PI3Kδ ( IC50 = 11 nM); PI3Kα ( IC50 = 280 nM); PI3Kβ ( IC50 = 480 nM); PI3Kγ ( IC50 = 2.57 μM); DNA-PK ( IC50 = 880 nM)

Leniolisib inhibits phosphorylation of AKT or S6, a pathway activity indicator that is elevated when APDS mutant p110 is expressed in cell lines and patient-derived lymphocytes, in a concentration-dependent manner[1].

The immune dysregulation is resolved by oral leniolisib, which also results in a dose-dependent decrease in PI3K/AKT pathway activity, normalization of circulating transitional and naive B cells, and a decrease in PD-1+CD4+ and senescent CD57+CD8+ T cells. All patients exhibit improvement in lymphoproliferation after 12 weeks of therapy, with decreases in lymph node sizes and spleen volumes of 39% (mean, range, 26-57%) and 40% (mean, range, 13-65%), respectively[1].

Biochemical assays for PI3K isoforms and other lipid kinases [2]
KinaseGlo format [2]
The inhibitory kinase activities on PI3Kα and PI3Kβ were determined using phosphatidyl inositol (PI) as substrate in n-Octyl-Glucoside (OG) using a luminescence assays based on ATP consumption (KinaseGlo). Some 50 nl of compound dilutions were dispensed onto black 384-well plates (Greiner Cat. No.784076). L-α-phosphatidylinositol (PI), provided as 10 mg/ml solution in methanol, was transferred into a glass tube and dried under nitrogen beam. It was then resuspended in 3% (v/v) Octylglucoside by vortexing and stored at 4°C. Some 4.5μl of a mix of PI/OG with 10 nM PI3Kα or 0.75 μg/ml PI3Kβ was added. The kinase reactions were started by addition of 4.5 μl of ATP-mix containing in a final volume of 9 μl the following: 10 mM TRIS-HCl pH 7.5, 3 mM MgCl2 or 3 mM MnCl2, 50 mM NaCl, 0.05% CHAPS, 1m MAKT DTT and 1 μM ATP. The reactions were carried out at room temperature (RT) for either 30 or 60 minutes (PI3Kα or PI3Kβ, respectively) stopped with 9 μl of Kinase Glo and plates were read 10 minutes later in a Synergy2 reader using an integration time of 0.1 seconds per well. To generate the 100% inhibition of the kinase reaction PI/OG in kinase buffer without addition of recombinant kinases was used while the 0% inhibition was given by the solvent vehicle (90% v/v) DMSO) in water in the presence of recombinant kinases.

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ADAPTA format [2]
The kinase assay format used for measuring the activity on PI3Kγ and PI3Kδ is non-radioactive and monitors the formation of ADP TR-FRET (ADAPTA). Some 50 nl of compound dilutions were dispensed onto white 384-well small volume polystyrene plates. Then, 4.5 μl of PI3K substrate PI followed by 4.5 μl of ATP (final assay volume 9 μL) are incubated at RT. The standard reaction buffer for the Adapta™ TR-FRET assay contained 10 mM Tris-HCl pH 7.5, 3 mM MgCl2, 50 mM NaCl, 1 mM DTT, and 0.05% (v/v) CHAPS. Reactions were stopped with 4.5 μl of a mixture of EDTA containing the Eu-labeled anti-ADP antibody and the Alexa Fluor® 647- labeled ADP tracer in TR-FRET dilution buffer. Plates are read 30 to 60 minutes (PI3Kγ or PI3Kδ, respectively) later in a Synergy2 reader using an integration time of 0.4 seconds and a delay of 0.05 seconds. Control for the 100% inhibition of the kinase reaction was performed by replacing the PI3K by the standard reaction buffer. The control for the 0% inhibition was given by the solvent vehicle of the compounds (90% DMSO in H2O). IC50 values obtained via KinaseGlo and ADAPTA values have been shown to be equivalent/comparable.

