Real-World Disproportionality Analysis of the Food and Drug Administration Adverse Event Reporting System Database for Asciminib.

INTRODUCTION: Asciminib is primarily utilized for treating Philadelphia chromosome-positive chronic myeloid leukemia in its chronic phase among patients harboring the T315I mutation or those who have been previously treated with at least two tyrosine kinase inhibitors. The safety profile of asciminib across a broad patient population over an extended timeframe remains unverified. This study uses a real-world pharmacovigilance database to evaluate the adverse events (AEs) linked with asciminib, providing valuable insights for clinical drug safety. METHODS: Data from the FDA Adverse Event Reporting System (FAERS) database, spanning from October 2021 to December 2023, served as the basis for this analysis. The extent of disproportional events was assessed using sophisticated metrics such as the reporting odds ratio, proportional reporting ratio, information component, and empirical Bayesian geometric mean. RESULTS: Within the specified period, the FAERS database documented 3,913,574 AE reports, with asciminib being associated with 966 incidents. Reactions to asciminib spanned 27 system organ categories. Utilizing four distinct analytical algorithms, 663 significant preferred terms exhibiting disproportional frequencies were identified. Notably, this investigation uncovered 26 significant AEs linked to off-label asciminib use, encompassing conditions such as gynecomastia, nephrotic syndrome, orchitis, pyelonephritis, hepatotoxicity, and pancreatitis. The median onset time for asciminib-related AEs was 52.5 days, ranging from 17 to 122.75 days. CONCLUSION: The study sheds light on additional potential AEs associated with asciminib use, warranting further research to confirm these findings.

Outcomes of chronic myeloid leukemia patients after therapeutic failure to conventional tyrosine kinase inhibitors and asciminib.

Patients with chronic myeloid leukemia (CML) who have failed conventional tyrosine kinase inhibitors (cTKIs) and asciminib constitute a complex group of patients with few therapeutic options. A retrospective descriptive multicenter observational study was carried out including patients with CML who had presented a therapeutic failure to ≥ 2 cTKIs and asciminib, and had received or were not candidates to ponatinib. Of the 19 patients enrolled, 8 patients failed asciminib due to intolerance and 11 due to resistance. The most common strategy for intolerant patients was to initiate a previously administered cTKI (6/8) with dose adjustments or symptomatic management of adverse events (AEs). Of the patients who failed due to resistance, only patients who underwent progenitor transplantation achieved profound long-term responses. A frequent strategy was to use ponatinib (4/11) in patients previously considered non-candidates, with poor responses in patients in whom dose reductions were used, and severe AEs in patients at standard doses. In the remaining patients, cTKIs and other agents (interferon, hydroxyurea, citarabine, busulfan) were used with poor responses. Patients who progressed to advanced stages had a dismal prognosis. With a median follow-up of 11.2 months, overall survival of the global cohort was 73%; 100% for intolerant patients, 71% for resistant patients and 25% for those who discontinue asciminib in accelerated/blastic phase.

Dose-escalation of second-line and first-line asciminib in chronic myeloid leukemia in chronic phase: the ASC2ESCALATE Phase II trial.

Up to 40% of newly diagnosed patients with chronic myeloid leukemia in chronic phase (CML-CP) discontinue treatment by 5 years, primarily due to resistance or intolerance. Rates of resistance to second-line (2L) treatment are also high. Some patients with resistance respond with dose escalation of tyrosine kinase inhibitors (TKIs). Asciminib demonstrated safety and efficacy across a broad dosage range. ASC2ESCALATE is an ongoing, Phase II, multicenter, single-arm, dose-escalation study of asciminib in 2L and first-line treatment of CML-CP. The primary end point is major molecular response at 12 months in 2L. Secondary end points include molecular responses at and by scheduled time points, survival, and safety. ASC2ESCALATE is the first study investigating asciminib in CML-CP following failure of one prior TKI.Clinical Trial Registration: NCT05384587 (ClinicalTrials.gov).

ASC2ESCALATE is an ongoing, Phase II, multicenter, single-arm, #dose-escalation study of oral asciminib in first-line and second-line treatment for patients with #CML in chronic phase.

