficient review process.5

To learn more about Amgen and how the speed of innovation is bringing new oncology treatments to patients with high unmet needs, visit Amgen.com/KnowKRAS.  

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1 Ryan MB, et al. Nat Rev Clin Oncol. 2018;15:709-720.

2 Cox AD, et al. Nat Rev Drug Discov. 2014;13:828-851.

3 Kim D, et al. Cell. 2020. doi:10.1016/j.cell.2020.09.044.

4 Ostrem JM, et al. Nature. 2013 ; 503 :548-551.

5 AMGEN, 2020. Retrieved January 8, 2021, from https://www.amgen.com/stories/2020/12/rapidly-advancing-development-of-amgens-investigational-kras-g12c-inhibitor

6 Pakkala S, et al. JCI Insight. 2018;3:e120858.

7 Arbour KC, et al. Clin Cancer Res. 2018;24:334-340.

8 Amgen, Data on File. 2020.

9 ClinicalTrials.gov. NCT04185883, NCT04380753, NCT03600883, NCT04303780. https://clinicaltrials.gov/ct2/. Accessed January 20, 2020.

 
 
Members of the editorial and news staff of the USA TODAY Network were not involved in the creation of this content.
 
 
 

Additional References:

Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism.

Lito P et al. Science. (2016)

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

Janes MR et al. Cell. (2018)

Potent and Selective Covalent Quinazoline Inhibitors of KRAS G12C.

Zeng M et al. Cell Chem Biol. (2017)

Campbell, J.D., Alexandrov, A., Kim, J., Wala, J., Berger, A.H., Pedamallu, C.S., Shukla, S.A., Guo, G., Brooks, A.N., Murray, B.A., et al.; Cancer Genome Atlas Research Network (2016). Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat. Genet.48, 607–616

Jordan, E.J., Kim, H.R., Arcila, M.E., Barron, D., Chakravarty, D., Gao, J., Chang, M.T., Ni, A., Kundra, R., Jonsson, P., et al. (2017). Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov. 7, 596–609.

Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.M., Gingras, M.C., Miller, D.K., Christ, A.N., Bruxner, T.J., Quinn, M.C., et al.; Australian Pancreatic Cancer Genome Initiative (2016). Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52.

Giannakis, M., Mu, X.J., Shukla, S.A., Qian, Z.R., Cohen, O., Nishihara, R., Bahl, S., Cao, Y., Amin-Mansour, A., Yamauchi, M., et al. (2016). Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 15, 857–865.

Read Full Post »

MDM2 inhibitor for the treatment of cancers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

AM 7209

ANTHONY MELVIN CRASTO, PhD

 

AM 7209

STR1

Amgen Inc. INNOVATOR

MF 747.700043 g/mol, C37H41Cl2FN2O7S

cas 1623432-51-8

US8952036

 

p53 is a tumor suppressor and transcription factor that responds to cellular stress by activating the transcription of numerous genes involved in cell cycle arrest, apoptosis, senescence, and DNA repair. Unlike normal cells, which have infrequent cause for p53 activation, tumor cells are under constant cellular stress from various insults including hypoxia and pro-apoptotic oncogene activation. Thus, there is a strong selective advantage for inactivation of the p53 pathway in tumors, and it has been proposed that eliminating p53 function may be a prerequisite for tumor survival. In support of this notion, three groups of investigators have used mouse models to demonstrate that absence of p53 function is a continuous requirement for the maintenance of established tumors. When the investigators restored p53 function to tumors with inactivated p53, the tumors regressed.

p53 is inactivated by mutation and/or loss in 50% of solid tumors and 10% of liquid tumors. Other key members of the p53 pathway are also genetically or epigenetically altered in cancer. MDM2, an oncoprotein, inhibits p53 function, and it is activated by gene amplification at incidence rates that are reported to be as high as 10%. MDM2, in turn, is inhibited by another tumor suppressor, p14ARF. It has been suggested that alterations downstream of p53 may be responsible for at least partially inactivating the p53 pathway in p53WT tumors (p53 wildtype). In support of this concept, some p53WT tumors appear to exhibit reduced apoptotic capacity, although their capacity to undergo cell cycle arrest remains intact. One cancer treatment strategy involves the use of small molecules that bind MDM2 and neutralize its interaction with p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting as an E3 ubiquitin ligase to promote p53 degradation; 2) binding to and blocking the p53 transcriptional activation domain; and 3) exporting p53 from the nucleus to the cytoplasm. All three of these mechanisms would be blocked by neutralizing the MDM2-p53 interaction. In particular, this therapeutic strategy could be applied to tumors that are p53WT, and studies with small molecule MDM2 inhibitors have yielded promising reductions in tumor growth both in vitro and in vivo. Further, in patients with p53-inactivated tumors, stabilization of wildtype p53 in normal tissues by MDM2 inhibition might allow selective protection of normal tissues from mitotic poisons.

The present invention relates to a compound capable of inhibiting the interaction between p53 and MDM2 and activating p53 downstream effector genes. As such, the compound of the present invention would be useful in the treatment of cancers, bacterial infections, viral infections, ulcers and inflammation. In particular, the compound of the present invention is useful to treat solid tumors such as: breast, colon, lung and prostate tumors; and liquid tumors such as lymphomas and leukemias. As used herein, MDM2 means a human MDM2 protein and p53 means a human p53 protein. It is noted that human MDM2 can also be referred to as HDM2 or hMDM2.

 

PATENT

US8952036

http://www.google.com/patents/US20140243372

 

Patent

WO 2015070224

Another particular MDM2 inhibitor is AM-7209 (Compound C herein), which is disclosed in U.S. provisional patent application number 61/770,901, filed February 28, 2013. (See Example No. 5 therein and below). AM-7209 has the following chemical name and structure: 4- (2-((3i?,5i?,65)-l-((5)-2-(tei’i-butylsulfonyl)-l-cyclopropylethyl)-6-(4-chloro-3-fluorophenyl)- 5-(3-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetamido)-2-methoxybenzoic acid

 

Discovery of AM-7209, a Potent and Selective 4-Amidobenzoic Acid Inhibitor of the MDM2–p53 Interaction

J. Med. Chem., 2014, 57 (24), pp 10499–10511    http://dx.doi.org:/10.1021/jm501550p
Abstract Image
Structure-based rational design and extensive structure–activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2–p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209(25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50= 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1
Yosup Rew, Principal Scientist,
Amgen

March 2013 – December 2014 (1 year 10 months)San Francisco Bay Area

Medicinal Chemistry (oncology)
1. Led optimization of small molecule inhibitors targeting protein-protein interactions in oncology programs
2. Discovered AM-7209, a back-up clinical candidate of AMG 232 featuring improved potency (KD from ITC competition = 38 pM), by replacing the carboxylic acid with an 4-amidobenzoic acid

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Curator: Aviva Lev-Ari, PhD, RN

For IP and Legal aspects of Biosimilars, go to:

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-intellectual-property-creation-and-protection-by-pioneer-and-by-biosimilar-manufacturers/

For CMC and Regulatory Affairs of Biosimilars, go to:

Biosimilars: CMC Issues and Regulatory Requirements

http://pharmaceuticalintelligence.com/2012/07/29/biosimilars-cmc-issues-and-regulatory-requirements/

The patent provisions of the Biosimilar Act, 2009 establish demanding and time-sensitive disclosure requirements. ObamaCare upheld by the Supreme Court is a victory for future development of pathways for biosimilar regulatory approval and eventually biosimilar generic drugs.

With the upheld ObamaCare, critical parts of the PPACA constitutional, and with it the BPCIA giving the FDA authority to approve biosimilars.

Had the PPACA been stricken in part or in its entirety, it would have presented obstacles to the BPCIA surviving in its present form. The US government has been critical of the 12-year data exclusivity period for Pioneer Innovators, calling for it to be shortened to 7 years (12 years is favorable to Pioneer Innovators and less favorable for Biosimilar manufacturers). The upheld ObamaCare, PPACA and BPCIA, constitutional, has prevented a multiyear delay in biosimilar approval. Thus, it was the best scenario for the biologics industry.

Thus, projection of Sales for Biosmilars as % of top 100 U.S. Pharmaceutical will receive a special meaning and an expected enhanced market share for 2012 year end and beyond 2012.

