Promising Tyrosine Kinase Inhibitor Research Articles

Common epidermal growth factor receptor mutations in north Indian patients with non-small cell lung carcinoma: evidence from real-time polymerase chain reaction.

Lung carcinoma was the ace cause of cancer deaths globally in 2022, with non-small cell lung carcinoma (NSCLC) accounting for 81% of the burden. Due to promising tyrosine kinase inhibitor (TKI) trials, NSCLC patients harboring epidermal growth factor receptor (EGFR) gene mutations are of interest. Our aim was to determine EGFR mutation prevalence in north India and its histologic and demographic correlations. We investigated the frequency of EGFR mutations in 40 patients with histologically confirmed NSCLC using real-time polymerase chain reaction. A 15% mutation frequency was observed in the study sample, involving 32 males and 8 females with a median age of 59 years. Squamous cell carcinoma (SCC) patients had only EXON20 (T790M, exon20 insertion) mutations, while adenocarcinoma patients had mutations in both EXON20 (T790M) and 21 (L858R) with mutation frequencies of 22% and 10%, respectively. 28% of the SCC patients were non-smokers, and 60% of these non-smokers had an EGFR mutation. South Indian and Asian studies have identified EXON19 (19-Del) and EXON21 (L858R) mutations as "common mutations" that account for nearly 80-90% of all mutations and respond well to TKIs. Interestingly, "common mutations" were found seldom in our study population, while the uncommon variants constitute 83% of all mutations, which we assume is due to diverse Indian genetics and ethnicity and co-existing signature mutations that involve the tyrosine kinase domain of EXON20. We suggest future genome-wide association studies to identify plausible genetic polymorphisms responsible for interethnic differences in EGFR mutation, which will contribute to better treatment and prevention of NSCLCs.

FLT3-CD3 Bispecific Antibody Specifically Eliminates Normal Hematopoietic Progenitors and AML in Humanized Mouse Models

Acute myeloid leukemia (AML) is a blood cancer arising from clonal proliferation of myeloid progenitors harboring oncogenic mutations. AML patients have poor clinical prognosis and limited therapeutic options, with myeloablation followed by hematopoietic stem cell transplantation (HSCT) as the only curative treatment. The conditioning regimens used indiscriminately kill all highly proliferative cell types, leading to life threatening side effects, and are also potentially ineffective against quiescent AML subpopulations. To address the challenges of efficacy and resistance in targeting AML, we developed a humanized bispecific antibody (CDX) that recruits T cells through CD3 to target and kill Fms-like Tyrosine Kinase 3 (FLT3) expressing cells. Normal FLT3 expression is mostly restricted to hematopoietic stem and progenitor cells (HSPCs) and its activation, through binding of FLT3 ligand (FLT3L), promotes normal differentiation of downstream blood lineages. FLT3 is also expressed on AML blasts in a majority of patients and is thought to promote survival and proliferation of AML. Although multiple promising tyrosine kinase inhibitors (TKIs) have been and are being developed to specifically target FLT3, secondary mutations leading to resistance against FLT3-TKIs remain a major obstacle. Surface expression of FLT3 in leukemic cells, as well as in HSCs, makes it an excellent target for T cell mediated conditioning that specifically eliminates both AML blasts and HSPCs, allowing for subsequent HSCT. In addition, HDX mass spectrometry revealed that our CDX binds an extracellular, membrane proximal FLT3 domain, which does not compete with FLT3L and is outside the regions commonly mutated in AML, therefore, unlike other therapies, targets all FLT3-expressing cells, regardless of mutations in the receptor. We found that in vitro treatment with CDX redirected primary human T cells to kill multiple AML-derived FLT3+ cell lines, some harboring common FLT3 mutations, at EC50 ranging from 0.3 pM to 15 pM. Additionally, the presence of FLT3L (10 nM) had no effect on the EC50 of CDX binding to FLT3+ target cells. To test CDX in vivo, we generated xenograft mice engrafted with primary human peripheral blood mononuclear cells (PBMCs) and MV4;11 cells (FLT3+ AML cell line), which were engineered to stably express EGFP. Mice were treated intravenously with 3 doses of CDX at a concentration of 0.1 mg/kg or left untreated over 1 week (n=5). Progression of AML was tracked by monitoring the frequency of EGFP-MV4;11 cells in peripheral blood (PB) of xenografted mice, which were euthanized if they presented with cachexia, hind-leg paralysis, and severe weight loss (symptoms of AML progression). Treatment with CDX significantly delayed the proliferation of MV4;11 cells, based on lower PB frequencies of EGFP-MV4;11, and increased median survival by 28 days relative to untreated mice. To more stringently model CDX efficacy in a clinical setting, we injected EGFP-MV4;11 cells into humanized mice that were generated by transplantation of human cord blood derived, CD34+ HSPCs and displayed stable multi-lineage engraftment (~20% human CD45+) with significant populations of T cells at 35 weeks. We observed that T cells in CDX-treated mice over-expressed the immune checkpoint receptor PD1, relative to untreated mice, and hypothesized that PD1 inhibition could enhance MV4;11 clearance via CDX treatment by preventing T cell exhaustion. Treatment regimens consisted of 3 doses of CDX (0.1 mg/kg), anti-PD1 antibody (100 μg), or a combination of CDX and anti-PD1 (n=5), followed by transplant with autologous mouse bone marrow cells. Treatment with CDX alone led to decreased PB frequencies of EGFP-MV4;11 cells, but only modestly improved median survival relative to anti-PD1 alone (9 days). In contrast, co-treatment with CDX and anti-PD1 antibody significantly increased median survival (16 days) and was associated with decreased PD1 expression on T cells. In addition, administration of CDX alone or in combination with PD1 in humanized mice resulted in efficient elimination of the human hematopoietic compartment from the mouse bone marrow. Based on our findings, CDX shows promise for treatment of AML with concurrent conditioning for HSCT with improved specificity compared to standard of care and even displays synergy with checkpoint inhibition of PD1. Disclosures Sirochinsky: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang:Hemogenyx Pharmaceuticals LLC: Current Employment. Shrestha:Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda:Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler:Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company.

