Fourth-generation ALK inhibitors are currently under investigation. Biomedical technology is constantly advancing and drug development is constantly evolving. The rapid pace of ALK-targeted drug development, along with the concurrent knowledge gained about resistance mechanisms, illustrates the pivotal role of an iterative, systematic discovery process in advancing oncology research and transforming patients' lives.
Medicilon has established a one-stop platform for preclinical biopharmaceutical R&D services, including drug discovery, pharmacological research, and preclinical research. The one-stop R&D platform strongly integrates advantageous resources, promotes efficient collaboration across departments, gives full play to the synergistic effect of 1+1>2, and accelerates the development of new drugs.
Lung cancer is one of the leading causes of cancer deaths worldwide, and it ranks first in terms of incidence and mortality in China. Lung cancer is classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) according to its biological characteristics, treatment, and prognosis. In China, NSCLC accounts for 80-85% of patients, and about 3%-7% of NSCLC patients have ALK gene mutations. Brain metastases occur in 20-40% of ALK-positive advanced NSCLC patients at the time of initial diagnosis, and the incidence increases with time.
ALK is found in anaplastic large cell lymphoma (ALCL). Given the close relationship between ALK mutations and tumors, many researchers have developed a series of anti-tumor drugs that mainly target ALK. ALK is a tyrosine kinase closely related to tumors, and the ALK gene is found in a range of malignancies, including NSCLC, mesenchymal large cell lymphoma, and neuroblastoma, with rearrangements, point mutations, or amplifications. Because of their rarity and precision, ALK fusion mutations are known as "diamond mutations".
As the number of therapeutic agents for ALK-positive patients increases, it is crucial to achieving precise treatment, and there is a general clinical consensus that the foundation of precise treatment is precise diagnosis. Currently, several precise diagnostic methods are available to detect the expression of ALK fusion genes in tumor tissues, which is revolutionary for the molecular diagnosis of NSCLC in recent years.
ALK (mesenchymal lymphoma kinase): also known as ALK tyrosine kinase receptor or CD246, is synthesized by the ALK gene encoding it. ALK is a receptor tyrosine kinase that belongs to the insulin receptor superfamily and is highly homologous to leukocyte tyrosine kinase (LTK). The human ALK gene is located on chromosome segment 2p23 and encodes a 1620 amino acid polypeptide that is post-translationally modified to produce a mature ALK protein of approximately 200-220 kDa. ALK maturation protein is a classical receptor tyrosine kinase that includes an extracellular ligand binding structural domain, a transmembrane structural domain, and an intracellular tyrosine kinase structural domain. The kinase structural domain shares a 3-tyrosine motif (Tyr1278, Tyr1282, and Tyr1283) with other kinases of the same family, which is located in the activation loop and represents the major autophosphorylation site for kinase activity. ALK is activated only upon ligand-induced homodimerization and is inactivated in the absence of ligand by dephosphorylation of the receptor protein tyrosine phosphatase β and zeta complex (PTPRB/PTPRZ1).
ALK protein structure [1]
ALK activates multiple pathways, including phospholipase C γ (PLCγ), JAK-STAT, PI3K-AKT, mTOR, sonic hedgehog, JUNB, CRKL-C3G (also known as RAPGEF1)-RAP1 GTPase and MAPK signaling cascades, affecting cell growth, transformation, and anti-apoptotic signaling. ALK is activated only upon ligand-induced homodimerization and is inactivated in the absence of ligand by dephosphorylation of the receptor protein tyrosine phosphatase β and zeta complex (PTPRB/PTPRZ1).
ALK signaling pathway [1]
In 2011, Crizotinib was approved by the FDA for the treatment of advanced ALK-positive NSCLC, only 4 years after Soda et al. identified ALK rearrangement as a potential oncogenic driver in NSCLC. Crizotinib is an oral, small-molecule ATP-competitive ALK inhibitor that was initially used as a MET TKI and then rapidly switched to target ALK after the discovery of the role of ALK rearrangement in NSCLC. Thus began the continuous iterative development of ALK inhibitors.