Biosimilars are occupying the Following ranking in the U.S. Pharmacuetical Sales – 2012: Top 100 Drugs for Q1 2012 by Sales: 10, 11, 12 13, 15, 24, 27, 29, 33, 35, 39, 57, 58, 62, 65, 70,  72, 74, 90, 98, 99. In addition the following biosimilars did not make the Top 100 list:

Biosimilar Drugs by US Sales – not included in the Top 100 Drug List 

Recombinate $2.9 1998 — Antihemophilic Factor VIII (Recombinant) by Baxter 5.7 Billion in 2012

Cerezyme $1.5 1994 —  Gaucher disease and Fabrazyme for Fabry disease by Genzyme 200 millions in sales

TYSABRI(R) (natalizumab) revenues were $280 million, in-line with the second quarter of 2011 by Elan and Biogen

NovoSeven $1.4 1999 —  Anti-fibrinolytics by Novo Nordisk – $1.5Billion

Synagis $1.3 1998 — Generic Name:  palivizumab     Anti-virals by AstraZeneca  $570 millions

Humulin $1.1 1992 Insulin Human by Eli Lilly $ 1.2 Billion

Kogenate FS $1.1 1993 — octocog alfa    Anti-fibrinolytics By Bayer $1.4 billion

U.S. Pharmacuetical Sales – 2012: Top 100 Drugs for Q1 2012 by Sales – Small Molecule Drugs (in green) and Biosimilars (in red)

The following is a list of the top 100 pharmaceutical drugs by retail sales in 2012, listed by U.S. sales value and drug name. Last updated: July 2012 (updated quarterly)

http://www.drugs.com/stats/top100/sales

Rank Drug

Sales ($000)

   
1 PlavixBristol-Myers Squibb Company

1,620,790

Stats

2 NexiumAstraZeneca Pharmaceuticals

1,395,981

Stats

3 AbilifyOtsuka Pharmaceutical Co.

1,340,200

Stats

4 SingulairMerck & Co., Inc.

1,238,134

Stats

5 SeroquelAstraZeneca Pharmaceuticals

1,161,141

Stats

6 Advair DiskusGlaxoSmithKline

1,139,182

Stats

7 CrestorAstraZeneca Pharmaceuticals

1,117,904

Stats

8 CymbaltaEli Lilly and Company

1,029,262

Stats

9 atorvastatinGeneric Drug

952,407

Stats

10 HumiraAbbott Laboratories

928,124

 

Stats

11 RemicadeCentocor Ortho Biotech, Inc

899,453

 

Stats

12 EnbrelAmgen Inc.

890,135

 

Stats

13 NeulastaAmgen Inc.

849,971

 

Stats

14 LipitorPfizer Inc

840,715

Stats

15 RituxanGenentech, Inc

756,875

 

Stats

16 CopaxoneTeva Pharmaceuticals

748,585

Stats

17 AtriplaGilead Sciences, Inc.

694,901

Stats

18 OxyContin

662,876

Stats

19 SpirivaBoehringer Ingelheim Pharmaceuticals, Inc

659,818

Stats

20 AvastinGenentech, Inc

632,183

Stats

21 ActosTakeda Pharmaceuticals North America, Inc

630,970

Stats

22 JanuviaMerck & Co., Inc.

583,603

Stats

23 TruvadaGilead Sciences, Inc.

546,098

Stats

24 LantusSanofi-Aventis

520,584

Stats

25 DiovanNovartis Corporation

509,615

Stats

26 LexaproForest Pharmaceuticals, Inc

491,053

Stats

27 EpogenAmgen Inc.

489,570

 

Stats

28 LyricaPfizer Inc

458,171

Stats

29 Lantus SolostarSanofi-Aventis

448,388

 

Stats

30 enoxaparinGeneric Drug

442,263

Stats

31 EloxatinSanofi-Aventis

431,928

Stats

32 CelebrexPfizer Inc

430,993

Stats

33 HerceptinGenentech, Inc

425,687

 

Stats

34 Diovan HCTNovartis Corporation

415,475

Stats

35 LucentisGenentech, Inc

409,547

 

Stats

36 SynagisMedImmune, Inc

396,556

Stats

37 NamendaForest Pharmaceuticals, Inc

391,638

Stats

38 GleevecNovartis Corporation

391,072

Stats

39 AvonexBiogen Idec

388,623

 

Stats

40 VyvanseShire US Inc

387,167

Stats

41 olanzapineGeneric Drug

385,867

Stats

42 IncivekVertex Pharmaceuticals

371,349

Stats

43 One Touch Ultra

366,294

Stats

44 SuboxoneReckitt Benckiser Pharmaceuticals Inc.

338,840

Stats

45 methylphenidateGeneric Drug

337,211

Stats

46 ZetiaMerck & Co., Inc.

328,653

Stats

47 AndroGelAbbott Laboratories

311,850

Stats

48 ProvigilCephalon, Inc.

303,029

Stats

49 LidodermEndo Pharmaceuticals

301,354

Stats

50 TriCorAbbott Laboratories

298,834

Stats

51 SymbicortAstraZeneca Pharmaceuticals

290,669

Stats

52 CombiventBoehringer Ingelheim Pharmaceuticals, Inc

285,487

Stats

53 ProAir HFATeva Pharmaceuticals

284,647

Stats

54 Seroquel XRAstraZeneca Pharmaceuticals

282,416

Stats

55 amphetamine/dextroamphetamineGeneric Drug

275,447

Stats

56 NasonexMerck & Co., Inc.

274,748

Stats

57 NovologNovo Nordisk Inc.

266,305

 

Stats

58 ProcritJanssen Pharmaceuticals, Inc

264,190

 

Stats

59 AlimtaEli Lilly and Company

263,024

Stats

60 ViagraPfizer Inc

260,678

Stats

61 GeodonPfizer Inc

260,514

Stats

62 Rebif

258,088

 

Stats

63 budesonideGeneric Drug

257,243

Stats

64 NiaspanAbbott Laboratories

255,383

Stats

65 HumalogEli Lilly and Company

244,587

 

Stats

66 Flovent HFAGlaxoSmithKline

241,552

Stats

67 LovazaGlaxoSmithKline

239,845

Stats

68 LevemirNovo Nordisk Inc.

239,576

Stats

69 Adderall XRShire US Inc

239,097

Stats

70 NeupogenAmgen Inc.

238,427

 

Stats

71 ReyatazBristol-Myers Squibb Company

238,151

Stats

72 AranespAmgen Inc.

231,643

 

Stats

73 metoprololGeneric Drug

231,395

Stats

74 NovoLog FlexPenNovo Nordisk Inc.

227,228

 

Stats

75 VytorinMerck & Co., Inc.

218,215

Stats

76 JanumetMerck & Co., Inc.

212,596

Stats

77 IsentressMerck & Co., Inc.

211,526

Stats

78 escitalopramGeneric Drug

210,171

Stats

79 CialisEli Lilly and Company

206,996

Stats

80 AciphexEisai Corporation

203,097

Stats

81 PradaxaBoehringer Ingelheim Pharmaceuticals, Inc

201,065

Stats

82 SolodynMedicis Pharmaceutical Corporation

198,909

Stats

83 fentanylGeneric Drug

197,350

Stats

84 ZyprexaEli Lilly and Company

194,460

Stats

85 VelcadeTakeda Pharmaceuticals North America, Inc

188,583

Stats

86 RestasisAllergan, Inc

188,501

Stats

87 LunestaSunovion Pharmaceuticals Inc.

187,941

Stats

88 acetaminophen/hydrocodoneGeneric Drug

185,374

Stats

89 PrezistaJanssen Pharmaceuticals, Inc

182,859

Stats

90 PegasysGenentech, Inc

181,693

 

Stats

91 ZyvoxPfizer Inc

179,523

Stats

92 Prevnar 13Wyeth

179,085

Stats

93 LovenoxSanofi-Aventis

178,957

Stats

94 BenicarDaiichi Sankyo

174,619

Stats

95 VESIcareAstellas Pharma US

174,524

Stats

96 Ventolin HFAGlaxoSmithKline

172,707

Stats

97 OrenciaBristol-Myers Squibb Company

172,202

Stats

98 BetaseronBayer Healthcare Pharmaceuticals

172,143

 

Stats

99 ErbituxBristol-Myers Squibb Company

171,513

 

Stats

100 DexilantTakeda Pharmaceuticals North America, Inc

171,179

Stats

Source: IMS Health (Midas).

Biosimilars Drugs by US Sales – not included in the Top 100 Drug List 

Recombinate $2.9 1998 — Antihemophilic Factor VIII (Recombinant) by Baxter 5.7 Billion in 2012

Cerezyme $1.5 1994 —  Gaucher disease and Fabrazyme for Fabry disease by Genzyme 200 millions in sales

TYSABRI(R) (natalizumab) revenues were $280 million, in-line with the second quarter of 2011 by Elan and Biogen

NovoSeven $1.4 1999 —  Anti-fibrinolytics by Novo Nordisk – $1.5Billion

Synagis $1.3 1998 — Generic Name:  palivizumab     Anti-virals by AstraZeneca  $570 millions

Humulin $1.1 1992 Insulin Human by Eli Lilly $ 1.2 Billion

Kogenate FS $1.1 1993 — octocog alfa    Anti-fibrinolytics By Bayer $1.4 billion

2011 US Sales vs. 2008 US Sales (in Billions) for Top Selling Biologics

Source for 2008 Sales

http://www.tbiweb.org/tbi/file_dir/TBI2009/Bao-lu%20Chen.pdf 

Source for 20011, Q1 2012 Sales

http://www.drugs.com/stats/top100/sales

Drug Name,  2008 Sales, Year approved , Indication

[i.e. Drug Name Enbrel,  2008 Sales $8.0B, Year approved 1998 , Indication RA]

Enbrel $8.0 1998 — RA, psoriatic arthritis, or ankylosing spondylitis indication

Q1 2012 12 (1) $890,135 1.92% 823 -4.63%
Q4 2011 11 (1) $873,343 1.67% 863 1.77%
Q3 2011 12 (1) $858,997 1.27% 848 -2.97%
Q2 2011 13 (2) $848,230 3.77% 874 3.19%
Q1 2011 11 $817,401 847

http://www.drugs.com/stats/enbrel

Remicade $7.9 1998 — RA & Chron’s Disease

Q1 2012 11 (2) $899,453 10.04% 1,556 10.04%
Q4 2011 13 (3) $817,365 -7.02% 1,414 -9.82%
Q3 2011 10 $879,054 1.52% 1,568 1.03%
Q2 2011 10 (2) $865,903 7.61% 1,552 7.11%
Q1 2011 12 $804,699 1,449

http://www.drugs.com/stats/remicade

Humira $7.3 2002  — treat rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis

Q1 2012 10 $928,124 2.50% 546 -2.85%
Q4 2011 10 (1) $905,527 3.18% 562 2.55%
Q3 2011 11 (3) $877,641 3.95% 548 3.01%
Q2 2011 14 $844,296 6.32% 532 2.31%
Q1 2011 14 $794,076 520

http://www.drugs.com/stats/humira

Rituxan $7.3 1997 — cancer medicines to treat non-Hodgkin’s lymphoma or chronic lymphocytic leukemia.