Ensartinib (X-396) Effectively Modulates Pharmacokinetic Resistance Mediated by ABCB1 and ABCG2 Drug Efflux Transporters and CYP3A4 Biotransformation Enzyme.

Ensartinib (X-396) is a promising tyrosine kinase inhibitor currently undergoing advanced clinical evaluation for the treatment of non-small cell lung cancer. In this work, we investigate possible interactions of this promising drug candidate with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs), which play major roles in multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). Accumulation studies showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 transporters. Additionally, incubation experiments with recombinant CYPs showed that ensartinib significantly inhibits CYP3A4 and CYP2C9. Subsequent molecular docking studies confirmed these findings. Drug combination experiments demonstrated that ensartinib synergistically potentiates the antiproliferative effects of daunorubicin, mitoxantrone, and docetaxel in ABCB1, ABCG2, and CYP3A4-overexpressing cellular models, respectively. Advantageously, ensartinib’s antitumor efficiency was not compromised by the presence of MDR-associated ABC transporters, although it acted as a substrate of ABCB1 in Madin-Darby Canine Kidney II (MDCKII) monolayer transport assays. Finally, we demonstrated that ensartinib had no significant effect on the mRNA-level expression of examined transporters and enzymes in physiological and lung tumor cellular models. In conclusion, ensartinib may perpetrate clinically relevant pharmacokinetic DDIs and modulate ABCB1-, ABCG2-, and CYP3A4-mediated MDR. The in vitro findings presented here will provide a valuable foundation for future in vivo investigations.

Metabolic profiling of tyrosine kinase inhibitor nintedanib using metabolomics

Nintedanib is a promising tyrosine kinase inhibitor for clinically treating idiopathic pulmonary fibrosis (IPF). Some clinical cases reported that nintedanib treatment can cause hepatotoxicity and myocardial toxicity. U. S. FDA warns the potential drug-drug interaction when it is co-administrated with other drugs. In order to understand the potential toxicity of nintedanib and avoid drug-drug interaction, the metabolism of nintedanib was systematically investigated in human liver microsomes and mice using metabolomics approach, and the toxicity of metabolites was predicted by ADMET lab. Nineteen metabolites were detected in vivo and in vitro metabolism, and 8 of them were undescribed. Calculated partition coefficients (Clog P) were used to distinguish the isomers of nintedanib metabolites in this study. The major metabolic pathways of nintedanib majorly included hydroxylation, demethylation, glucuronidation, and acetylation reactions. The ADMET prediction indicated that nintedanib was a substrate of the cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). And nintedanib and most of its metabolites might possess potential hepatotoxicity and cardiotoxicity. This study provided a global view of nintedanib metabolism, which could be used to understand the mechanism of adverse effects related to nintedanib and its potential drug-drug interaction.