History of ALK inhibitor development in NSCLC [2]
❖First-generation ALK inhibitors
Crizotinib
The first FDA-approved ALK-TKI drug, approved by the FDA in August 2011, filled the gap of targeted drugs in ALK-positive NSCLC at that time. Crizotinib is an effective oral tyrosine kinase inhibitor for ALK fusion genes or locally advanced or metastatic ROS1 fusion-positive NSCLC. Crizotinib is more effective than chemotherapy and has fewer toxic side effects.
❖Second-generation ALK inhibitors
Alectinib
Approved by the FDA in December 2015 for the treatment of locally advanced or metastatic ALK-positive NSCLC whose disease has progressed or failed after treatment with Crizotinib. Alectinib is a potent and selective ALK inhibitor with good efficacy in patients with Crizotinib-refractory ALK-positive NSCLC with CNS metastasis. It has good brain-entry properties.
Ceritinib
Approved by the FDA in May 2017 for the treatment of ALK-positive metastatic NSCLC. Indicated in ALK-positive, ROS1-positive locally advanced or metastatic NSCLC. Doubling of progression-free survival and significant efficacy in brain metastases.
Ensartinib
Approved for marketing by NMPA in November 2020. The first new domestic class 1 drug for the treatment of ALK-mutated advanced NSCLC, providing a new treatment option for NSCLC patients. It is indicated for ALK-positive locally advanced or metastatic NSCLC with high selectivity. For ALK-positive locally advanced or metastatic NSCLC that has progressed after prior treatment with Crizotinib or is intolerant to Crizotinib. It compares favorably with similar imported drugs in terms of efficacy, has a higher response rate, especially in patients with brain metastases, and has a good and controlled safety profile.
Brigatinib
Approved by the FDA in April 2017 for the treatment of ALK-positive NSCLC that is intolerant after treatment with Crizotinib. Targeted inhibition of both ALK and EGFR. Brigatinib is an orally effective selective ALK and ROS1 tyrosine kinase inhibitor. Brigatinib has better overall and intracranial efficacy compared to Crizotinib in patients with ALK-positive NSCLC treated with primary ALK inhibitors. Brigatinib was well tolerated for long-term use.
❖Third-generation ALK inhibitors
Lorlatinib
Approved for marketing by the FDA in November 2018 for the treatment of patients with ALK-positive NSCLC whose disease continues to progress, who have received Crizotinib and at least one other ALK inhibitor for metastatic disease, or whose disease progresses to metastatic disease after treatment with Alectinib or Ceritinib. More brain entry than Alectinib. Lorlatinib has excellent blood-brain barrier penetration, and the treatment of patients without brain metastases is effective in preventing brain metastases from occurring. Lorlatinib has a unique chemical structure with a large cyclic amide structure, which is more penetrating and binding to ALK, and therefore shows better results in clinical practice.
❖Quadruple-generation ALK inhibitors
Double mutation-active ALK TKI
Quad-generation ALK TKI is being developed to overcome ALK mutations, mainly due to the sequential use of "mono-mutation-active" ALK TKI, including in particular the ALK G1202R-based double mutation. Four generations of ALK TKI are currently in development, including TPX-0131 and NVL-655. In addition to broad-spectrum single ALK mutations, TPX-0131 and NVL-655 also inhibit acquired double "compound" ALK mutations. TPX-0131 and NVL-655 can overcome multiple double mutations in vitro.
Inhibitory activity of TPX-0131 and NVL-655 [3]
TPX-0131 is a potent CNS-permeable macrocyclic molecule that inhibits ALK fusion proteins. In cellular assays, TPX-0131 was more effective than the five currently approved ALK inhibitors against WT ALK and many types of ALK-resistant mutations (e.g., G1202R, L1196M, and compound mutations). In biochemical assays, TPX-0131 effectively inhibited WT ALK and 26 ALK mutants (single and compound mutations) with an IC50<10 nM. TPX-0131 acted on ALK (G1202R) and ALK compound mutation-dependent xenograft tumor models, leading to complete tumor regression. After oral administration of TPX-0131 to rats, TPX-0131 levels in the brain were observed to be approximately 66% of those in plasma.