Q1 2012 15 (1) $756,875 -1.91% 547 -0.91%
Q4 2011 14 (2) $771,622 6.96% 552 4.74%
Q3 2011 16 $721,408 -1.77% 527 -1.86%
Q2 2011 16 (4) $734,378 7.26% 537 5.09%
Q1 2011 20 $684,666

http://www.drugs.com/stats/rituxan

Second Quarter 2012 Highlights: RITUXAN(R) (rituximab) revenues from our unconsolidated joint business arrangement were $285 million for the quarter, an increase of 31% year-over-year. As previously disclosed, during the second quarter of 2011 our share of RITUXAN revenues from unconsolidated joint business was reduced by approximately $50 million to reflect our share of damages and interest that might be awarded in relation to an intermediate decision in Genentech, Inc.’s ongoing arbitration with Hoechst GmbH

http://www.marketwatch.com/story/correcting-and-replacing-biogen-idec-increases-revenue-18-to-14-billion-in-the-second-quarter-2012-07-24

Herceptin $5.7 1998 —  treat metastatic breast cancer that has progressed after treatment with other chemotherapy

Q1 2012 33 $425,687 -0.06% 155
Q4 2011 33 (2) $425,931 7.61% 155 4.73%
Q3 2011 31 (1) $395,804 -0.64% 148 -0.67%
Q2 2011 32 (4) $398,348 3.62% 149 1.36%
Q1 2011 36 $384,428 147

http://www.drugs.com/stats/herceptin

Lantus $5.1 2000 — long-acting form of the hormone insulin.

Q1 2012 29 (5) $448,388 9.81% 3,737 7.32%
Q4 2011 34 $408,336 8.54% 3,482 7.07%
Q3 2011 34 (2) $376,208 4.53% 3,252 6.00%
Q2 2011 36 (5) $359,907 7.80% 3,068 8.30%
Q1 2011 41 $333,878 2,833

http://www.drugs.com/stats/lantus-solostar

Epogen/Procrit $5.1 1989Anemia, low RBC

Worldwide, sales of the two drugs – sold under the brand names Epogen, Procrit and Aranesp – exceeded $9 billion in 2005 for Amgen and Johnson & Johnson, their makers.  Johnson & Johnson, which sells epoetin under the brand names Procrit in the United States and Eprex everywhere else, reported sales of $2.4 billion in the first nine months of 2006, down slightly from 2005.

Amgen Recalls Anemia Medications for Glass Fragments09/24/2010 – Drug-makers Amgen (AMGN) and Johnson & Johnson (JNJ) are voluntarily recalling two brandsof an injectable anemia medication because vials containing the drug may have tiny glass flakes. The drug, Epoetin alfa, is marketed under the brand names Epogen and Procrit.Known as lamellae, the glass fragments are created by the interaction of the drug with glass vials during storage, Amgen said in a statement announcing the recall. The recall is being conducted in cooperation with the Food and Drug Administration, Amgen said.

Latest study shows anemia drugs Epogen, Aranesp and Procrit cause strokes, says FDA

Posted on January 7, 2010

Anemia drugs sold by Amgen and Johnson & Johnson have been reported to cause strokes when prescribed in high doses, according to an article from the FDA, recently published in the The New England Journal of Medicine. The law firm of Aylstock, Witkin, Kreis & Overholtz is investigating the FDA’s recent announcement.

The FDA commentary said the latest study and previous studies “raise major concerns” about the use of these drugs to treat anemia caused by kidney disease. The drugs are also used to treat anemia caused by chemotherapy. Studies over the past several years have revealed a link between the drugs and heart attacks, strokes, and other problems.

Amgen’s anemia drugs include Epogen and Aranesp. Johnson & Johnson sells anemia drug Procrit, which is produced by Amgen. The drugs are designed to raise red blood cell levels, to promote delivery of oxygen to body tissues.

http://www.awkolaw.com/news/heart-attacks/anemia-drugs-epogen-aranesp-procrit-cause-strokes-says-fda/

Epogen / Procrit / Aranesp: The July 2012 News Report Which Tells Story Of Big Pharma Profits Over Patient Safety And Drug Efficacy

Once The FDA Started Paying Attention The Writing On The Wall Became Apparent, Albeit Too Late For Some

(Posted by  at DrugInjuryWatch.com)

This lengthy and well-presented news report, “Anemia drugs made billions, but at what cost?”, written by Peter Whoriskey and published July 19, 2012 by The Washington Post (free registration required), is a must-read for anyone with a concern or interest in how larger pharmaceutical companies might put corporate profits ahead of patient safety and drug efficacy.

Here is an excerpt from this Washington Post article which will give you a sense of what went on that, in hindsight, is so disturbing:

For years, a trio of anemia drugs known as Epogen, Procrit and Aranesp ranked among the best-selling prescription drugs in the United States, generating more than $8 billion a year for two companies, Amgen and Johnson & Johnson. Even compared with other pharmaceutical successes, they were superstars. For several years, Epogen ranked as the single costliest medicine under Medicare: U.S. taxpayers put up as much as $3 billion a year for the drugs.

The trouble, as a growing body of research has shown, is that for about two decades, the benefits of the drug — including “life satisfaction and happiness” according to the FDA-approved label — were wildly overstated, and potentially lethal side effects, such as cancer and strokes, were overlooked.

Last year, Medicare researchers issued an 84-page study declaring that among most kidney patients, the original and largest market for the drugs, there was no solid evidence that they made people feel better, improved their survival or had any “clinical benefit” besides elevating a statistic for red blood cell count.

As for some of the key events which led up to this revelation of sorts, we start with a June 24, 2011 FDA press release, “FDA modifies dosing recommendations for Erythropoiesis-Stimulating Agents — Cites increased risk of cardiovascular events when used to treat chronic kidney disease”, which included the following:

The U.S. Food and Drug Administration today recommended more conservative dosing guidelines for Erythropoiesis-Stimulating Agents (ESAs) when used to treat anemia in patients with chronic kidney disease (CKD) because of the increased risks of cardiovascular events such as stroke, thrombosis, and death….

Procrit —  (epoetin alfa) is a man-made form of a protein that helps your body produce red blood cells

Q1 2012 58 (3) $264,190 -2.13% 295 -4.22%
Q4 2011 55 (2) $269,937 3.58% 308 3.01%
Q3 2011 53 (12) $260,610 -21.61% 299 -21.32%
Q2 2011 41 (7) $332,466 7.04% 380 5.56%
Q1 2011 48 $310,606 360

http://www.drugs.com/stats/procrit

Epogen —  (epoetin alfa) is a man-made form of a protein that helps your body produce red blood cells

Q1 2012 27 (7) $489,570 -24.54% 555 -17.04%
Q4 2011 20 (2) $648,794 4.67% 669 3.40%
Q3 2011 22 (2) $619,828 -13.96% 647 -18.41%
Q2 2011 20 (1) $720,376 3.32% 793 4.48%
Q1 2011 19 $697,224 759

http://www.drugs.com/stats/epogen

Neulasta $4.2 2002 — used to prevent neutropenia, a lack of certain white blood cells caused by receiving chemotherapy. stimulates the bone marrow and promotes the growth of white blood cells called neutrophils

Q1 2012 13 (1) $849,971 3.33% 331 1.53%
Q4 2011 12 (2) $822,578 4.59% 326 3.49%
Q3 2011 14 (1) $786,464 -3.86% 315 -5.69%
Q2 2011 15 $818,068 4.04% 334 3.41%
Q1 2011 15 $786,288 323

http://www.drugs.com/stats/neulasta

Novolog $3.7 2000 —  Insulin aspart is a fast-acting form of insulin. NovoLog is used to treat type 1 (insulin-dependent) diabetes in adults and children who are at least 2 years old. It is usually given together with a long-acting insulin.

Q1 2012 57 (6) $266,305 5.67% 2,980 3.72%
Q4 2011 63 (3) $252,015 0.97% 2,873 -0.48%
Q3 2011 60 (1) $249,591 -0.96% 2,887 -2.66%
Q2 2011 61 (5) $252,010 3.16% 2,966 -0.70%
Q1 2011 66 $244,297 2,987

http://www.drugs.com/stats/novolog

Erbitux $3.6 2004 — used to treat cancers of the colon and rectum. It is also used to treat head and neck cancer.

Q1 2012 99 (2) $171,513 2.30% 266 3.91%
Q4 2011 97 (7) $167,657 -0.15% 256 0.79%
Q3 2011 90 (3) $167,909 -2.48% 254 -1.93%
Q2 2011 93 (2) $172,185 -0.89% 259 -0.38%
Q1 2011 95 $173,735 260

http://www.drugs.com/stats/erbitux

Aranesp $3.2 2001 — Anemia, low RBC,  (darbepoetin alfa) is a man-made form of a protein that helps your body produce red blood cells. 