Brivanib Exhibits Potential for Pharmacokinetic Drug-Drug Interactions and the Modulation of Multidrug Resistance through the Inhibition of Human ABCG2 Drug Efflux Transporter and CYP450 Biotransformation Enzymes.

Brivanib, a promising tyrosine kinase inhibitor, is currently undergoing advanced stages of clinical evaluation for solid tumor therapy. In this work, we investigated possible interactions of this novel drug candidate with ABC drug efflux transporters and cytochrome P450 (CYP450) drug-metabolizing enzymes that participate in cancer multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). First, in accumulation experiments with various model substrates, we identified brivanib as an inhibitor of the ABCB1, ABCG2, and ABCC1 transporters. However, in subsequent combination studies employing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide proliferation assays in both Madin-Darby canine kidney II (MDCKII) and A431 cellular models, only ABCG2 inhibition was revealed to be able to synergistically potentiate mitoxantrone effects. Advantageous to its possible use as MDR antagonist, brivanib's chemosensitizing properties were not impaired by activity of any of the MDR-associated ABC transporters, as observed in comparative viability assay in the MDCKII cell sublines. In incubation experiments with eight recombinant CYP450s, we found that brivanib potently inhibited CYP2C subfamily members and the CYP2B6 isoform. Finally, in induction studies, we demonstrated that brivanib upregulated ABCB1 and CYP1A2 messenger RNA levels in systemic cell models, although this interaction was not significantly manifested at a functional level. In conclusion, brivanib exhibits potential to cause clinically relevant pharmacokinetic DDIs and act as a modulator of ABCG2-mediated MDR. Our findings might be used as an important background for subsequent in vivo investigations and pave the way for the safe and effective use of brivanib in oncological patients.

Synthesis of Substituted Aminopyrimidines as Novel Promising Tyrosine Kinase Inhibitors

A procedure has been proposed for the synthesis of a series of substituted N-(1,3-thiazol-2-yl)-pyrimidin-2-amines and N-(pyrimidin-2-yl)thioureas by reactions of diethyl 2-(ethoxymethylidene)malonate and ethyl 2-(ethyoxymethylidene)-3-oxobutanoate with 1,3-thiazol-2-ylguanidines and amidinothiourea, respectively. Preliminary screening has revealed high inhibitory activity of ethyl 2-(carbamothioylamino)-4-methylpyrimidine-5-carboxylate and ethyl 2-(carbamothioylamino)-6-oxo-1,6-dihydropyrimidine-5-carbox-ylate toward several protein kinases.

Novel Tetrahydrobenzo [b] Thiophene Compounds Exhibit Anticancer Activity through Enhancing Apoptosis and Inhibiting Tyrosine Kinase.

Developing new chemotherapeutic agents with molecular targets, larger margin of safety against normal cells and low cost is the target many scientists try to achieve. The present study was undertaken to investigate the anticancer activity of a novel series of thiophene compounds and the molecular mechanisms associated. A series of novel heterocyclic compounds including pyrimidine derivatives (2, 3, 4, 5 8, 11, 12, 13, 14, and 15), thiophene derivatives (6, 7, and 10) and oxoisothiazolidine derivative (9) was synthesized from 4,5,6,7- tetrahydrobenzo[b] thiophene (1). The newly synthesized derivatives along with the parent compound were evaluated for their anticancer activity against human HepG2, MCF7 and HCT116 cell lines and compared to doxorubicin as a reference drug. Compound 7 was very selective in targeting only the colon cells. Compounds 1, 5, and 12 showed strong cytotoxic activities against the 3 cell lines at 6-16 µM without any apparent toxicity to the normal fibroblasts WI-38. They had DNA affinity at 29-36 µM. The three compounds enhanced apoptosis to varying degrees elevating the expression of Bax, caspase 9 and caspase 3 in HepG2. Compound 5 was the most potent analogue and was superior to the standard drug used in upregulating the apoptotic genes and inhibiting tyrosine kinase at 1 µM. The IC50 value for compound 5 against TK was 296 nM. Taken together, this study presents some thiophene scaffolds as auspicious hits for further optimization as specific antiproliferative agents against cancer cells and promising tyrosine kinase inhibitors at nanomolar concentrations.