Clinical outcomes of chronic myeloid leukaemia patients taking asciminib through a Managed Access Programme (MAP) in Australia.

Asciminib is a novel allosteric STAMP (specifically targets the ABL myristoyl pocket) inhibitor of the BCR::ABL1 oncogene. Real-world clinical outcomes of patients with tyrosine kinase inhibitor (TKI)-resistant/intolerant chronic myeloid leukaemia (CML) in Australia on the Managed Access Programme for asciminib showed higher molecular responses for those with intolerance versus resistance ± intolerance to their last TKI. There remains a clinical need to improve outcomes in patients with CML who have resistance to multiple TKIs, especially in the ponatinib-pretreated cohort.

[Chronic myeloid leukemia: 2024 update on novel therapeutic strategies].

Many patients with chronic myeloid leukemia (CML) can now maintain response thanks to the advent of tyrosine kinase inhibitors (TKIs) and STAMP inhibitors, but adverse events associated with prolonged TKI therapy have become a problem. Adequate management of adverse events is key to successful treatment, as some can significantly impact the patient's prognosis. The goal of CML treatment was once to prevent acute transformation, but now that many patients achieve deep remission and long-term survival, the goal has shifted to achieving long-term treatment free remission (TFR). It is essential to carefully consider disease risk, patient background, and adverse events of each therapeutic agent in order to make the appropriate choice. This article reviews the treatment of chronic phase CML (CML-CP) as described in the 2023 edition of the Guidelines for Hematopoietic Tumors, focusing on treatment options for first-line CML-CP, dose optimization of ponatinib, outcomes with the new CML drug asciminib, and TFR.

Top advances of the year: Leukemia.

In the year 2021, there were three new Food and Drug Administration approvals for all leukemia types: asciminib (Scemblix) for chronic myeloid leukemia, brexucabtagene autoleucel (Tecartus) for relapsed/refractory B-cell acute lymphocytic leukemia, and asparaginase erwinia chrysanthemi (recombinant)-rywn (Rylaze) for acute lymphocytic leukemia. This is down from 2017-2018 when eight new therapies were approved for acute myeloid leukemia alone. However, this decrease from prior years does not imply that little progress was made in our understanding or treatment of leukemias in 2021. Asciminib and brexucabtagene autoleucel, in particular, are representative of major developing trends. Asciminib, a targeted therapy, is only one of many drugs in development that are products of a bedside-to-bench approach fueled by new sequencing and other genetic technologies that have greatly increased our understanding of the biology behind hematologic diseases. Brexucabtagene autoleucel, an adoptive cell therapy, is the newest of several similar treatments for B cell-associated neoplasms, and it is representative of a massive push to develop novel immunotherapies for a broad range of hematologic malignancies. This commentary reviews the development of asciminib and brexucabtagene autoleucel and describes other major advances in the associated fields of targeted therapy and immunotherapy for leukemias.

The EMA Assessment of Asciminib for the Treatment of Adult Patients With Philadelphia Chromosome-Positive Chronic Myeloid Leukemia in Chronic Phase Who Were Previously Treated With at Least Two Tyrosine Kinase Inhibitors.

Asciminib is an allosteric high-affinity tyrosine kinase inhibitor (TKI) of the BCR-ABL1 protein kinase. This kinase is translated from the Philadelphia chromosome in chronic myeloid leukemia (CML). Marketing authorization for asciminib was granted on August 25, 2022 by the European Commission. The approved indication was for patients with Philadelphia chromosome-positive CML in the chronic phase which have previously been treated with at least 2 TKIs. Clinical efficacy and safety of asciminib were evaluated in the open-label, randomized, phase III ASCEMBL study. The primary endpoint of this trial was major molecular response (MMR) rate at 24 weeks. A significant difference in MRR rate was shown between the asciminib treated population and the bosutinib control group (25.5% vs. 13.2%, respectively, P = .029). In the asciminib cohort, adverse reactions of at least grade 3 with an incidence ≥ 5% were thrombocytopenia, neutropenia, increased pancreatic enzymes, hypertension, and anemia. The aim of this article is to summarize the scientific review of the application which led to the positive opinion by the European Medicines Agency's Committee for Medicinal Products for Human Use.

