PI3Kδ; PI3Kγ

Tenalisib is a dual PI3K δ/γ inhibitor, increases Ruxolitinib activity in JAK2-V617F mutant erythroleukemia cell lines[1].
Resistance to ruxolitinib was confirmed by a right-ward shift in EC50 of ruxolitinib in a HEL cell proliferation assay (0.82 μM Vs. 12.2 μM). Endogeous pAKT expression was 3.7-fold higher in HEL-RR compared to HEL-RS cells indicating activation of the AKT signaling pathway. While single-agent activity of RP6530 was modest (33-46% inhibition @ 10 μM) in both HEL-RS and HEL-RR cells, addition of 10 μM RP6530 to ruxolitinib was synergistic resulting in a near-complete inhibition of proliferation (>90% for HEL-RS and >70% for HEL-RR). While the order of addition did not affect the potency of RP6530, addition of 5 μM RP6530, 4 h prior to the addition of ruxolitinib resulted in a significant reduction in EC50 of ruxolitinib (5.8 μM) in HEL-RR cells. On lines with cell proliferation data, incubation of 10 μM RP6530 with ruxolitinib for 72 h increased the percent of apoptotic cells (55% in HEL-RS and 37% in HEL-RR) compared to either agent alone (16-27% in HEL-RS and 17-21% in HEL-RR). Conclusions: Ruxolitinib resistance in the V617F JAK-2 mutant HEL cells is accompanied by an increase in pAKT expression. Inhibition of pAKT via the addition of RP6530, a dual PI3K δ/γ inhibitor, resulted in a reversal of ruxolitinib resistance. Complementary activity was also observed in HEL-RS cells indicating that a combination of ruxolitinib and RP6530 could have a positive bearing on the clinical outcome in MF patients[1].

RP6530 has an excellent pharmacokinetics with plasma concentrations reaching well above the EC75 at doses as low as 3 mg/kg in rat and dog for 6-12 h. In addition, RP6530 shows >70 and >100% oral bioavailability with a half-life of 2 and 3 h in rat and dog respectively. The predicted T1/2, Cmax, and AUC0-t at 10 mg dose in human are 9.5 h, 14.0 μM, and 342.0 μM respectively.

Passive resistance was conferred by incubating HEL cells with increasing concentrations of ruxolitinib over an 8-10-week period. Endogenous JAK2, PI3Kδ, PI3Kδ, and pAKT were estimated by Western Blotting. RP6530, ruxolitinib, and the combination of RP6530 + Ruxolitinib were tested for their effect on viability and apoptosis. Cell viability was assessed by a MTT assay. Induction of apoptosis was analyzed by Annexin V/PI staining[1].

[1]. Abstract 2704: RP6530, a dual PI3K δ/γ inhibitor, potentiates ruxolitinib activity in the JAK2-V617F mutant erythroleukemia cell lines.

Background: Myelofibrosis (MF) represents a life-threatening neoplasm that manifests particularly in the elderly population and is characterized by bone marrow fibrosis and extramedullary hematopoeisis. While ruxolitinib, a JAK1/2 inhibitor, has recently been approved by the USFDA for its disease modifying potential in MF patients, it is still not considered as a curative option. Targeting another kinase such as PI3K, downstream of JAK, could therefore be a more efficient way of treating myelofibrotic neoplasms. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor that demonstrated high potency against PI3Kδ (IC50 = 25 nM) and γ (IC50 = 33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. The objective of this study was to evaluate the effect of a combination of ruxolitinib and RP6530 in the JAK2-V617F mutant Human Erythroleukemia (HEL) cell line. Methods: Passive resistance was conferred by incubating HEL cells with increasing concentrations of ruxolitinib over an 8-10-week period. Endogenous JAK2, PI3Kδ, PI3Kδ, and pAKT were estimated by Western Blotting. RP6530, ruxolitinib, and the combination of RP6530 + Ruxolitinib were tested for their effect on viability and apoptosis. Cell viability was assessed by a MTT assay. Induction of apoptosis was analyzed by Annexin V/PI staining. Results: Resistance to ruxolitinib was confirmed by a right-ward shift in EC50 of ruxolitinib in a HEL cell proliferation assay (0.82 μM Vs. 12.2 μM). Endogeous pAKT expression was 3.7-fold higher in HEL-RR compared to HEL-RS cells indicating activation of the AKT signaling pathway. While single-agent activity of RP6530 was modest (33-46% inhibition @ 10 μM) in both HEL-RS and HEL-RR cells, addition of 10 μM RP6530 to ruxolitinib was synergistic resulting in a near-complete inhibition of proliferation (>90% for HEL-RS and >70% for HEL-RR). While the order of addition did not affect the potency of RP6530, addition of 5 μM RP6530, 4 h prior to the addition of ruxolitinib resulted in a significant reduction in EC50 of ruxolitinib (5.8 μM) in HEL-RR cells. On lines with cell proliferation data, incubation of 10 μM RP6530 with ruxolitinib for 72 h increased the percent of apoptotic cells (55% in HEL-RS and 37% in HEL-RR) compared to either agent alone (16-27% in HEL-RS and 17-21% in HEL-RR). Conclusions: Ruxolitinib resistance in the V617F JAK-2 mutant HEL cells is accompanied by an increase in pAKT expression. Inhibition of pAKT via the addition of RP6530, a dual PI3K δ/γ inhibitor, resulted in a reversal of ruxolitinib resistance. Complementary activity was also observed in HEL-RS cells indicating that a combination of ruxolitinib and RP6530 could have a positive bearing on the clinical outcome in MF patients.[1]

C23H18FN5O2

415.42

415.144

C, 66.50; H, 4.37; F, 4.57; N, 16.86; O, 7.70

1639417-54-1

Tenalisib;1639417-53-0

117729511

White to off-white solid

5.11

2

7

5

31

704

1

CC[C@H](C1=C(C(=O)C2=CC=CC=C2O1)C3=CC(=CC=C3)F)NC4=NC=NC5=C4NC=N5

HDXDQPRPFRKGKZ-MRXNPFEDSA-N

InChI=1S/C23H18FN5O2/c1-2-16(29-23-19-22(26-11-25-19)27-12-28-23)21-18(13-6-5-7-14(24)10-13)20(30)15-8-3-4-9-17(15)31-21/h3-12,16H,2H2,1H3,(H2,25,26,27,28,29)/t16-/m1/s1

3-(3-fluorophenyl)-2-[(1R)-1-(7H-purin-6-ylamino)propyl]chromen-4-one

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)

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.)