Q1 2012 72 (6) $231,643 -5.86% 293 -7.86%
Q4 2011 66 (15) $246,056 -6.07% 318 -3.64%
Q3 2011 51 (3) $261,967 -10.25% 330 -11.29%
Q2 2011 48 (3) $291,873 -1.03% 372 -1.33%
Q1 2011 51 $294,912 377

http://www.drugs.com/stats/aranesp

The article reports on the decline of worldwide sales of Aranesp drug from Thousand Oaks, California-based Amgen Inc. as of the second quarter of 2007. According to Amgen, the 10% decrease of Aranesp worldwide sales was due to the reimbursement issues related to the anemia drug and the drop of U.S. demand for drug, in which the U.S. Aranesp reported sales in the second quarter of 2007 was only $578 million from $713 million in 2006.

http://connection.ebscohost.com/c/articles/26375335/amgen-posts-lower-aranesp-sales

1/24/2011, Amgen boosts prices to offset Aranesp sales

Amgen is hiking prices to make up for the shrinking sales volume of its anemia drug Aranesp. Bloomberg reports that Amgen raised the price tag on Aranesp itself by 4.4 percent, but also marked up the white-blood-cell-boosting meds Neulasta and Neupogen by 2.9 percent.

http://www.fiercepharma.com/story/amgen-boosts-prices-offset-aranesp-sales/2011-01-24

Recombinate $2.9 1998 — Antihemophilic Factor VIII (Recombinant)

BioScience core franchises include: Hemophilia, Biotherapeutics, BioSurgery and Vaccines. BioScience products represent approximately 45 percent of Baxter’s annual sales, totaling $5.7 billion in 2010.

2007 Outlook – Sales within Baxter’s BioScience business totaled $1.2 billion, an increase of 18 percent from the same period last year. This growth was driven by record sales of ADVATE, Antihemophilic Factor (Recombinant), Plasma/Albumin Free Method (rAHF-PFM) for the treatment of hemophilia A, antibody therapy products, including GAMMAGARD LIQUID(TM) [Immune Globulin Intravenous (Human)] (IVIG) 10% Solution for the treatment of primary immunodeficiencies, specialty plasma therapeutics and biosurgery products. Medication Delivery sales increased 7 percent to $1.0 billion, with increased sales of infusion systems, intraveneous solutions and parenteral nutrition products, along with accelerated growth in the company’s drug delivery business. Renal sales increased 6 percent to $537 million reflecting accelerating gains in peritoneal dialysis patients globally.

Lucentis $2.7 2006 intraocular injection. (ranibizumab injection) is a recombinant humanized IgG1 kappa isotype monoclonal antibody fragment designed for intraocular use. Ranibizumab binds to and inhibits the biologic activity of human vascular endothelial growth factor A (VEGF-A).

Date Range Sales Rank Sales ($000) Units (000)
Q1 2012 35 (5) $409,547 -6.89% 224 -5.88%
Q4 2011 30 (2) $439,867 2.44% 238 2.59%
Q3 2011 28 (2) $429,393 1.13% 232 0.87%
Q2 2011 30 (3) $424,611 0.95% 230 1.32%
Q1 2011 33 $420,635 227

http://www.drugs.com/stats/lucentis

http://www.gene.com/gene/about/ir/historical/product-sales/lucentis.html

Lucentis brought in $1.7 billion for Roche last year, according to data compiled by Bloomberg.Alimera Sciences Inc. (ALIM), based in Alpharetta, Georgia, and Psivida Corp. (PSDV) also are developing a diabetic macular edema treatment known as Iluvien. The FDA has twice rejected Iluvien, most recently in November.

The FDA pooled results from two Roche clinical trials and found 39 percent of patients who used the 0.3 milligram dose were able to read three additional lines of letters on an eye chart after two years compared to 41 percent who had the same effect on the 0.5 milligram dose, according to an FDA staff report released July 24.

Genentech recommended approval of the 0.3 milligram dose in its application to the FDA since there isn’t evidence of additional benefit of the higher dose, Terence Hurley, a spokesman for the company, said in an e-mail.

Patients who received the monthly injection also were significantly more likely than those who received fake doses of the drug to achieve 20/40 vision, enough eyesight to drive.

http://www.bloomberg.com/news/2012-07-26/roche-s-lucentis-backed-by-fda-panel-for-diabetic-blindness-1-.html

Avonex $2.6 1996 —  Multiple Sclerosis, a form of protein called beta interferon that occurs naturally in the body. Interferons help the body fight viral infections. Avonex is used to treat patients with relapsing forms of multiple sclerosis to slow the accumulation of physical disability. This medication will not cure MS, it will only decrease the frequency of relapse symptoms.

Q1 2012 39 (1) $388,623 2.22% 130 -3.70%
Q4 2011 38 (5) $380,189 0.19% 135 -2.17%
Q3 2011 33 $379,457 -0.05% 138 -1.43%
Q2 2011 33 (4) $379,639 2.45% 140 -1.41%
Q1 2011 37 $370,570 142

http://www.drugs.com/stats/avonex

Second-quarter net income surged 34 percent to $386.8 million, or $1.61 a share, from $288 million, or $1.18, a year earlier, the Weston, Massachusetts-based company said today in a statement. Earnings excluding some items of $1.82 topped by 26 cents the average of 21 analysts’ estimates (BIIB) compiled by Bloomberg. Revenue beat estimates by about $90 million.

Biogen said profit this year is expected to be more than $6.20 a share, 5 cents higher than its May 1 forecast (BIIB). The company has been increasing sales of Avonex, Rituxan and Tysabri, another MS therapy, while developing new medicines to introduce to the market.

http://www.businessweek.com/news/2012-07-24/biogen-second-quarter-profit-rises-as-avonex-sales-increase

AVONEX(R) (interferon beta-1a) revenues increased 16% year-over-year to $762 million.

Novolin $2.5 1991  —  Novolin R (insulin regular) is a short-acting form of human insulin, Diabetes, Type 1 Type 2

Date Range Sales Rank Sales ($000) Units (000)
Q1 2012 74 (2) $227,228 8.96% 2,489 13.81%
Q4 2011 76 (4) $208,552 10.19% 2,187 6.73%
Q3 2011 80 (6) $189,267 4.15% 2,049 4.92%
Q2 2011 86 (7) $181,733 3.71% 1,953 29.60%
Q1 2011 93 $175,235 1,507
http://www.drugs.com/stats/novolog-flexpen
Novo Nordisk launches iPhone app Posted 17th September 2010, 15:11:54
An iPhone app has been launched by Novo Nordisk in the US which lets healthcare staff check dosage guidelines for diabetes patients.

Novo Dose provides product-specific data for the company’s insulin analog agents Levemir (insulin detemir), NovoLog (insulin aspart) and NovoLog Mix (insulin aspart protamine/insulin aspart injectable).

Combined sales of the three medications increased by 24% last year, feeding a double-digit growth in Novo Nordisk sales and profits.

Novo Dose, the second diabetes app created by the industry, tells professionals when and how to dose the drugs, how to titrate and provides information on the blood glucose goals of patients.

Commenting on the new technology, Anup Kumar Sabharwal, an endocrinologist at the University of Miami Clinics’ Diabetes Research Institute, said: “This is where modern medicine is headed.”

Humalog $2.2 1996  Humalog is used to treat type 1 (insulin-dependent) diabetes in adults. Insulin lispro is a fast-acting form of insulin. It is usually given together with another long-acting insulin. It works by lowering levels of glucose in the blood. Humalog is also used together with oral (taken by mouth) medications to treat type 2 (non insulin-dependent) diabetes in adults.

Q1 2012 65 $244,587 -2.70% 2,570 -3.85%
Q4 2011 65 (2) $251,367 3.78% 2,673 2.81%
Q3 2011 63 (4) $242,208 -0.75% 2,600 -1.78%
Q2 2011 67 (5) $244,050 4.83% 2,647 1.15%
Q1 2011 72 $232,809 2,617

http://www.drugs.com/stats/humalog

Pegasys $2.0 2002 — (peginterferon alfa-2a) is made from human proteins that help the body fight viral infections. Pegasys is used to treat chronic hepatitis B or C. It is often used together with another medication called ribavirin (Copegus, Rebetol, RibaPak, Ribasphere, RibaTab).

Q1 2012 90 (1) $181,693 3.92% 87 3.57%
Q4 2011 91 () $174,833 84

http://www.drugs.com/stats/pegasys

Rebif $1.7 2002 — (interferon beta-1a) is a protein identical to one found in the body. Interferon beta-1a is made from human proteins. Interferons help the body fight viral infections. Rebif is used to treat relapsing multiple sclerosis (MS). This medication will not cure MS, it will only decrease the frequency of relapse symptoms.

Q1 2012 62 (1) $258,088 -0.21% 540 -9.09%
Q4 2011 61 (7) $258,643 0.43% 594 -0.34%
Q3 2011 54 (5) $257,535 1.48% 596 -1.49%
Q2 2011 59 (2) $253,780 0.25% 605 -0.66%
Q1 2011 61 $253,143 609

http://www.drugs.com/stats/rebif

Cerezyme $1.5 1994 —  Gaucher disease and Fabrazyme for Fabry disease.