Effects of the STAMP-inhibitor asciminib on T cell activation and metabolic fitness compared to tyrosine kinase inhibition by imatinib, dasatinib, and nilotinib.

T cell function is central to immune reconstitution and control of residual chronic myeloid leukemia (CML) cells after treatment initiation and is associated with achieving deep molecular response as a prerequisite for treatment-free remission, the ultimate therapeutic goal in CML. ATP-pocket-binding tyrosine kinase inhibitors (TKIs) like imatinib, dasatinib, and nilotinib are widely used for treating CML, but they have shown to inhibit T cell function as an "off-target" effect. Therefore, we tested asciminib, the first-in-class BCR::ABL1 fusion protein inhibitor specifically targeting the ABL myristoyl pocket (STAMP) and compared its effects on T cell function with imatinib, dasatinib, and nilotinib. Whereas all four TKIs inhibited the expression of the co-stimulatory protein CD28, the amino acid transporter CD98, proliferation, and secretion of pro-inflammatory cytokines IFNγ, IL-6, and IL-17A upon T cell stimulation, asciminib had less impact on PD-1, activation markers, and IL-2 secretion. T cells treated with asciminib and the other TKIs maintained their ability to mobilize their respiratory capacity and glycolytic reserve, which is an important surrogate for metabolic fitness and flexibility. Overall, we found milder inhibitory effects of asciminib on T cell activation, which might be beneficial for the immunological control of residual CML cells.

Deucravacitinib is an allosteric TYK2 protein kinase inhibitor FDA-approved for the treatment of psoriasis.

Psoriasis is a heterogeneous, inflammatory, autoimmune skin disease that affects up to 2% of the world's population. There are many treatment modalities including topical medicines, ultraviolet light therapy, monoclonal antibodies, and several oral medications. Cytokines play a central role in the pathogenesis of this disorder including TNF-α, (tumor necrosis factor-α) IL-17A (interleukin-17A), IL-17F, IL-22, and IL-23. Cytokine signaling involves transduction mediated by the JAK-STAT pathway. There are four JAKS (JAK1/2/3 and TYK2) and six STATS (signal transducer and activators of transcription). Janus kinases contain an inactive JH2 domain that is aminoterminal to the active JH1 domain. Under basal conditions, the JH2 domain inhibits the activity of the JH1 domain. Deucravacitinib is an orally effective N-trideuteromethyl-pyridazine derivative that targets and stabilizes the TYK2 JH2 domain and thereby blocks TYK2 JH1 activity. Seven other JAK inhibitors, which target the JAK family JH1 domain, are prescribed for the treatment of neoplastic and other inflammatory diseases. The use of deuterium in the trimethylamide decreases the rate of demethylation and slows the production of a metabolite that is active against a variety of targets in addition to TYK2. A second unique aspect in the development of deucravacitinib is the targeting of a pseudokinase domain. Deucravacitinib is rather specific for TYK2 and its toxic effects are much less than those of the other FDA-approved JAK inhibitors. The successful development of deucravacitinib may stimulate the development of additional pseudokinase ligands for the JAK family and for other kinase families as well.

Properties of FDA-approved small molecule protein kinase inhibitors: A 2023 update.

Owing to the dysregulation of protein kinase activity in many diseases including cancer, this enzyme family has become one of the most important drug targets in the 21st century. There are 72 FDA-approved therapeutic agents that target about two dozen different protein kinases and three of these drugs were approved in 2022. Of the approved drugs, twelve target protein-serine/threonine protein kinases, four are directed against dual specificity protein kinases (MEK1/2), sixteen block nonreceptor protein-tyrosine kinases, and 40 target receptor protein-tyrosine kinases. The data indicate that 62 of these drugs are prescribed for the treatment of neoplasms (57 against solid tumors including breast, lung, and colon, ten against nonsolid tumors such as leukemia, and four against both solid and nonsolid tumors: acalabrutinib, ibrutinib, imatinib, and midostaurin). Four drugs (abrocitinib, baricitinib, tofacitinib, upadacitinib) are used for the treatment of inflammatory diseases (atopic dermatitis, psoriatic arthritis, rheumatoid arthritis, Crohn disease, and ulcerative colitis). Of the 72 approved drugs, eighteen are used in the treatment of multiple diseases. The following three drugs received FDA approval in 2022 for the treatment of these specified diseases: abrocitinib (atopic dermatitis), futibatinib (cholangiocarcinomas), pacritinib (myelofibrosis). All of the FDA-approved drugs are orally effective with the exception of netarsudil, temsirolimus, and trilaciclib. This review summarizes the physicochemical properties of all 72 FDA-approved small molecule protein kinase inhibitors including lipophilic efficiency and ligand efficiency.