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Radiometric format[2]
The radiometric protein kinase assay (33PanQinase® Activity Assay) was performed in 96-well Flash-PlatesTM from Perkin Elmer (Boston, MA, USA) in a 50 μl reaction volume. The reaction cocktail was pipetted in 4 steps in the following order:
• 10 μl of non-radioactive ATP solution (in H2O)
• 25 μl of assay buffer/ [γ-33P]-ATP mixture
• 5 μl of test sample in 10% DMSO
• 10 μl of enzyme/substrate mixture
The assay contained 70 mM HEPES-NaOH pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Naorthovanadate, 1.2 mM DTT, 1.0 μM ATP, [γ-33P]-ATP (approx. 8 x 1005 cpm per well), 2.4 nM protein kinase, and 20 μg/ml substrate (casein).

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All protein kinases provided by ProQinase were expressed in Sf9 insect cells or in E.coli as recombinant GST-fusion proteins or His-tagged proteins, either as full-length or enzymatically active fragments. All kinases were produced from human cDNAs. Kinases were purified by either GSH-affinity chromatography or immobilized-metal affinity chromatography. Affinity tags were removed from a number of kinases during purification. The purity of the protein kinases was examined by SDS-PAGE/Coomassie staining; the identity was checked by mass spectroscopy. The reaction cocktails were incubated at 30 °C for 60 minutes. The reaction was stopped with 50 μl of 2 % (v/v) H3PO4, plates were aspirated and washed two times with 200 μl 0.9 % (w/v) NaCl. Kinase activity dependent transfer of 33Pi (counting of “cpm”) was determined with a microplate scintillation counter (Microbeta. Wallac). All assays were performed with a BeckmanCoulter Biomek 2000/SL robotic system. For each kinase, the median value of the cpm of three wells with complete reaction cocktails, but without kinase, was defined as "low control" (n=3). This value reflects unspecific binding of radioactivity to the plate in the absence of protein kinase but in the presence of the substrate. Additionally, for each kinase the median value of the cpm of three other wells with the complete reaction cocktail, but without any compound, was taken as the "high control", i.e. full activity in the absence of any inhibitor (n=3). The difference between high and low control was taken as 100 % activity for each kinase. As part of the data evaluation the low control of each row of a particular plate was subtracted from the high control value as well as from their corresponding 10 "compound values". The residual activity (in %) for each well of each row of a particular plate was calculated by using the following formula: Res. Activity (%) = 100 X [(cpm of compound – low control) / (high control – low control)] Since 10 distinct concentrations of each test compound were tested against each kinase, the evaluation of the raw data resulted in 10 values for residual activities per kinase. Based on each 10 corresponding residual activities, IC50 values were calculated using Prism 5.04 for Windows (Graphpad, San Diego, California, USA; www.graphpad.com). The mathematical model used was "Sigmoidal response (variable slope)“ with parameters "top“ fixed at 100% and "bottom" at 0 %. The in vitro biochemical activity on Vps34 and PI4Kβ were determined using PI as substrate in n-Octyl- Glucoside (OG) luminescence assays based on ATP consumption (KinaseGlo). Inhibitory activity on mTOR was assessed in an antibody-dependent TR-FRET assay to determine the phosphorylation of 4EBP-1 catalyzed by recombinant mTOR. The biochemical activity on DNA-PK was measured by the incorporation of radioactive 33P by DNA-PK into a peptide substrate and was determined by liquid scintillation counting.

Cellular assays for B and T cell activation in vitro [2]
Mouse B cell activation (CD86 expression) and proliferation after B cell receptor stimulation.[2]
CDZ173 was first dissolved and diluted in DMSO followed by a 1:50 dilution in medium. Splenocytes from Balb/c mice were isolated, re-suspended and transferred to 96 well plates (200 μl/well). The diluted compound or solvent were added to the plates (25 μl) and incubated at 37 °C for 1 hour. Then the cultures were stimulated with 25 μl anti-IgM mAb/well (final concentration 30 μg/ml) for 24 hours at 37 °C and stained with anti-mouse CD86-FITC and anti-mouse CD19-PerCP (2 μl of each antibody/well, both Becton Dickinson). CD86 expression on CD19 positive B cells was quantified by flow cytometry. Based on the reduction of CD86 expression IC50 values were determined. To assess effects on proliferation, murine B cells were stimulated via the B cell receptor by anti-IgM antibody in the presence of titrated amounts of compound and proliferation was assessed by incorporation of radioactive 3H-Thymidine over the last 16 hours of an 88 hour incubation period as described (Julius et al 1984).

