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) is a life-threatening cancer, particularly common in the elderly, characterized by myelofibrosis and extramedullary hematopoiesis. Although the JAK1/2 inhibitor ruxolitinib was recently approved by the US FDA for the treatment of MF patients, it is not considered a curative therapy. Therefore, targeting other downstream kinases of JAK, such as PI3K, may be a more effective approach to treating myelofibrotic tumors. RP6530 is a novel, potent, and selective PI3Kδ/γ inhibitor, exhibiting potent inhibitory activity against both PI3Kδ (IC50 = 25 nM) and γ (IC50 = 33 nM) enzymes, with selectivity for both α (>300-fold) and β (>100-fold) subtypes. This study aimed to evaluate the effect of ruxolitinib in combination with RP6530 on the JAK2-V617F mutant human erythroleukemia (HEL) cell line. Methods: HEL cells were incubated with escalating concentrations of ruxolitinib for 8–10 weeks to induce passive resistance. The expression levels of endogenous JAK2, PI3Kδ, and pAKT were detected using Western blotting. The effects of RP6530, ruxolitinib, and the combination of RP6530 and ruxolitinib on cell viability and apoptosis were examined. Cell viability was assessed using the MTT assay. The induction of apoptosis was analyzed using Annexin V/PI double staining. Results: In HEL cell proliferation assays, a rightward shift in the EC50 value of ruxolitinib (0.82 μM vs. 12.2 μM) confirmed the development of ruxolitinib resistance. Compared to HEL-RS cells, HEL-RR cells showed a 3.7-fold higher expression level of endogenous pAKT, indicating activation of the AKT signaling pathway. RP6530 monotherapy showed low activity in both HEL-RS and HEL-RR cells (inhibition rate of 33-46% at 10 μM), but 10 μM RP6530 in combination with ruxotinib had a synergistic effect, almost completely inhibiting cell proliferation (>90% in HEL-RS cells, >70% in HEL-RR cells). Although the order of addition did not affect the efficacy of RP6530, adding 5 μM RP6530 4 hours before adding ruxotinib significantly reduced the EC50 value of ruxotinib in HEL-RR cells (5.8 μM). In cell lines with cell proliferation data, co-incubation of 10 μM RP6530 with ruxotinib for 72 hours increased the percentage of apoptotic cells compared to RP6530 or ruxotinib alone (55% in HEL-RS cells and 37% in HEL-RR cells; while the percentages of apoptotic cells with RP6530 or ruxotinib alone were 16-27% and 17-21%, respectively). Conclusion: Ruxotinib resistance in V617F JAK-2 mutant HEL cells is accompanied by increased pAKT expression. Ruxotinib resistance can be reversed by inhibiting pAKT with the addition of the dual PI3K δ/γ inhibitor RP6530. Complementary activity was also observed in HEL-RS cells, suggesting that the combined use of ruxotinib and RP6530 may have a positive impact on the clinical outcomes of 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.)