Genetic Landscape of Chronic Myeloid Leukemia and a Novel Targeted Drug for Overcoming Resistance.

Tyrosine kinase inhibitors (TKIs) exemplify the success of molecular targeted therapy for chronic myeloid leukemia (CML). However, some patients do not respond to TKI therapy. Mutations in the kinase domain of BCR::ABL1 are the most extensively studied mechanism of TKI resistance in CML, but BCR::ABL1-independent mechanisms are involved in some cases. There are two known types of mechanisms that contribute to resistance: mutations in known cancer-related genes; and Philadelphia-associated rearrangements, a novel mechanism of genomic heterogeneity that occurs at the time of the Philadelphia chromosome formation. Most chronic-phase and accelerated-phase CML patients who were treated with the third-generation TKI for drug resistance harbored one or more cancer gene mutations. Cancer gene mutations and additional chromosomal abnormalities were found to be independently associated with progression-free survival. The novel agent asciminib specifically inhibits the ABL myristoyl pocket (STAMP) and shows better efficacy and less toxicity than other TKIs due to its high target specificity. In the future, pooled analyses of various studies should address whether additional genetic analyses could guide risk-adapted therapy and lead to a final cure for CML.

The Importance of the Pyrazole Scaffold in the Design of Protein Kinases Inhibitors as Targeted Anticancer Therapies.

The altered activation or overexpression of protein kinases (PKs) is a major subject of research in oncology and their inhibition using small molecules, protein kinases inhibitors (PKI) is the best available option for the cure of cancer. The pyrazole ring is extensively employed in the field of medicinal chemistry and drug development strategies, playing a vital role as a fundamental framework in the structure of various PKIs. This scaffold holds major importance and is considered a privileged structure based on its synthetic accessibility, drug-like properties, and its versatile bioisosteric replacement function. It has proven to play a key role in many PKI, such as the inhibitors of Akt, Aurora kinases, MAPK, B-raf, JAK, Bcr-Abl, c-Met, PDGFR, FGFRT, and RET. Of the 74 small molecule PKI approved by the US FDA, 8 contain a pyrazole ring: Avapritinib, Asciminib, Crizotinib, Encorafenib, Erdafitinib, Pralsetinib, Pirtobrutinib, and Ruxolitinib. The focus of this review is on the importance of the unfused pyrazole ring within the clinically tested PKI and on the additional required elements of their chemical structures. Related important pyrazole fused scaffolds like indazole, pyrrolo[1,2-b]pyrazole, pyrazolo[4,3-b]pyridine, pyrazolo[1,5-a]pyrimidine, or pyrazolo[3,4-d]pyrimidine are beyond the subject of this work.

Real-life analysis on safety and efficacy of asciminib for ponatinib pretreated patients with chronic myeloid leukemia.

