BTK (IC50 = 3 nM)

Acalabrutinib inhibits tyrosine phosphorylation of downstream targets of ERK, IKB, and AKT in the in vitro signaling assay on primary human CLL cells. IC50 values on nine kinases with a cysteine residue in the same location as BTK show that acalabrutinib has a higher selectivity for BTK. Crucially, acalabrutinib does not inhibit EGFR, ITK, or TEC like ibrutinib does. EGFR phosphorylation at tyrosine residues Y1068 and Y1773 is unaffected by acalabrutinib. Acalabrutinib exhibits nearly no inhibition or a much higher IC50 (>1000 nM) on the kinase activities of ITK, EGFR, ERBB2, ERBB4, JAK3, BLK, FGR, FYN, HCK, LCK, LYN, SRC, and YES1 when compared to ibrutinib[1].

ACP-196, when given orally to mice, inhibits anti-IgM-induced CD86 expression in CD19+ splenocytes in a dose-dependent manner; the ED50 for ACP-196 is 0.34 mg/kg, while that of ibrutinib is 0.91 mg/kg. The length of Btk inhibition following a single oral dose of 25 mg/kg is compared using a comparable model. At three hours after dosage, ACP-196 inhibits CD86 expression by more than 90%[3].

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Acalabrutinib (ACP-196) in preclinical research[1]
Acalabrutinib was evaluated in several animal models of B cell non-Hodgkin lymphoma (NHL). These studies provided preclinical in vivo data necessary to move acalabrutinib into human trials. In a study of canine model of B cell NHL, 12 dogs with B cell NHL were orally administered acalabrutinib at escalating dosages of 2.5 mg/kg every 24 h (6 dogs), 5 mg/kg every 24 h (5 dogs), or 10 mg/kg every 12 h (1 dog). As a result, 3 dogs achieved a partial remission (PR), 3 dogs had stable disease (SD), whereas the remaining 6 dogs had progression of disease (PD). This study therefore showed that acalabrutinib has single agent biologic activity in a spontaneous large animal model of NHL.[1]

The in vivo effects of acalabrutinib against CLL cells were demonstrated in the NSG mouse model with xenografts of human CLL. Acalabrutinib significantly inhibited proliferation of human CLL cells in the spleens of NSG mice at all dose levels, as measured for the expression of Ki67 (P = 0.002). Tumor burden decreased with the treatment of acalabrutinib in a dose-dependent manner. Acalabrutinib inhibited BCR signaling by reduced phosphorylation of PLCγ2. Acalabrutinib transiently increased CLL cell counts in the peripheral blood. Therefore, the novel BTK inhibitor acalabrutinib shows in vivo efficacy against human CLL cells xenografted to the NSG mouse model.

Two murine models were used in another in vivo study. In the TCL1 adoptive transfer model, acalabrutinib inhibited BCR signaling by decreased autophosphorylation of BTK and reduction in surface expression of the BCR activation markers CD86 and CD69. Most interestingly, acalabrutinib treatment increased survival significantly over mice receiving vehicle (median 81 vs 59 days, P = 0.02). The second murine model was the NSG xenograft model. Acalabrutinib treatment significantly decreased the phosphorylation of PLCγ2 and ERK (P = 0.02), reduced tumor cell proliferation (P = 0.02), and tumor burden (P = 0.04). Acalabrutinib was shown to be a potent inhibitor of BTK in both murine models of human CLL.

ACP-196's decreased intrinsic reactivity of its electrophile was linked to its high selectivity for Btk when tested against a panel of 395 non-mutant kinases, according to a previous study. In contrast to ibrutinib, ACP-196 was unable to inhibit EGFR, Itk, or Txk. Further confirmation of ibrutinib's EGFR inhibition without ACP-196 inhibition was obtained through phosphoflow assays conducted on EGFR-expressing cell lines.

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Compared to ibrutinib and CC-292, ACP-196 demonstrated higher selectivity for Btk when profiled against a panel of 395 non-mutant kinases (1 μM) in a competitive binding assay. IC50 determinations on 9 kinases with a Cys in the same position as Btk showed ACP-196 to be the most selective. The improved selectivity is related to the reduced intrinsic reactivity of ACP-196's electrophile. Importantly, unlike ibrutinib, ACP-196 did not inhibit EGFR, Itk or Txk. Phosphoflow assays on EGFR expressing cell lines confirmed ibrutinib's EGFR inhibition (EC50: 47-66 nM) with no inhibition observed for ACP-196 at 10 μM. These data may explain the ibrutinib-related incidence of diarrhea and rash. Ibrutinib's potency on Itk and Txk may explain why it interferes with cell-mediated anti-tumor activities of therapeutic CD20 antibodies and immune-mediated killing in the tumor microenvironment. In human whole blood, ACP-196 and ibrutinib showed robust and equipotent inhibitory activity on B-cell receptor induced responses in the low nM range, whereas CC-292 was 10-20 fold less potent.[3]

The EGFR-expressing cell lines used in the phosphoflow tests EGFR inhibition by ibrutinib was further validated without any ACP-196 inhibition being seen.

