Ki: 4.9 pM (hCD73)[1]

AB-680 ammonium reduces CD73 in human, mouse CD8+ T cells, and hPBMC, with IC50s of 0.070, 0.008, 0.66, and 0.011 nM, respectively. It also inhibits soluble hCD73 with an IC50 of 0.043 nM in CHO cells[1].

On the basis of the extraordinary potency of AB680 in vitro and excellent pharmacokinetic profile in preclinical species, AB680 was advanced to a phase I clinical study in healthy volunteers. This placebo-controlled study assessed the safety, tolerability, and PK/PD profile of AB680. Here we present the initial pharmacokinetic data of AB680 in humans. AB680 was well tolerated when administered as a single iv dose across the range of 0.1–25 mg (six cohorts). All treatment-emergent adverse events were mild or moderate in severity, with no clear pattern of toxicity at these dose levels. AB680 displayed low clearance and a long half-life (67–74 h) following an iv infusion of 16 or 25 mg over 30–60 min. The human PK profile of AB680 is consistent with the intended Q2W dosing schedule and validates the design strategy employed to discover a long-acting drug for iv-administration.[1]

CYP inhibition procedure: [1] Test compounds were evaluated in-vitro for its potential to inhibit major human drug metabolizing enzymes of the cytochrome P450 family. The test compounds were incubated separately over a concentration range of 0 to 40 µM with 0.1 mg/mL human liver microsomal protein suspension in 0.1 M potassium phosphate buffer at pH 7.4, 1 mM NADPH, and a probe substrate (Phenacetin for CYP1A2, Diclofenac for CYP2C9, S-Mephenytoin for CYP2C19, Dextromethorphan for CYP2D6, and Midazolam for CYP3A4). Each substrate was incubated at 37 °C for 5 to 20 minutes as defined by the previous assay validation. Samples for each substrate were collected and pooled with samples from other substrate incubations for determination of product formation by LC-MS/MS. IC50 values were calculated using variable slope (4-parameter) model. Furafylline (1A2), sulfaphenazole (2C9), (+)-N-3-Benzylnirvanol (2C19), quinidine (2D6), and ketoconazole (3A4) were used as reference controls.

Cellular Human CD73 Assay. [1] Generation and Expansion of Human CD73 Stable CHO Cell Line. Stable cell lines were generated by Lake Pharma using a standard protocol to transform CHO cells with a pcDNA3.1(+) vector carrying the human NT5E (CD73) gene. Antibiotic selection was performed in CD OptiCHO cell media containing 5 μg / mL Puromycin and 200 μg / mL Hygromycin B. Pools of surviving CHO-CD73 were collected and frozen in 7.5% DMSO in CD OptiCHO cell media. Cryopreserved cells were defrosted in a water bath at 37°C by agitating the vial until the cells were completely thawed. Cells were then transferred to a 15 mL Falcon tube prior to centrifuging at 225xg for 5 minutes to pellet the cells. The cell pellet was resuspended in fresh warm CD OptiCHO Growth Medium supplemented with 2 mM Glutamax and transferred to a T175 flask. After two days and on reaching ~80% confluence (~20 million cells/flask) cells were split 1:3 into three fresh T175 flasks. After a further three days, cells were transferred to a 15 mL Falcon tubes and centrifuged at 250xg for 5 minutes to pellet. Cells were re-suspended at a density of 3 million cells per mL in CellBanker2 cryopreservation media and aliquoted into cryogenic vials. Cell aliquots were stored at -80°C until needed.

Clinical Study[1] The phase I clinical study was a first-in-human, double-blind, randomized, placebo-controlled combined single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to evaluate the safety, tolerability, PK, and potential PD effects of AB680 in healthy volunteers. Participants were randomly selected to receive AB680 (n = 6) or matching placebo (n = 2) in each of seven dosing cohorts in the SAD part and a single dose cohort in the MAD part. In the SAD part of the study, participants received a single iv infusion of 0.1, 0.6, 2, 4, 8, 16, 25 mg of AB680 or placebo. In the MAD part of the study, participants received iv infusion of 8 mg of AB680 or placebo once daily on 3 days (days 1, 8, and 15).

[1]. Discovery of AB680: A Potent and Selective Inhibitor of CD73. J Med Chem . 2020 Oct 22;63(20):11448-11468.

[2]. Discovery and Characterization of AB680, a Potent and Selective Small-Molecule CD73 Inhibitor for Cancer Immunotherapy.