Structural design of TPX-0131 [4]
❖Project example: ALK degradation agent SIAIS164018
SIAIS164018 is an orally effective degradation agent that degrades not only ALK or mutant EGFR but also other tumor proteins involved in metastasis. Particularly potent targeting of L858R+T790M mutant EGFR, the two most important targets in non-small cell lung cancer. SIAIS164018 inhibits cell migration and invasion of Calu-1 and MDA-MB-231. SIAIS164018 also degrades several important tumor proteins associated with metastasis, such as FAK, PYK2, and PTK6.
The multi-targeted degradation agent SIAIS164018[5]
In this study, Medicilon’s researchers evaluated pharmacokinetic (PK) data of SIAIS164018 in rats administered intravenously and orally The data showed that SIAIS164018 was well bioavailable and tolerated in vivo.
Pharmacokinetic study of SIAIS164018 [5]
❖Project example: ALK inhibitor CGT-9475
On July 22, 2022, CGT-9475, a new generation ALK inhibitor developed by CGeneTech, was approved by FDA to enter clinical trials. ALK inhibitors have significant advantages in clinical application, but the use of ALK inhibitors will inevitably generate secondary drug resistance and brain metastasis problems. CGT-9475 aims to overcome drug resistance and solve the problem of CNS metastasis with quality and differentiated development. CGT-9475 showed significant inhibitory effects against drug-resistant mutations such as L1196M and RET in NSCLC cell lines in preclinical research. Meanwhile, CGT-9475 demonstrated a good blood-brain barrier penetration effect in preclinical research, which will bring new hope for NSCLC brain metastasis patients. Medicilon provided a complete preclinical development service for CGT-9475 (two years of preclinical candidate screening, followed by systematic pharmacological studies, pharmacodynamic studies, pharmacokinetic studies, safety evaluation studies, and IND filing). CGT-9475 is expected to be used for follow-up treatment after resistance to existing ALK inhibitors.
❖Project case: 4th generation ALK inhibitor innovation drug development
Lorlatinib, as a 3rd generation ALK inhibitor, has shown excellent clinical efficacy but also faces the problem of treatment resistance, in which compound mutations are an important cause of resistance. The 4th generation, represented by TPX-0131 and NVL-655, can effectively inhibit compound mutations, especially NVL-655, which significantly improves the off-target effect on TRKB and is expected to solve the neurotoxicity problem of existing drugs. Medicilon has used representative 3rd generation ALK inhibitors such as TPX-0131 and NVL-655 as positive references to design and synthesize highly active 4th generation ALK target small molecules with certain novelty, and to investigate and study them in depth.
In recent years, with the implementation and expansion of China's "major new drug creation" science and technology major projects, China's local pharmaceutical R&D innovation capacity has greatly improved, which has injected a strong impetus into the global pharmaceutical R&D field, meaning that more innovative drugs are expected to be accelerated to market. In the direction of drug development, more and more pharmaceutical R&D workers are devoting themselves to targeted drug development to meet more patients' needs and keep gathering efforts to achieve breakthroughs!
References
[1] Carminia Maria Della Corte, et al. Role and targeting of anaplastic lymphoma kinase in cancer. Mol Cancer. 2018 Feb 19;17(1):30.
[2] Brandon Golding, et al. The function and therapeutic targeting of anaplastic lymphoma kinase (ALK) in non-small cell lung cancer (NSCLC). Mol Cancer. 2018 Feb 19;17(1):52.
[3] Sai-Hong Ignatius Ou, et al. Will the clinical development of 4th-generation "double mutant active" ALK TKIs (TPX-0131 and NVL-655) change the future treatment paradigm of ALK+ NSCLC? Transl Oncol. 2021 Nov;14(11):101191. doi: 10.1016/j.tranon.2021.101191.
[4] Brion W Murray, et al. TPX-0131, a Potent CNS-penetrant, Next-generation Inhibitor of Wild-type ALK and ALK-resistant Mutations. Mol Cancer Ther. 2021 Sep;20(9):1499-1507. doi:
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