Last year Genzyme was forced to temporarily close its manufacturing plant in Boston due to a viral contamination. The interruption lead to shortages of two key drugs: Cerezyme for Gaucher disease and Fabrazyme for Fabry disease.

That crisis sent the company’s stock price plummeting from nearly $84 in 2008 to a low earlier this year of $45.39. Sanofi’s offer to acquire the company for $18.5 billion, or $69 a share — along with a 14 percent rise in the NYSE Arca Biotech Index since late July — have helped the shares rebound.

But Genzyme is now on a mission to prove to shareholders that it is worth more than Sanofi is offering, and executives told investors on a conference call that the third quarter marks the beginning of its financial turnaround.

Third-quarter sales of Cerezyme, the company’s top drug, rose to $179.8 million from $93.6 million a year earlier, beating analysts’ average forecast of $175 million.

“In the third quarter we saw our financial recovery start to take effect, and we expect that this will accelerate during the fourth quarter as Cerezyme patients are able to return to normal dosing levels and we begin to increase shipments of Fabrazyme,” Genzyme CEO Henri Termeer said in a statement.

Cerezyme is the principal drug for Gaucher patients. In the first quarter of 2012 Genzyme (now part of Sanofi (SNY))reported Cerezyme sales of 149 million euros (approx. $194 million), up 5.8% from the same quarter of the previous year. The other supplier Shire (SHPGY) reported $72 million in Vpriv sales, up 22%. There is now a third supplier, Pfizer (PFE), teamed up with the Israeli company Protalix Biotherapeutics (PLX), whose product was approved by the FDA in May 2012. Elelyso (taliglucerase alfa) is now available in the US.
Product Cerezyme
2009 2010 2011
 Total 793 720 885
 Ann. Growth Total -9% 23%

http://www.evaluatepharma.com/Universal/View.aspx?type=Entity&entityType=Product&lType=modData&id=15461&componentID=1002

Tysabri $1.4 2004 — Multiple Sclerosis by Elan and Biogen

Global in-market sales of TYSABRI in the second quarter of 2012 were $395 million, an increase of 2% over the second quarter of 2011. The total was comprised of $211 million in U.S. sales and $184 million in sales outside the U.S.

TYSABRI(R) (natalizumab) revenues were $280 million, in-line with the second quarter of 2011.
ITALIAN DISPUTE

Elan derives its revenue almost exclusively from Tysabri and it reported total sales for the three months to June 30 of $288 million, up 6 percent on a year ago once sales from its since-divested drug delivery business are omitted.

That compared to the $299 million forecast by four analysts surveyed by Reuters and was driven by in-market sales of Tysabri that rose 2 percent year-on-year to $395 million, also shy of the $419 million expected by analysts.

Biogen, which detailed the sales numbers when it reported second quarter results on Tuesday, attributed the softer-than-expected Tysabri sales to a dispute with the Italian government over pricing.

The number of patients on Tysabri rose 4 percent to 69,100, maintaining Elan and Biogen’s 10 to 12 percent share of the MS drug market in the face of competition from Swiss drugmaker Novartis AG’s Gilenya treatment, the first multiple sclerosis pill to come on the market.

The average addition of 185 new patients per week was the highest quarterly run-rate since the fourth quarter of 2009.

http://in.reuters.com/article/2012/07/25/elan-idINL6E8IP1VV20120725

NovoSeven $1.4 1999 —  Anti-fibrinolytics by Novo Nordisk —

Generic Name:   eptacog alfa
Product NovoSeven
2009 2010 2011
 Total 1,324 1,431 1,559
 Ann. Growth Total 8% 9%

http://www.evaluatepharma.com/Universal/View.aspx?type=Entity&entityType=Product&id=13483&lType=modData&componentID=1002

Synagis $1.3 1998 — Generic Name:  palivizumab     Anti-virals by AstraZeneca

Product Synagis
2009 2010 2011
 Total 1,042 906 570
 Ann. Growth Total -13% -37%

http://www.evaluatepharma.com/Universal/View.aspx?type=Entity&entityType=Product&lType=modData&id=91&componentID=1002

Neupogen $1.3 1991 —  (filgrastim) is a man-made form of a protein that stimulates the growth of whiteblood cells in your body. White blood cells help your body fight against infection. Neupogen is used to treat neutropenia, a lack of certain white blood cells caused by cancer,bone marrow transplant, receiving chemotherapy, or by other conditions.

Q1 2012 70 $238,427 0.06% 170 -2.86%
Q4 2011 70 (5) $238,289 0.16% 175 10.76%
Q3 2011 65 (5) $237,915 0.69% 158 0.64%
Q2 2011 70 (4) $236,294 2.51% 157 0.64%
Q1 2011 74 $230,515 156

http://www.drugs.com/stats/neupogen

Betaseron $1.2 1993 — (interferon) is made from human proteins. Interferons help the body fight viral infections. Betaseron is used to treat relapsing multiple sclerosis (MS). Betaseron will not cure MS, it will only decrease the frequency of relapse symptoms.

Q1 2012 98 (1) $172,143 2.93% 67 -10.67%
Q4 2011 99 (12) $167,236 -3.76% 75 -5.06%
Q3 2011 87 (2) $173,769 -2.89% 79 -2.47%
Q2 2011 89 (4) $178,938 -2.17% 81 -7.95%
Q1 2011 85 $182,908 88

http://www.drugs.com/stats/betaseron

Humulin $1.1 1992 Insulin Human by Eli Lilly 

Product Humulin R
2009 2010 2011
 Total 1,022 1,089 1,249
 Ann. Growth Total 7% 15%

http://www.evaluatepharma.com/Universal/View.aspx?type=Entity&entityType=Product&lType=modData&id=12399&componentID=1002

Kogenate FS $1.1 1993 — octocog alfa    Anti-fibrinolytics By Bayer

Product Kogenate
2009 2010 2011
 Total 1,238 1,332 1,496
 Ann. Growth Total 8% 12%

http://www.evaluatepharma.com/Universal/View.aspx?type=Entity&entityType=Product&lType=modData&id=11681&componentID=1002

Conclusion

Biosimilars are defined as biological products similar, but not identical, to the reference biological products that are submitted for separate marketing approval following patent expiration of the reference biological products. As one of the ICH members, the US needs to catch up with the EU and Japan as those two countries have already issued regulatory guidelines for biosimilars.

Once Congress establishes a legal framework, FDA is expected to set up a biosimilar approval pathway which will be similar to those in the EU and Japan and harmonized under ICH. The biosimilar will need a full CMC development package plus demonstration of comparable quality attributes and comparable efficacy and safety to the innovator’s product. Table 5 provides a comparison summary between small-molecule generics and biosimilars. It will take a much bigger effort to develop a biosimilar than a generic drug. Automatic substitution between the innovator product and a biosimilar is not appropriate as a biosimilar is not a generic version of the innovator product and is approved based on comparability to the innovator product.

REFERENCES

http://www.wolfgreenfield.com/files/2426_biosimilars_2_final_pdf.pdf

Read Full Post »

Biosimilars: CMC Issues and Regulatory Requirements

Reporter: Aviva Lev-Ari, PhD, RN

Updated on 6/30/2015

Biosimilars in the US: How much can we learn from Europe?

http://www.xcenda.com/Insights-Library/HTA-Quarterly-Archive-Insights-to-Bridge-Science-and-Policy/HTA-Quarterly-Spring-2015/Biosimilars-in-the-US-How-much-can-we-learn-from-Europe/#.VY-99uehlRA.mailto

Updated on 2/10/2014

Cambridge Healthtech    Institute’s Fifth  Annual Biotherapeutics Analytical Summit
Hyatt Regency    Baltimore | Baltimore,  MD | BiotherapeuticsAnalyticalSummit.com

<http://wec.chi-lifescience.com/t/20344782/1039908871/3650297/1006/>

The    Science and  Regulation of Process Changes for Biologics (Comparability)
Thursday,  March 27, 2014 | 5:30 – 8:30 PM | More Information 

http://wec.chi-lifescience.com/t/20344782/1039908871/4704449/1007/?fb8dc108=YXZpdmFsZXYtYXJpQGFsdW0uYmVya2VsZXkuZWR1&x=9c418447

Manufacturing changes can impact on quality     attributes of biologics, and may affect efficacy and/or safety of the product.   For that reason, a thorough comparability exercise is required, to assess the   impact of the change and whether CMC data alone will suffice to support the     change. This interactive short course will consider comparability exercises     during development, as well as post-approval, addressing regulatory and     technical requirements. This should provide the attendee with the knowledge on   how to prepare a comparability package for discussion with regulatory  agencies,   towards acceptance of the proposed change to the process/product.  Attendees   will  be contacted before the event and asked about topics on which  they would   like to  focus.

Topics covered     include:

  • Ways that manufacturing changes can impact    on quality attributes
  • Features of a thorough comparability exercise
  • Critical evaluation of quality data
  • The comparability exercise during development
  • Post-approval comparability, ICH Q5E, Comparability Protocols (US) and Change Management Protocols (EU)
  • Regulatory requirements in the EU and US: guidelines, their interpretation and application
  • Discussion with Q&A


Course    Instructors:
       
Christopher J. Holloway, Ph.D., Group     Director, Regulatory Affairs &  CSO, ERA Consulting Group        
Kazumi Kobayashi, Ph.D., Director, Bioprocess Development, Biogen Idec,    Inc.       
Marjorie Shapiro, Ph.D., Chief, Laboratory of Molecular and Developmental  Immunology, Division of Monoclonal Antibodies,            FDA/CDER
 

Comparability and Developability conference program at Biotherapeutics Analytical Summit.