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Recent recognition of B-cell receptor (BCR) signaling as a critical factor in the progression of B-cell malignancies, including non-Hodgkin lymphoma (NHL), has resulted in the development of numerous targeted therapeutics that inhibit this signaling pathway. Ibrutinib, a small molecule inhibitor of Bruton tyrosine kinase (Btk) a key signaling molecule in the BCR pathway, has demonstrated significant clinical activity in a broad range of B-cell cancers. ACP-196 is a second generation Btk inhibitor with increased target selectivity and enhanced in vivo potency compared with ibrutinib and, thus, may represent an improvement over its predecessor. With the following studies, we sought to evaluate ACP-196 in canine models of B-cell NHL with the ultimate goal of providing the preclinical data necessary to move ACP-196 into human clinical trials. Using two immunophenotypically confirmed canine B-cell lymphoma cell lines, CLBL-1 and 17-71, we demonstrate potent in vitro inhibition of activation of Btk and the downstream effectors ERK 1/2 and PLCγ2 following 1 hour of treatment with ACP-196 at concentrations as low as 10nM.[2]

canine model of B cell NHL
2.5, 5, 10 mg/kg.
orally administered

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In vivo studies were performed in companion dogs as part of an ongoing clinical trial. Twelve dogs with immunophenotypically confirmed, spontaneously occurring B-cell NHL were orally administered ACP-196 at dosages of 2.5mg/kg every 24 hours (6 dogs), 5mg/kg every 24 hours (5 dogs), or 10mg/kg every12 hours (1 dog). Btk occupancy in peripheral blood and lymphoma cells was assessed using a biotin-tagged probe derived from ACP-196. Using this assay we found that at 2.5mg/kg full Btk occupancy was achieved in peripheral B cells 3h after dosing for all dogs, except for a single dog with high peripheral B-cell count. At 24 hours after dosing, 83-99% Btk target occupancy was observed for all dogs. Partial response, as assessed by a modified RECIST scheme, was achieved in 2 dogs in the 2.5mg/kg group and the dog in the 10mg/kg group. Upon relapse, one of the responders in the 2.5mg/kg group was dose escalated to 10mg/kg q12 on day 42 and partial response from relapse was reestablished. Of the remaining 9 dogs, 3 achieved stable disease for > 28 days and 6 discontinued the study after developing progressive disease within 28 days of starting treatment. In total, to date, 3 dogs achieved a partial response, 3 dogs stable disease, and 6 dogs progressive disease. ACP-196 was well tolerated with only mild anorexia noted in some dogs. These data demonstrate that ACP-196 has single agent biologic activity in a spontaneous large animal model of human NHL. Studies in dogs with NHL are ongoing to define regimens prior to initiation of human phase I clinical trials. Additional cohorts are planned combining ACP-196 with a phosphatidylinositide 3-kinase (PI3K) delta-specific inhibitor.[2]

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In vivo, oral administration of ACP-196 in mice resulted in dose-dependent inhibition of anti-IgM-induced CD86 expression in CD19+ splenocytes with an ED50 of 0.34 mg/kg compared to 0.91 mg/kg for ibrutinib. A similar model was used to compare the duration of Btk inhibition after a single oral dose of 25 mg/kg. ACP-196 and ibrutinib inhibited CD86 expression >90% at 3h and ∼50% at 24h postdose. In contrast, CC-292 inhibited ∼50% at 3h and ∼20% at 24h postdose. An ELISA based Btk target occupancy assay was developed to measure target coverage in preclinical and clinical studies. In healthy volunteers, ACP-196 at an oral dose of 100 mg QD showed >90% target coverage over a 24h period. Btk occupancy and regulation of the PD markers (CD69 and CD86) correlated with PK parameters for exposure. In CLL patients, after 7 days of dosing with ACP-196 at 200 mg QD, 94% Btk target occupancy was observed compared with ∼80% reported for ibrutinib at 420 mg QD [3].