Quemliclustat is a small-molecule competitive inhibitor that inhibits the extracellular enzyme CD73 (differentiation cluster 73; 5'-extracellular nucleotidase; 5'-NT; extracellular 5'-nucleotidase), possessing potential immunomodulatory and antitumor activity. Upon administration, quemliclustat targets and binds to CD73, leading to CD73 aggregation and internalization. This prevents CD73-mediated conversion of adenosine monophosphate (AMP) to adenosine and reduces the level of free adenosine in the tumor microenvironment (TME). This prevents adenosine-mediated lymphocyte suppression and enhances the activity of CD8-positive effector cells and natural killer (NK) cells. Furthermore, it activates macrophages and reduces the activity of myeloid-derived suppressor cells (MDSCs) and regulatory T lymphocytes (Tregs). CD73 inhibits tumor cell growth by eliminating the suppression of the immune system and enhancing cytotoxic T cell-mediated anti-cancer immune responses. In addition, CD73 aggregation and internalization reduce cancer cell migration and prevent metastasis. CD73 is a plasma membrane protein belonging to the 5'-nucleotidase (NTase) family, upregulated in various cancer cell types. It catalyzes the conversion of extracellular nucleotides (such as AMP) into membrane-permeable nucleosides (such as adenosine); it plays a crucial role in adenosine-mediated immunosuppression in the tumor microenvironment (TME). High concentrations of extracellular adenosine (ADO) in the TME suppress immune function by inhibiting the activation of T cells and NK cells. The generation of intratumoral adenosine methionine (ADO) depends on the sequential catabolism of ATP by two extracellular nucleotidases, CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 blocks the main pathway of ADO generation in the TME and reverses ADO-mediated immunosuppression. Through in-depth research on structure-activity relationship (SAR), structure-based drug design, and optimization of pharmacokinetic properties, AB680, a highly potent (Ki = 5 pM), reversible, and selective CD73 inhibitor, was ultimately discovered. AB680 also exhibited extremely low clearance and a long half-life in preclinical animal models, making it suitable for long-acting parenteral administration. AB680 is currently undergoing a phase I clinical trial. Preliminary data show that AB680 is well tolerated and its pharmacokinetic profile is suitable for intravenous administration every two weeks (Q2W). [1]

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Introduction. Extracellular adenosine (ADO) suppresses immune function by inhibiting T cell and NK cell activation and is present in high concentrations in the tumor microenvironment (TME). The generation of ADO in tumors depends on the sequential catabolic metabolism of ATP by two extracellular nucleotidases, CD39 (ATP→AMP) and CD73 (AMP→ADO). Inhibition of CD73 eliminates the major and irreplaceable pathway for ADO generation in the TME and reverses ADO-mediated immunosuppression. This article introduces the characteristics of AB680, a novel, highly effective, reversible, and selective small molecule inhibitor of CD73, currently in preclinical development and a promising anti-tumor drug.
Methods. The hydrolysis of AMP by CHO-CD73 cells was measured using the malachite green assay to evaluate the efficacy of the CD73 inhibitor. Efficacy was also assessed using human T cells and soluble recombinant CD73. Furthermore, selectivity for related extracellular nucleotidases was evaluated. CD73 inhibition was determined by quantitative AMP hydrolysis using a luminescent method in the presence of human serum. The ability of AB680 to reverse AMP-mediated immunosuppression of human CD4+/CD8+ T cells was determined by adding exogenous AMP during T cell activation using anti-CD3/CD28/CD2 magnetic beads. The pharmacokinetic properties of AB680 were evaluated in several preclinical animal models. The expected human dosing regimen for AB680 was determined using allometric scaling.
Results. AB680 is a potent, reversible, and selective CD73 inhibitor (IC50 < 0.01 nM on human CD8+ T cells), maintaining high activity even in the presence of human serum. AB680 exhibits selectivity exceeding 10,000-fold against relevant extracellular nucleotidases, as well as numerous unrelated enzymes, receptors, and ion channels. AB680 does not significantly inhibit major CYP450 isoenzymes or hERG potassium channels. AB680 effectively reverses AMP and ADO-mediated immunosuppression in vitro. In the presence of high concentrations of AMP, AB680 significantly restores CD25 expression, IFN-γ production, and proliferation in human CD4+ and CD8+ T cells. The pharmacokinetics (PK) of AB680 have been evaluated in rodents and non-rodents. The pharmacokinetic (PK) characteristics of AB680 are characterized by extremely low clearance and long half-life in preclinical animal models. It is expected that its PK characteristics in humans are suitable for intravenous (iv) administration, which is consistent with the dosing cycle of typical monoclonal antibodies (mAbs). In preclinical animal models, high-dose AB680 infusion was well tolerated. Conclusion: AB680 is a highly effective and selective CD73 small molecule inhibitor that can reduce AMP and ADO-mediated tumor immune responses by potently blocking the production of adenosine in the tumor microenvironment (TME). AB680 has good expected human PK characteristics and is suitable for parenteral administration. It is expected to enter the clinical development stage in 2018. [2]

614.12220

AB-680;2105904-82-1

162642790

Typically exists as solid at room temperature

VVDIOJBTSCUIEW-AJLCLCFCSA-N

InChI=1S/C20H24ClFN4O9P2.2H3N/c1-10(11-4-2-3-5-13(11)22)24-14-6-16(21)25-19-12(14)7-23-26(19)20-18(28)17(27)15(35-20)8-34-37(32,33)9-36(29,30)31;;/h2-7,10,15,17-18,20,27-28H,8-9H2,1H3,(H,24,25)(H,32,33)(H2,29,30,31);2*1H3/t10-,15+,17+,18+,20+;;/m0../s1

diazanium;[(2R,3S,4R,5R)-5-[6-chloro-4-[[(1S)-1-(2-fluorophenyl)ethyl]amino]pyrazolo[3,4-b]pyridin-1-yl]-3,4-dihydroxyoxolan-2-yl]methoxy-[[hydroxy(oxido)phosphoryl]methyl]phosphinate

AB-680 (ammonium); AB-680 ammonium

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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