The third track of the Biotherapeutics Analytical    Summit focuses on the practical application of analytical characterization for    Comparability, Biosimilarity and Development purposes. It covers case studies    with a variety of products and a range of analytical technologies. We have    invited the FDA and regulatory experts to advise and to discuss regulatory    challenges being experienced by the industry. This conference also covers the    link between the process and analytical technologies for innovator products and   for biosimilars.

BiotherapeuticsAnalyticalSummit.com/Comparability

http://wec.chi-lifescience.com/t/20344782/1039908871/4665296/1009/?fb8dc108=YXZpdmFsZXYtYXJpQGFsdW0uYmVya2VsZXkuZWR1&x=833e704b>

SOURCE

From: Biotherapeutics Analytical Summit Short Course <lauran@healthtech.com>
Date: Mon, 10 Feb 2014 12:40:59 -0500
To: <avivalev-ari@alum.berkeley.edu>
Subject: The Science and Regulation of Process Changes for Biologics (Comparability)

Comparability: The Final Frontier of Protein Therapies

The rapid expansion of protein therapeutics has crossed into all major disease classes (cancer, metabolic disease, inflammatory disease, infectious disease, immune disorders, etc.).  Due to the lengthy learning curve and high cost of developing these complex products, the development of therapies has been traditionally limited to highly specialized companies.  As protein therapeutics become more mainstream, these products are finding new applications in disease treatment and commercial application beyond the range of the traditional biotech companies.  Unfortunately, the expansion of medical applications for these therapies is outpacing the rate of innovation in product development and, as a result, market availability is becoming constrained by the ability to characterize and control product characteristics.

The Future Opportunity:

The key to most effectively and efficiently developing a biopharmaceutical or biologic to marketing approval is to have a clear understanding of the unique properties of proteins and to use that knowledge to design appropriate manufacturing processes, preclinical pharmacology-toxicology and clinical programs.  It is essential in dealing with complex comparability issues related to the types of manufacturing processes or changes in manufacturing to understand the scientific reasons for requiring a demonstration of comparability and the relationships between bioanalytical differences or changes in a protein and potential alterations in protein functionality in terms of specificity, potency, pharmacological activity, pharmacokinetics, toxicity and ultimately clinical safety and efficacy.  This understanding requires in depth knowledge of protein chemistry, manufacturing processes, pharmacology, immunology and toxicology that comes with extensive training and experience.  The same scientific and regulatory expertise and experience that is required for a successful demonstration of comparability is also applicable to the development of a biosimilar. This type of experience and expertise should prove invaluable as the expansion in the area of protein therapies continues and the development of biosimilars grows in the coming years.

How to Get There from Here:

Product developers can begin today to capitalize on this opportunity.   A number of technology advancements are being explored that will enhance our understanding of relationships between process, product, and clinical safety and efficacy.   One must think through how to integrate the future product comparability into early stage product development.  For new product development, these issues will include more in-depth analysis of the product structure and relationship of various structural features to function andin vivo activity, increased knowledge about the effect of process conditions on the types and mix of final product variants, and careful choice of in vitro binding and functional assays that clearly relate to the proposed mechanism of action of the product and can often also be used as a potency assays.  New technologies are being developed for these assays, and appropriate in vitro functional assays as relates to pharmacological mechanism of action can be very useful for demonstrating comparability.

A careful determination of appropriate animal models of disease for demonstration of proof-of-concept pharmacology is also important early in development. Identification of appropriate biomarkers of efficacy or safety should also be examined in these early animal models for future use in clinical development as well as demonstration of comparability.  The discovery of appropriate biomarkers can sometimes be carried over to the clinic and used in clinical trials with the appropriate validation.  Finally, as the safety database for the various classes of biopharmaceuticals and biologics expands, the understanding of safety issues associated with each of these various product classes will make it easier to more efficiently demonstrate comparability as well as to develop biosimilar products.

For developers who are trying to bridge comparability on products that lack a complete process/product history due to legacy issues or as in the case for biosimilar development, companies must think about the pharmacology of the product as relates to the proposed indication of either the previous iteration of the product or the innovator product, depending on whether this is a comparability issue or development of a biosimilar. It is the pharmacological activity of a given protein product that determines the efficacy and to a great extent the toxicity of the product.   The pharmacological activity of a product is driven by protein structure, mix of product variants, binding kinetics, dose, dosing regimen, route of administration, and final product formulation, among others.

As the development of biopharmaceuticals and biologics continues to expand, more and more information accrues on potential safety issues related to each of the various product classes, and this information will also prove quite useful to demonstration of comparability and development of biosimilars.

Biologics Consulting Group can assist developers in designing and implementing each of their comparability programs with the greatest chance of rapid regulatory approval.  Our staff has both FDA and industry experience, with a track record of success in helping academic institutions, start-ups, and established biotech and pharma companies.  Our direct knowledge and contemporary experience with all possible regulatory pathways – and every associated nuance — and can provide the requisite preclinical, clinical, quality, analytical, and manufacturing support to increase your chances for success.

Contributors: T. CarrierD. BarngroverJ. JessopV. NarbutJ. HumphriesB. FraserR. WolffN. RitterL. Winberry 

SOURCE

http://www.biologicsconsulting.com/perspectives/comparability-protein-therapies/

For IP and Legal aspects of Biosimilars, go to:

Biosimilars: Intellectual Property Creation and Protection by Pioneer and by Biosimilar Manufacturers

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-intellectual-property-creation-and-protection-by-pioneer-and-by-biosimilar-manufacturers/

For Financial Aspects of Biosimilars, go to:

Biosimilars: Financials 2012 vs. 2008

http://pharmaceuticalintelligence.com/2012/07/30/biosimilars-financials-2012-vs-2008/

Tr e n d s  i n  B i o / P h a r m a c e u t i c a l I n d u s t r y , 1 9 -26

Special Report on Biosimilars

About the Author: Dr. Bao-Lu has over 18 years of experience in product development, CMC regulatory, manufacturing management and quality oversight. He is currently the Director of Manufacturing and Process Development at Sangamo BioSciences. In this role, he oversees outsourced GMP production and testing of Sangamo’s gene therapy products and is responsible for the release and disposition of final drug product. He also provides CMC regulatory support and manages the in-house quality system by maintaining GMP database and implementing quality SOPs. Previously, Bao-Lu served as an Associate Director of Formulation at Xencor and Chiron and a Formulation Scientist at Amgen. Bao-Lu graduated with a BS degree from Fudan University and was selected as one of the forty chemistry students in the first year CGP Doering program. Bao-Lu earned his Ph.D. in Chemistry from University of Oregon and performed postdoctoral research in Biology at Massachusetts Institute of Technology.

http://www.tbiweb.org/tbi/file_dir/TBI2009/Bao-lu%20Chen.pdf

CMC Issues and Regulatory Requirements for Biosimilars

Abstract

Chemistry, Manufacturing and Controls (CMC), preclinical and clinical are three critical pieces in biosimilars development. Unlike a small-molecule generic drug, which is approved based on “sameness” to the innovator’s drug; a biosimilar is approved based on high similarity to the original approved biologic drug. This is because biologics are large and complex molecules. Many functional-, safety- and efficacy-related characteristics of a biologic depend on its manufacturing process. A biosimilars manufacturer won’t be able to exactly replicate the innovator’s process. The traditional abbreviated pathway for generic drug approval through the Hatch- Waxman Act of 1984 doesn’t apply for biosimilars as drugs and biologics are regulated under different laws. New laws and regulations are needed for biosimilars approval in the US. The EU has issued biosimilars guidelines based on comparative testing against the reference biologic drug (the original approved biologic). A full scale CMC development is required including expression system, culture, purification, formulation, analytics and packaging. The manufacturing process needs to be developed and optimized using state-of-the-art technologies. Minor differences in structure and impurity profiles are acceptable but should be justified. Abbreviated clinical testing is required to evaluate surrogate markers for efficacy and demonstrate no immunogenic response to the product.

We anticipate the package for a biosimilars approval in the US will be similar to that in the EU and contain a full quality dossier with a comparability program including detailed product characterization comparison and reduced preclinical and clinical requirements.

Biosimilars Become Inevitable

 Biologics developed through biotechnology constitute an essential part of the pipeline for medicines available to patients today. Biologic drugs are quite expensive and many of them are top-selling medicines (see Table 1). Since they come at extremely high prices to consumers, some patients may not be able to afford the use of biologics as the best-available treatments to their conditions. The patent protection on a large number of biologics has expired since 2001. These off-patent biologics include Neupogen, Novolin, Protropin, Activase, Epogen or Procrit, Nutropin, Humatrope, Avonex, Intron A, and Humulin. Traditionally, when a drug patent expires, a generic drug will be quickly developed and marketed. Similarly, generic version of off-patent biologic drugs (also referred to biosimilars or follow-on biologics or biogenerics) represents an extraordinary opportunity to companies that want to seize the potentially great commercial rewards in this unexploited territory. Biosimilars not only benefit the biosimilar manufacturers but also can save patients, and insurance companies, substantial cost and allow patients to gain access to more affordable biologics resulting in market expansion. The government can use biosimilars to reduce healthcare costs. Therefore, development and marketing of bosimilars are supported by both manufacturers and consumers.