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Mouse and human mixed lymphocyte reaction (MLR) [2]
The MLR is considered a simple model for allogeneic T cell activation in vitro. For mouse MLR, equal amounts of spleen cells (4 x 105 cells per well) from C57/Bl6 and DBA/2 mice are mixed and incubated with serial dilutions of compound in 200 ml complete RPMI medium in flat bottom tissue culture microtiter plates (Falcon, Becton Dickinson, Basel, Switzerland) at 37oC in 5% CO2. After 4 days, 1 mCi 3H-thymidine (Amersham, UK) was added to each well and incubated for additional 5 hours. Subsequently, cells are harvested with a BetaplateTM 96-well harvester on filter paper and radioactivity measured with a BetaplateTM counter. The degree of inhibition was calculated in percent according to the equation Inhibition [%] = (high control - sample) / high control x 100 and the concentration leading to half-maximal inhibition (IC50 values) was determined.[2]

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Human T cell proliferation[2]
The effect of CDZ173 on human T cell proliferation induced by T cell receptor engagement with or without CD28 co-stimulation was measured as described in brief: Flat bottomed 96 well tissue culture plates were coated with either 5 μg/ml anti-CD3 (OKT3) or 5 μg/ml anti-CD3 (OKT3) and 1 μg/ml anti-CD28 (clone 15E8, Novartis) antibody solutions overnight at 2-8°C. Human PBMCs were isolated as described above and were taken up in tissue culture medium at a cell concentration of 2.5x105 cells/ml. PBMCs were then incubated with titrated amounts of CDZ173 at 37°C, 5%CO2 for 1 h. Antibody solution was flicked off the pre-coated 96 well plates, plates were washed 3 times with 200μl/well PBS and PBS was removed. The cell suspension was then plated out (5x104 cells/well) and incubated for 56 hrs at 37°C, 5% CO2. Cells were then pulsed with 1μCi/well 3HThymidine, incubated for an additional 16-20 hrs at 37°C + 5%CO2 and incorporated radioactivity was measured as described above.

PK/PD study in rats[2]
Animals[2]
PK/PD experiments were performed with adult male Lewis rats (LEW/Han/Hsd, Charles River, Germany, weighing 225-236 g. Studies were performed according to the Swiss federal law for animal protection and approved by the Veterinary Office Basel (BS No. 425). Animals were housed under conventional hygienic conditions (2 animals /cage, temperature 20-24°C, relative humidity minimum 40%, light/dark cycle 12 hrs) and fed a standard diet (NAFAG 890 25W16) and drinking water ad libitum. They were allowed unrestricted access to food and water before and during the experiment.
A suspension vehicle consisting of 0.5% Tween80 (Fluka 93781) and 0.5% Carboxymethyl-cellulose in Water was used. Suspensions for oral administration were prepared by adding the vehicle to the compound and stirring at RT overnight. All solvents and reagents used were of analytical grade. Compound dosing, PK/PD blood collection CDZ173 was administered p.o. in a volume of 4 ml/kg body weight. For blood collection, animals were anaesthetized with isoflurane using a Fluvac airflow system. Whole blood was collected sublingually 24 hours pre-dose and at 1, 2, 4, 6, 8, 10, 12 and 24 hours post-dose. For pharmacodynamic analysis 100 μl rat blood was collected per time point in Eppendorf tubes with 30 IU sodium heparin (5000 I.U./ml). For pharmacokinetic analysis 150 μl rat blood was collected per time point in EDTA coated Eppendorf tubes.