Quantitative bioanalytical assay for the tropomyosin receptor kinase inhibitor larotrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry

Larotrectinib is a promising tyrosine kinase inhibitor for solid tumors harboring tropomyosin receptor kinase gene fusions. A bioanalytical assay was developed for this drug in small volume samples using a 96-well format to efficiently support multiple mouse studies. The assay was completely validated for mouse plasma and partially for homogenates of eight different tissues: brain, heart, kidneys, liver, lungs, small intestine, spleen, and testes. Proteins in 10-μl samples were precipitated using acetonitrile containing momelotinib as internal standard. Chromatographic separation of analyte and internal standard from endogenous interferences was performed on an ethylene bridged octadecyl silica column using 0.1% (v/v) formic acid (in water) and methanol for gradient elution. Electrospray ionization and selected reaction monitoring on a triple quadrupole mass spectrometer were used for detection. In the range 1–2000 ng/ml the drug could be quantified in all 9 matrices with precisions (within-day and between-day) in the range 2.7–11.1% and accuracies in the range 87.4–101.4%. Compounds were sufficiently stable under all investigated conditions except for kidney homogenate. A pilot pharmacokinetic and tissue distribution study in mice demonstrated the applicability of the new presented assay for larotrectinib.

Photosensitization by imatinib. A photochemical and photobiological study of the drug and its substructures.

Imatinib (IMT) is a promising tyrosine kinase inhibitor used in the treatment of some types of human cancer. It constitutes a successful example of rational drug design based on the optimization of the chemical structure to reach an improved pharmacological activity. Cutaneous reactions, such as increased photosensitivity or pseudoporphyria, are among the most common nonhematological IMT side effects; however, the molecular bases of these clinical observations have not been determined. Thus, to gain insight into the IMT photosensitizing properties, we addressed its photobehavior together with that of its potentially photoactive anilino-pyrimidine and pyridyl-pyrimidine fragments. In this context, steady-state and time-resolved fluorescence as well as laser flash photolysis experiments have been conducted, and the DNA photosensitization potential has been investigated by means of single-strand break detection using agarose gel electrophoresis. The obtained results reveal that the drug itself and its anilino-pyrimidine fragment are not DNA photosensitizers. By contrast, the pyridyl-pyrimidine substructure displays a marked phototoxic potential, which has been associated with the generation of a long-lived triplet excited state. Interestingly, this reactive species is efficiently quenched by benzanilide, another molecular fragment of IMT. Clearly, integration of the photoactive pyridyl-pyrimidine moiety in a more complex structure strongly modifies its photobehavior, which in this case is fortunate as it leads to an improved toxicological profile. Thus, on the bases of the experimental results, direct in vivo photosensitization by IMT seems unlikely. Instead, the reported photosensitivity disorders could be related to indirect processes, such as the previously suggested impairment of melanogenesis or the accumulation of endogenous porphyrins.

256 Do combination and sequential therapies have increased side-effects?

Recent developments in molecular biology have increased our understanding of the biology and genetics of renal cell carcinoma (RCC) and identified pathways for novel targeted therapy. Several targeted therapies are now available that show promising activity in this disease. Sunitinib, an oral multitargeted tyrosine kinase inhibitor (TKI), has recently been approved for first-line treatment of metastatic RCC (mRCC) and is the new reference standard for the treatment of clear-cell disease. Three other promising TKIs are temsirolimus, approved for the treatment of advanced RCC, sorafenib, approved for the treatment of patients with advanced RCC who have failed or are considered unsuitable for cytokine therapy and bevacizumab, effective in combination with immunotherapy for first-line therapy of mRCC. Several other agents are under investigation as single-agent or combination therapy for mRCC. These include the TKIs axitinib, pazopanib, everolimus, erlotinib, gefitinib and lapatinib. Use of these agents is leading to the development of treatment paradigms that will transform the management of mRCC.

Microenvironmental Factors Determine the Sensitivity of Acute Myeloid Leukemia Cells to Tyrosine Kinase Inhibitors.