Differences between Generic Drugs and Biosimilars

Enacted in 1984, the US Drug Price Competition and Patent Term Restoration Act, informally known as the “Hatch-Waxman Act of 1984” standardized US procedures for an abbreviated pathway for the approval of small-molecule generic drugs. The generic drug approval

is based on “sameness”. In comparison to the innovator’s drug, a generic drug is a product that has the same active ingredient, identical in dose, strength, route of administration, safety, efficacy, and intended use. For approval, the generic companies can go through the Abbreviated

New Drug Application (ANDA) process with reduced requirement in comparison to approval for a new drug entity. The generic drugs need to show bioequivalence to the innovator drugs typically based on pharmacokinetic parameters such as the rate of absorption or bioavailability in 24 to 36 healthy volunteers. No large clinical trials for safety and efficacy are required. The generic companies can rely on the FDA’s previous findings of safety and effectiveness of the innovator’s drugs.

However, the abbreviated pathway for generic drugs legally doesn’t apply to biologics as small-molecule drugs and biologics are regulated under different laws and approved through different pathways in the US (Table 2). Small-molecule drugs are regulated under the Food, Drug and Cosmetic Act (FD&C) and require submission of a New Drug Application (NDA) to FDA for drug review and approval. Biologics are regulated under the Public Health Service Act (PHS) and require submission of a Biologic License Application (BLA) to FDA for review and approval. The Hatch-Waxman Act of 1984 doesn’t apply for biosimilars. New laws are needed to establish a pathway for biosimilar approval.

There are some crucial differences between biologics and small-molecule drugs (Table 3). Small-molecule drugs are made from chemical synthesis. They are not sensitive to process changes. The final product of a small-molecule drug can be fully characterized. The developmentand production of generic drugs are relatively straightforward. Biologics are made from living organisms so that its functional-, efficacy- and safety-related properties depend on its manufacturing and processing conditions. They are sensitive to process changes. Even minor modifications of the manufacturing process can cause variations in important properties of a biological product. Thus it is believed that a biologic product is defined by its manufacturing process. Biologics are 100- or 1,000-fold larger than small-molecule drugs, possess sophisticated three-dimensional structures, and contain mixtures of protein isoforms. A biological product is a heterogeneous mixture and the current analytical methods cannot characterize these complex molecules sufficiently to confirm structural equivalence with the reference biologics.

Laws and Regulatory Pathways for Drug Approval in the US

Law/Application           Small-molecule            Drug Biologics                     

Law                     Food, Drug and Cosmetic Act (FD&C)             Public Health Service Act (PHS)

Drug application                 New Drug Application (NDA)   Biologic License Application (BLA)

Generic application   Abbreviated New Drug   Application(ANDA)   No pathway yet

 Immunogenicity Poses a Concern

One of the major complications that biologics can produce is immunogenicity as therapeutic proteins are inherently immunogenic [2]. Immunogenicity is related to biologics structure and formulation and is dependent on dose, route of administration and frequency of administration.

Clinical implications of immunogenicity are not always predictable. Formation of antibodies can result in harmless clinical effect or produce significant adverse events or severe disease. Examples are provided below. The Eprex (Erythropoietin, EPO) has been marketed by Johnson & Johnson (J&J) in the European Union (EU) countries for 10 years with no noticeable

Differences between small-molecule drugs and biologics

Product characteristics

Small-molecule generics Small, simple molecule

(Molecular weight: 100-1,000 Da)

Biosimilars   Large, complex molecules, Higher order structures, Post-translational, modifications

(Molecular weight: 15,000-150,000 Da)

Production

Small-molecule generics Produced by chemical synthesis

Biosimilars  Produced in living organisms

Analytical testing

Small-molecule  Well-defined chemical structure, all its various components in the finished drug can be determined

Biosimilars  Heterogeneous mixture, difficult to characterize, some of the components of a finished biologic may be unknown

Process dependence

Small-molecule   Not sensitive to manufacturing process changes. The finished product can be analyzed to establish the sameness.

Biosimilars   Sensitive to minor changes in manufacturing process. The product is defined by the process

Identity and purity

Small-molecule Often meeting pharmacopeia or other standards of identity (e.g., minimums for purity and potency)

Biosimilars   Most have no pharmacopeia monographs

immunogenicity issues prior to 1998. When J&J made a change in the Eprex formulation by replacing human serum albumin (HAS) with polysobate 80 and glycine in response to the

request from European health authorities, some patients developed pure red-cell aplasia (PRCA), a severe form of anemia. Eprex induced antibodies neutralize all the exogenous rHuEPO and cross-react with endogenous erythropoietic proteins. As a result, serum EPO is undetectable

and erythropoiesis becomes ineffective. Upon investigation, J&J found that polysorbate 80 might have caused uncoated rubber stoppers in single-use Eprex syringes to leach plasticizers, which stimulated an immune response that resulted in PRCA. Replacing with Teflon coated stoppers resulted in 90% decrease in PRCA by 2003 [3,4]. The effect of neutralizing antibodies has not always resulted in serious clinical consequences. Three interferon beta products, Betaseron, Rebif and Avonex, are marketed by three different companies. These products induce neutralizing antibodies in multiple sclerosis patients from 5 to 50% after one year treatment. Although these antibodies might be associated with loss of efficacy of treatment resulting in some patients to withdraw from the treatment, it seems no other severe adverse effects were detected [5,6].

Regulatory Landscape

The US, the EU and Japan are the three cornerstonemembers of the International Conference on Harmonization (ICH), which intends to harmonize the regulatory requirements for drug or biologic approval in these three regions. With the other two members, the EU and Japan, already have established biosimilar approval procedures (see below), the US lags behind in the biosimilar race. There are no formal approval pathways for biosimilars in the US. Congress needs to establish a legal framework in order for FDA to develop guidelines. Legislation has been under discussion in Congress since 2007. The legislative debate is centered on patient safety and preserving incentives to innovate with introduction of biosimilars. Two bills introduced in March 2009 deserve attentions [7,8]. The Waxman bill (H.R. 1427) proposes 5 years of market exclusivity to the innovator companies and requires no clinical trials for biosimilar development. The Eshoo bill (H.R. 1548) proposes 12 years of market exclusivity to the innovator companies and requires clinical trials for biosimilar development. Obama administration appears to favor a 7-year market exclusivity [9]. Once a legal framework is established for biosimilars, the FDA will likely take a conservative approach using the comparability as an approval principle. Clinical proof of efficacy and safety will be required, probably in reduced scale.

In the EU, the European Medicines Agency (EMEA) issued regulatory guidelines for approving biosimilars in 2005 (Figure 1) [10-16]. These include two general guidelines for quality issues [11] and non-clinical and clinical issues [12] and four class-specific annexes for specific data requirements for Granulocyte-Colony Stimulating factor (G-CSF) [13], Insulin [14], Growth hormone [15] and Erythropoietin [16]. In addition, a concept paper on interferon alpha [17] is also available. So far, there are eleven biosimilar products which received market authorization in the EU and they are biosimilar versions of human growth hormone, Epoetin and filgrastim. It is estimated six to eight years on average for a biosimilar to be developed [18].

The EMEA treats a biosimilar medicine as a medicine which is similar to a biological medicine that has already been authorized (the “biological reference medicine”) in the EU, The active substance of a biosimilar medicine is similar to the one of the biological reference medicine.

A biosimilar and the biological reference medicine are used in general at the same dose to treat the same disease. A biosimilar and the biological reference medicine are not automatically interchangeable because biosimilar and biological reference medicine are only similar but not identical. A physician or a qualified healthcare professional should make the decision to treat a patient with a reference or a biosimilar medicine. Since the biosimilar may contain different inactive ingredients, the name, appearance and packaging of a biosimilar medicine differ to those of the biological reference medicine. In addition, a pharmacovigilance plan must be in place for post-marketing safety monitoring.

Japan’s Ministry of Health, Labor and Welfare (MHLW) issued guidelines for follow-on proteins or biosimilars approval in March 2009. The first biosimilar, Sandoz’ growth hormone Somatropin, was approved in June 2009. The MHLW’s guidelines consider biosimilars drugs which are equivalent and homogeneous to the original biopharmaceuticals in terms of quality, efficacy and safety. Biosimilars are also requested to be developed with updated technologies and knowledge. Biosimilars need to demonstrate enough similarity to guarantee the safety and efficacy instead of absolute identity to the original biologics. Biosimilars’ regulatory approval applications will be categorized separately from conventional generic drugs. In general, the applications should be submitted, as the new drug applications, with data from clinical trials, manufacturing methods, long-term stability and information on overseas use. The MHLW will assess the data on absorption, distribution, metabolism and excretion (ADME) on a case-by-case basis. The applications do not need to provide data on accessory pharmacology, safety pharmacology and genotoxicity.

Biosmilars are already thriving in Eastern Europe and Asia, where regulatory and intellectual property (IP) standards for biosimilars are more liberal. Biosimilars developed in these regions are primarily sold domestically. These markets are considered less controlled. The quality of the biosimilars may not be in full compliance with ICH guidelines although they are often developed through comparative quality testing and clinical trials against the biologics which are already approved in Western countries

CMC Development

The CMC requirements for biosimilars in the EU are those described in the ICH Common Technical Document (CTD) Quality Module 3 with supplemental information demonstrating comparability or similarity on quality attributes to the reference medicine product.