Failure of second-generation tyrosine kinase inhibitors (2GTKI) is a challenging situation in patients with chronic myeloid leukemia (CML). Asciminib, recently approved by the US Federal Drug Administration, has demonstrated in clinical trials a good efficacy and safety profile after failure of 2GTKI. However, no study has specifically addressed response rates to asciminib in ponatinib pretreated patients (PPT). Here, we present data on responses to asciminib from 52 patients in clinical practice, 20 of them (38%) with prior ponatinib exposure. We analyzed retrospectively responses and toxicities under asciminib and compared results between PPT and non-PPT patients.After a median follow-up of 30 months, 34 patients (65%) switched to asciminib due to intolerance and 18 (35%) due to resistance to prior TKIs. Forty-six patients (88%) had received at least 3 prior TKIs. Regarding responses, complete cytogenetic response was achieved or maintained in 74% and 53% for non-PPT and PPT patients, respectively. Deeper responses such as major molecular response and molecular response 4.5 were achieved in 65% and 19% in non-PPT versus 32% and 11% in PPT, respectively. Two patients (4%) harbored the T315I mutation, both PPT.In terms of toxicities, non-PPT displayed 22% grade 3-4 TEAE versus 20% in PPT. Four patients (20% of PPT) suffered from cross-intolerance with asciminib as they did under ponatinib.Our data supports asciminib as a promising alternative in resistant and intolerant non-PPT patients, as well as in intolerant PPT patients; the resistant PPT subset remains as a challenging group in need of further therapeutic options.

Targeting BCR-Abl in the treatment of Philadelphia-chromosome positive chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is an indolent malignant hematological disease that accounts for about 15% of all cases of leukemia. This disorder results from the formation of the Philadelphia chromosome that involves a reciprocal translocation that produces a lengthened chromosome 9 and shortened chromosome 22 - the Philadelphia chromosome. As a consequence of the translocation, the dysregulated BCR-Abl fusion oncoprotein is formed and it produces the abnormal proliferation of white blood cells. The treatment of CML with imatinib revolutionized the treatment of this disorder and led to the discovery and development of dozens of effective targeted protein kinase inhibitors. Imatinib (first generation), dasatinib, nilotinib, and bosutinib (second generation) have been FDA-approved for frontline therapy, and ponatinib (third generation) is approved for resistant disease with a T315I mutation. Each of these drugs is orally bioavailable. The BCR-Abl fusion protein lacks the physiological N-terminal myristoyl group that binds to a hydrophobic pocket in the large protein kinase lobe and inhibits enzyme activity. The absence of the myristoyl group leads to enhanced protein kinase catalytic activity. Asciminib was designed to bind to this binding pocket to reduce Abl kinase activity. Asciminib is orally effective and was FDA-approved as a third-line treatment for CML and a first-line treatment in patients with the T315I mutation. It blocks the activity of BCR-Abl by interacting with the myristate-binding site located 23 Å from the ATP-binding site and is the prototype of a type IV inhibitor. Asciminib is a so-called STAMP inhibitor that Specifically Targets the Abl Myristoyl Pocket.

A kinase inhibitor which specifically targets the ABL myristate pocket (STAMP), but unlike asciminib crosses the blood-brain barrier.

The ubiquitously expressed ABL1 and ABL2 protein kinases play many important roles in cell function. Although they have been implicated in neuron development, maintenance and signaling, there are no good tool compounds to evaluate the effects of ABL kinase inhibition in the brain. Asciminib is a recently approved drug that specifically and potently inhibits the tyrosine kinase activity of ABL1, ABL2 and that of the chimeric BCR-ABL1 oncoprotein which causes chronic myeloid leukemia. Herein we show that asciminib does not penetrate the intact blood-brain barrier (BBB) following administration to rats, which curtails its utility for assessing the in vivo effects of ABL kinase inhibition in the brain. However, we describe another specific ABL kinase inhibitor, possessing physicochemical characteristics suitable for BBB penetration, and which after administration (either i.v., i.p. or p.o.) to mice achieves substantial, pharmacologically relevant brain concentrations. This bipyridine compound (4) therefore has potential for elucidating the role of ABL kinases in the brain in non-clinical studies.

Asciminib monotherapy for newly diagnosed chronic myeloid leukemia in chronic phase: the ASC4FIRST phase III trial.

Asciminib is the first BCR::ABL1 inhibitor that works by Specifically Targeting the ABL Myristoyl Pocket (STAMP). Asciminib has shown favorable efficacy and safety in patients with chronic myeloid leukemia in chronic phase without the T315I mutation who have received ≥2 prior tyrosine kinase inhibitors (TKIs) in phase I and III clinical trials and in patients with the T315I mutation who have received ≥1 prior TKI in phase I. ASC4FIRST (NCT04971226) is a phase III, multicenter, open-label, randomized study of asciminib versus investigator-selected TKI in patients with newly diagnosed chronic myeloid leukemia in chronic phase. The primary end point is major molecular response at week 48. Secondary end points include responses at and by scheduled time points, safety, pharmacokinetics and patient-reported outcomes. Clinical Trial Registration: NCT04971226 (ClinicalTrials.gov).