There is an increasing body of evidence indicating that the bone marrow microenvironment can generate drug resistance in acute leukemia. The mechanisms underlying this effect have not yet been elucidated; signals triggered by direct contact with extracellular matrix components and by mesenchymal cell (MSC)-secreted factors have been implicated. The protective effect of the microenvironment has been primarily observed for classical chemotherapeutic drugs. Recent reports, however, indicate that this can also occur with molecularly targeted therapies. Thus, it was shown that interleukin (IL)-7 desensitizes BCR-ABL+ leukemic cells to imatinib (Williams RT et al. Genes Dev 2007) and MSC-conditioned media protects BCR-ABL+ cells to imatinib and nilotinib (Weisberg E at al. Mol Cancer Ther, 2008). Several tyrosine kinase inhibitors are in clinical development for the treatment of acute myeloid leukemia (AML). The aim of this study was to determine whether bone marrow MSC affected the sensitivity of AML cells to 3 promising tyrosine kinase inhibitors (sorafenib, sunitinib, and midostaurin) and, if so, to begin to elucidate the underlying mechanisms. Using proliferation assays, we found that 3 AML cells lines (MV4-11, U937, and THP1) were significantly less sensitive to the tyrosine kinase inhibitors when cultured in the presence of bone marrow-derived MSC for 24h before exposure to drugs for 72h. In experiments with MV4-11, IC50 increased from 4.7 nM to 55 nM for sorafenib, from 10 nM to 110 nM for sunitinib, and from 28 nM to 135 nM for midostaurin; in experiments with U937, IC50 increases were 5.1 μM to 11 μM, 6.2 μM to > 10 μM, and 230 to > 1000 nM for each drug; and in experiments with THP1, they were 6.3 μM to 11 μM, 2.2 μM to > 10 μM, and 211 nM to 996 nM. Coculture with MSC also reduced sorafenib- and sunitinib-induced apoptosis by > 60%. Interestingly, drug resistance increased even further after coculturing the cell lines with MSC for 4 weeks or longer: sunitinib had virtually no effect on the proliferation of MV4-11 cells at concentrations of up to 100 nM, and on THP-1 cells at 10 μM. To determine whether the induction of drug resistance was dependent on the direct contact of AML cells with MSC, we tested sensitivity to sorafenib after separating MV4-11cells from MSC with transwell inserts. Under these conditions, the protective effect of MSC was lessened but not abrogated. These results indicated that direct contact with MSC was not an absolute requirement for induction of drug resistance and that MSC-secreted soluble factors might be, at least in part, involved. We therefore determined the soluble factors secreted by MSC using a multiplex assay and tested whether their secretion was augmented by contact with AML cells. MSC secreted IL-6 (230 pg/mL), IL-8 (1880 pg/mL), and VCAM-1 (30 pg/mL). When cocultured with MV4-11, U937 and THP-1 cells for 24h, IL-6 secretion increased 1.3 to 1.8-fold, IL-8 increased 1.5 to 2.6-fold, and VCAM-1 increased 2.2 to 5.6-fold; after 72 of coculture, dramatically elevated levels of IL-6 (2140–3869 pg/mL), IL-8 (4296–8068 pg/mL), and VCAM-1 (5109–6389 pg/mL) were observed. The effects of these and other MSC-derived factors on the sensitivity of AML cells lines and primary AML cells to tyrosine kinase inhibitors are being tested. These results indicate that the anti-AML effect of tyrosine kinase inhibitors is strongly inhibited by bone marrow MSC cells, and support the concept that the microenvironment is an important determinant of resistance to these agents in leukemia. We suggest that the development of agents that interfere with the interaction between AML cells and MSC, and with the molecular mechanisms underlying this protective effect of MSC is a crucial step to improve cure rates.

Current options for the treatment of locally advanced and metastatic renal cell carcinoma: focus on sunitinib

ABSTRACT Recent developments in molecular biology have increased our understanding of the biology and genetics of renal cell carcinoma (RCC) and identified pathways for novel targeted therapy. Several targeted therapies are now available that show promising activity in this disease. Sunitinib, an oral multitargeted tyrosine kinase inhibitor (TKI), has recently been approved for first-line treatment of metastatic RCC (mRCC) and is the new reference standard for the treatment of clear-cell disease. Three other promising TKIs are temsirolimus, approved for the treatment of advanced RCC, sorafenib, approved for the treatment of patients with advanced RCC who have failed or are considered unsuitable for cytokine therapy and bevacizumab, effective in combination with immunotherapy for first-line therapy of mRCC. Several other agents are under investigation as single-agent or combination therapy for mRCC. These include the TKIs axitinib, pazopanib, everolimus, erlotinib, gefitinib and lapatinib. Use of these agents is leading to the development of treatment paradigms that will transform the management of mRCC.