Absorption, Distribution and Excretion
The geometric mean absolute bioavailability of acalabrutinib is 25% with a median time to peak plasma concentrations (Tmax) of 0.75 hours.
After administration of a single 100 mg radiolabelled acalabrutinib dose in healthy subjects, 84% of the dose was recovered in the feces and 12% of the dose was recovered in the urine. An irradiated dose of acalabrutinib was 34.7% recovered as the metabolite ACP-5862; 8.6% was recovered as unchanged acalabrutinub; 10.8 was recovered as a mixture of the M7, M8, M9, M10, and M11 metabolites; 5.9% was the M25 metabolite; 2.5% was recovered as the M3 metabolite.
The mean steady-state volume of distribution is approximately 34 L.
Acalabrutinib's mean apparent oral clearance (CL/F) is observed to be 159 L/hr with similar PK between patients and healthy subjects, based on population PK analysis.

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Metabolism / Metabolites
Acalabrutinib is mainly metabolized by CYP3A enzymes. ACP-5862 is identified to be the major active metabolite in plasma with a geometric mean exposure (AUC) that is about 2-3 times greater than the exposure of acalabrutinib. ACP-5862 is about 50% less potent than acalabrutinib in regards to the inhibition of BTK.

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Biological Half-Life
After administering a single oral dose of 100 mg acalabrutinib, the median terminal elimination half-life of the drug was found to be 0.9 (with a range of 0.6 to 2.8) hours. The half-life of the active metabolite, ACP-5862, is about 6.9 hours.

Hepatotoxicity
In open label clinical trials of acalabrutinib in patients with CLL and mantle cell lymphoma, serum aminotransferase elevations occurred in 19% to 23% of patients during therapy and rose to above 5 times ULN in 2% to 3%. These elevations were transient and resolved spontaneously but occasionally led to early drug discontinuation. Among the 610 patients treated with acalabrutinib in pre-registration trials, there were no instances of clinically apparent liver injury attributed to its use, but there was a single instance of acute liver failure and death due to reactivation of hepatitis B. Similar cases of reactivation have been reported with ibrutinib, another small molecule inhibitor of Bruton's tyrosine kinase. Experience with acalabrutinib has been limited and the frequency of clinically apparent liver injury and reactivation of hepatitis B are not known. The majority of cases have occurred in patients taking multiple immunosuppressive agents and not just acalabrutinib alone.
Likelihood score: D (possible rare cause of reactivation of hepatitis B).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of acalabrutinib during breastfeeding. Because acalabrutinib is over 97% bound to plasma proteins, and the half-life of the drug and metabolite are less than 7 hours, the amount in milk is likely to be low. However, the protein binding of the active metabolite is not known and the manufacturer recommends that breastfeeding be discontinued during acalabrutinib therapy and for at least 2 weeks after the final dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Reversible binding of acalabrutinib to human plasma protein is approximately 97.5%. The in vitro mean blood-to-plasma ratio is about 0.7. _In vitro_ experiments at physiologic concentrations show that acalabrutinib can be 93.7% bound to human serum albumin and 41.1% bound to alpha-1-acid glycoprotein.

[1]. J Hematol Oncol . 2016 Mar 9:9:21.

[2].Cancer Res (2014) 74 (19_Supplement): 1744.

[3]. Cancer Res (2015) 75 (15_Supplement): 2596.

Pharmacodynamics
Acalabrutinib is a Bruton Tyrosine Kinase inhibitor that prevents the proliferation, trafficking, chemotaxis, and adhesion of B cells. It is taken every 12 hours and can cause other effects such as atrial fibrillation, other malignancies, cytopenia, hemorrhage, and infection.

C26H23N7O2

465.51

465.191

C, 67.08; H, 4.98; N, 21.06; O, 6.87

1420477-60-6

Acalabrutinib-d4;2699608-18-7;Acalabrutinib-d3; 1420477-60-6; 2058091-99-7 (citrate); 2242394-65-4; 2058091-96-4 (phosphate); 2058091-93-1 (3 hydrate); 2058092-05-8 (sulfate); 2058091-94-2 (fumarate); 2058091-97-5 (tartrate)

71226662

Yellow solid powder

1.4±0.1 g/cm3

1.715

0.77

2

6

4

35

845

1

O=C(C#CC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C1=NC(C2C([H])=C([H])C(C(N([H])C3=C([H])C([H])=C([H])C([H])=N3)=O)=C([H])C=2[H])=C2C(N([H])[H])=NC([H])=C([H])N12

WDENQIQQYWYTPO-IBGZPJMESA-N

InChI=1S/C26H23N7O2/c1-2-6-21(34)32-15-5-7-19(32)25-31-22(23-24(27)29-14-16-33(23)25)17-9-11-18(12-10-17)26(35)30-20-8-3-4-13-28-20/h3-4,8-14,16,19H,5,7,15H2,1H3,(H2,27,29)(H,28,30,35)/t19-/m0/s1

4-[8-amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin-1-yl]-N-pyridin-2-ylbenzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+2% Tween 80+ddH2O: 6mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)