Since the US is a member of ICH and encourages submission using CTD format, once the legal framework for approving biosimilars is established in the US, the CMC development will be similar to those in the EU.

Biosimilar manufacturers will have no access to the manufacturing process and product specifications of the innovator’s products because these are proprietary knowledges. To develop a biosimilar, a biosimilar manufacturer will need to first identify a marketed biologic product to serve as the reference biologic product. Then a detailed characterization of the reference biologic product will be performed. The information obtained from the characterization of the reference biologic product will be utilized to direct the process development of the biosimilar product and comparative testing to demonstrate bioequivalence between the biosimilar product and the reference biologic product. A biosimilar will be manufactured from a completely new process, which may be based on different host/vector system with different process steps, facilities and equipment.

A flow chart for a typical work flow from production to drug use is shown in Figure 2. The CMC development starts with establishment of the expression system. A cell-line will be selected among bacterial, yeast and mammalian host strains and then the correct DNA sequence will be inserted. Elaborate cell-screening and selection methods are then used to establish a master cell bank. Extensive characterization on the master cell bank needs to be carried out to provide microbiological purity or sterility and identity [19].

Bulk protein production involves developing robust and scalable fermentation and purification processes. The goals for fermentation are to increase the expression level and efficiency without compromising the correct amino acid sequence and post translational modification. Achieving high expression requires optimizing culture medium and growth conditions, and efficient extraction and recovery procedures. Correct amino acid sequence and post translati0nal modification will need to be verified.

Cell Bank

Fermentation

Purification

Drug Substance

Formulation

Fill/finish

Drug Product

Shipment

Administration

Typical flow chart for a biologics from production to drug use, above

Solubilization and refolding of insoluble proteins are sometimes necessary for proteins which have tendency to aggregate under the processing condition. Differences in the cell bank and production processes may create impurities that are different from the innovator’s product. The purification process needs to remove impurities such as host-cell proteins, DNA, medium constituents, viruses and metabolic by-products as much as possible. It is important for biosimilar manufacturers to accept appropriate yield losses to achieve high purity, because any increase in yield at the expense of purity is unacceptable and can have clinical consequences.

The final product is produced by going through formulation, sterile filtration and fill/finish into the final containers. Selection of formulation components starts from basic buffer species for proper pH control and salt for isotonicity adjustment. Surfactants may be needed to prevent proteins from being absorbed onto container surface or water-air interface or other hydrophobic surfaces. Stabilizers are required to inhibit aggregation, oxidation, deamidation and other degradations. The container and closure system can be glass vials, rubber stoppers and aluminum seals or pre-filled syringes or IV bags. The container and closure integrity needs to be verified by sterility or dyeleak test.

Biologics are not pure substances. They are heterogeneous mixtures. Each batch of a biologic product for clinical or commercial use needs to be produced in compliance with current Good Manufacturing Practice (cGMP) and is typically tested by a panel of assays to ensure the product meets pre-defined specifications for quality, purity, potency, strength, identity and safety. The product purity is often measured by multiple assays, which measure different product related variants (biologically active) or product related impurities (biologically inactive). Biologics are parenteral drugs and filled into the final containers through the aseptic process so that microbiological control is critical. It is advisable to set up product specifications for a biosimilar within the variation of the reference biologic product. Product characterization can be performed on selected batches for primary sequence, high order structures, isoform profiles, heterogeneity, product variants and impurities and process impurity profiles. Physicochemical characterization tests include IEF, CE, HIC, LCMS, carbohydrate analysis, N & C terminal sequencing, amino acid analysis, analytical ultracentrifugation, CD and DSC [20,21]. Biologics are highly sensitive to environmental influences during storage, shipment and handling. Temperature excursion, movement, and exposure to UV light can lead to protein degradation. Product expiry needs to be based on the real time stability data. Stability program should also include accelerated or stress studies to gain insight of the degradation profiles. In-use stability studies are carried out to verify shipping conditions or handling procedures cause no detrimental effect to the drug product.

 Comparability Demonstration

 A comparability exercise based on the ICH guideline [22] needs to be performed to demonstrate that the biosimilar product and the reference biologic product have similar profiles with respect to product quality, safety, and efficacy. This is accomplished by comparative testing of the biosimilar product and the reference biologic product to demonstrate they have comparable molecular structure, in vitro and in vivo biological activities, pre-clinical safety and pharmacokinetics, and safety and efficacy in human patients. Comparison of quality attributes between the biosimilar and the reference biologic product employs physicochemical

Product release assays for biologics

Type                         Assays

Quality              Appearance, particulates, pH, osmolality

Purity                 SDS-PAGE, SEC-HPLC, IEX-HPLC, RP-HPLC

Potency             In vitro or in vivo bioactivity assays

Strength             Protein concentration by A280

Identity               Western blot, peptide mapping, isoelectric focusing

Safety                  Endotoxin, sterility, residual DNA, host cell proteins

and biological characterization. Comparability on physical properties, amino acid sequence, high order structures, post-translationally modified forms are evaluated by physicochemical tests. In vitro receptor-binding or cell-based (binding) assays or even the in vivo potency studies in animals need to be performed to demonstrate comparable activity despite they are often imprecise. Levels of product related impurities (aggregates, oxidized forms, deamidated forms) and process related impurities and contaminants (host cell proteins, residual genomic DNA, reagents, downstream impurities) need to be assessed and quantified. Stability profiles of the biosimilar product and the reference biologic product also need to be studies by placing the products under stressed conditions. The rate of degradation and degradation profiles (oxidation, deamidation, aggregation and other degradation reactions) will be compared. If unknown degradation species are detected, they need to be studied to determine if they affect safety and efficacy. If differences on product purities and stability profiles are present between the biosimilar product and the reference biologic product, these differences need to be justified using scientific knowledge or preclinical or clinical studies. Changes in the impurity profile should be justified as well.

The demonstration of comparability in quality attributes does not necessarily mean that the biosimilars and the reference biologics are identical, but that they are highly similar. In many cases, the relationship between specific quality attributes and safety and efficacy has not been fully established. For example, physicochemical characterization cannot easily predict immunogenicity and slight changes in manufacturing processes or product composition can give rise to unpredicted changes in safety and efficacy. Changes in bioavailability, pharmacokinetics, bioactivity bioactivity, and immunogenicity are the main risks associated with the manufacturing of biosimilars. In vivo studies should be designed to measure the pharmacokinetics and pharmacodynamics relevant to clinical studies. Such in vivo studies should be designed to detect response differences between the biosimilar and the reference biologic not just responses per se. In vivo studies of the biosimilar’s safety in animals may be used to research any concerns into the safety of the biosimilar in human patients. Although extensive clinical testing is not necessary for biosimilars, some degree of clinical testing is needed to establish therapeutic comparability on efficacy and safety between the biosimilar and the reference biologic product [23,24]. This includes using surrogate markers of specific biologic activity as endpoints for demonstrating efficacy, and showing that patients didn’t develop immunogenic responses to the product. In general, the approval of biosimilars will be based on the demonstration of comparable efficacy and safety to an innovator reference product in a relevant patient population. Clinical data requirement for each individual product will be different and will be determined on a case-by-case basis.

Small-molecule Generics versus Biosimilars

 Small-molecule

  • Approval based on “sameness”

Biosimilars

  • Approval based on “high similarity”

Small-molecule

  • Replicate the innovator’s process and product and perform a bioavailability study demonstrating similar pharmacokinetic properties

Biosimilars

  • Full CMC development with comparative testing, conduct substantial clinical trials for efficacy and safety including immunogenicity

Small-molecule

  • Abbreviated registration procedures in Europe and US

Biosimilars

  • Regulatory pathway is defined in EU on “Comparability” status, no pathway yet in US under BLA

Small-molecule

  • Therapeutically equivalent, thus interchangeable

Biosimilars

  • Lack of automatic substitutability

Small-molecule

  • $1 to $5 million to develop

Biosimilars

  • $100-$200 million to develop

Small-molecule

  • Brand-to-generic competition

Biosimilars

  • Brand-to-Brand competition

Conclusion

The patent provisions of the Biosimilar Act, 2009 establish demanding and time-sensitive disclosure requirements. ObamaCare upheld by the Supreme Court is a victory for future development of pathways for biosimilar regulatory approvaland eventually biosimilar generic drugs.

Biosimilars are defined as biological products similar, but not identical, to the reference biological products that are submitted for separate marketing approval following patent expiration of the reference biological products. As one of the ICH members, the US needs to catch up with the EU and Japan as those two countries have already issued regulatory guidelines for biosimilars. 2009 and 2012 represent milestones in the regulatory provisions for biosimilars in the US.

Once Congress establishes a legal framework, FDA is expected to set up a biosimilar approval pathway which will be similar to those in the EU and Japan and harmonized under ICH. The biosimilar will need a full CMC development package plus demonstration of comparable quality attributes and comparable efficacy and safety to the innovator’s product. Table 5 provides a comparison summary between small-molecule generics and biosimilars. It will take a much bigger effort to develop a biosimilar than a generic drug. Automatic substitution between the innovator product and a biosimilar is not appropriate as a biosimilar is not a generic version of the innovator product and is approved based on comparability to the innovator product.

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