Allosteric Inhibition of c-Abl to Induce Unfolded Protein Response and Cell Death in Multiple Myeloma.

Endoplasmic reticulum stress activates inositol-requiring enzyme 1α (IRE1α) and protein kinase, R-like endoplasmic reticulum kinase (PERK), the two principal regulators of the unfolded protein response (UPR). In multiple myeloma, adaptive IRE1α signaling is predominantly activated and regulates cell fate along with PERK. Recently, we demonstrated that GNF-2, an allosteric c-Abl inhibitor, rheostatically enhanced IRE1α activity and induced apoptosis through c-Abl conformational changes in pancreatic ß cells. Herein, we analyzed whether the pharmacological modulation of c-Abl conformation resulted in anti-myeloma effects. First, we investigated the effects of GNF-2 on IRE1α activity and cell fate, followed by an investigation of the anti-myeloma effects of asciminib, a new allosteric c-Abl inhibitor. Finally, we performed RNA sequencing to characterize the signaling profiles of asciminib. We observed that both GNF-2 and asciminib decreased cell viability and induced XBP1 mRNA splicing in primary human myeloma cells and myeloma cell lines. RNA sequencing identified the induction of UPR- and apoptosis-related genes by asciminib. Asciminib re-localized c-Abl to the endoplasmic reticulum, and its combination with a specific IRE1α inhibitor, KIRA8, enhanced cell death with the reciprocal induction of CHOP mRNA expression. Together, the allosteric inhibition of c-Abl-activated UPR with anti-myeloma effects; this could be a novel therapeutic target for multiple myeloma.

Investigating Potential Cardiovascular Toxicity of Two Anti-Leukemia Drugs of Asciminib and Ponatinib in Zebrafish Embryos.

BCR-ABL, a fusion protein kinase, is a druggable target exclusively expressed in patients with chronic myeloid leukemia (CML). Several anti-leukemia medicines targeting this protein have been developed in recent years. However, therapeutic options are limited for CML patients bearing multiple BCR-ABL1 mutations. Ponatinib (PON), a potent tyrosinase inhibitor, was one of the approved drugs for managing BCR-ABL1 T315I mutant disease. However, treatment of patients with PON reported severe side effects related to cardiovascular events. Asciminib (ASC) was the first allosteric inhibitor approved to target the myristoyl pocket of BCR-ABL protein to inhibit protein activity. The different mechanism of inhibition opens the possibility of co-exposure with both medicines. Reports on cardiovascular side effects due to the combination use of PON + ASC in pre-clinical and clinical studies are minimal. Thus, this study aimed to observe the potential cardiovascular-related side effect after co-exposure to ASC and PON using zebrafish as an animal model. In this study, zebrafish were acutely exposed to both compounds. The cardiovascular physiology parameters and gene expression related to cardiovascular development were evaluated. We demonstrate that combining ASC with PON at no observed effect concentration (NOEC) did not cause any significant change in the cardiac performance parameter in zebrafish. However, a significant increase in nkx2.5 expression level and a substantial decrease in blood flow velocity were recorded, suggesting that combining these compounds at NOEC can cause mild cardiovascular-related side effects.

[Novel therapies for chronic myeloid leukemia].

Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myeloid leukemia (CML). Five types of TKIs, including the third-generation TKI, ponatinib, are available in Japan, and TKI resistance has almost been overcome. However, TKI-related adverse events, such as vascular occlusive diseases that are frequently associated with ponatinib use, have become a critical concern. A recent dose optimization study of ponatinib demonstrated a dosing regimen that balances its risks and benefits in CML therapy. Furthermore, asciminib, a CML therapeutic drug with a new mechanism of action, has become available and is being applied clinically in Europe and the USA. This article outlines the latest treatments developed for CML in the chronic phase with prior therapy.

Reply to Correspondence on "Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase".

Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 µM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (Cmax =1.6-3.7 µM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.