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PK/PD study in monkey[2]
Animals[2]
PK/PD experiments were performed with cynomolgus monkeys all 8-9 years old, weighing 4.4-6.2 kg, captive-bred, from SICONBREC Inc (n=3 non naïve: #5517♀, 5518♀, 5528♂). They showed normal hematology, serum/urine chemistry and were negative for tuberculosis, salmonella/shigella, viral infections (Herpes B, STLV, SIV, SRV type D, Hepatitis B), and ectoand endo-parasites. Animals have access to food and water ad libitum at any time (excepting time in chair and two hours feeding restriction post-dosing) during the course of the study. Each animal is identified by its leg tattoo number or the chest tattoo number assigned by the NHP-team at NIBR-ATI. The bodyweight of the animals was obtained before dosing with test compound. During the 7 hour time course, the monkeys were housed individually in properly marked cages in a room with controlled temperature (22 ± 2ºC) and humidity not less than 40%. Afterwards they returned to the group housing facility housed under natural day-light conditions. They were fed according to the Novartis Standards (Primate Pellets [Kliba Nafag 3446 pellets]), fruits and vegetables) and had free access to water whenever animals were unrestrained. Animal handling, care, drug treatments and blood sampling were performed according to the Swiss Federal Law for animal protection (animal license BS No. 1495).
Drug preparation and administration [2]
CDZ173 was prepared as a suspension one day before dosing the first animal and 2 days before dosing the 2 remaining animals and stored at RT. Some 225.6 mg CDZ173 were dissolved in 42.864 ml 0.5% CMC, 2.256 ml 10% Tween80 in H2O. CDZ173 was administered p.o. at 10 mg/kg body weight. CDZ173 was administered p.o. in a volume of 4 ml/kg body weight. For blood collection, animals were anaesthetized with isoflurane using a Fluvac airflow system. Whole blood was collected from the abdominal aorta using a 10 ml syringe with hypodermic needle pre-coated with sodium heparin. Blood was transferred into 50 ml Falcon tubes and the anticoagulant concentration was adjusted to 100 U/ml final concentrations.

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Pharmacokinetic studies[2]
In vivo pharmacokinetic studies in rats[2]
Female wild-type Sprague Dawley rats (Iffa Credo, France) were kept in standard cages and conditions according to Swiss Animal Welfare guidelines (12h light/dark cycles, RT at 22-24 °C, humidity at least 45 % but <70 %) with free access to Ringer solution (glucose 5%, NaCl 0.9% and KCl 0.5%) and pelleted rodent chow. 96-120 hours before administration of the test substance the animals were anesthetized with isoflurane and catheters were surgically implanted under aseptic precautions (use of sterile instruments and surgical material in combination with local antibiotic prophylaxis) into the femoral artery and vein. The catheters were exteriorized in the neck region, connected to a Harvard swivel system (Harvard Instruments) and filled with 0.9% saline containing 100 U·mL-1 heparin. After recovery from anesthesia the animals were housed individually in special cages with free access to food and tap water until and throughout the experiment. Analgesic treatment with Temgesic (10 μg/kg s.c., application volume 1 mL/kg) was performed in the evening following surgery and in the next morning. Compound administration was in the morning (6-8 AM). Blood samples were collected at various time points from the femoral artery catheter into Eppendorf tubes coated with sodium EDTA. Blood samples were immediately frozen at –20 °C until final processing (maximum storage was 8 days). Intravenous and oral dosing was performed in the same animals after a 48 h wash-out interval between the single dose applications. The test substance was administered intravenously as a solution in 1-methyl-2-pyrrolidone and polyethylene glycol 200 (30:70, v/v) at a dose of 1 mg/kg and orally as a homogenous aqueous suspension in Tween 80 and carboxy methyl cellulose sodium 0.5/0.5/99 (w/w) at a dose of 3 mg/kg.
In vivo pharmacokinetic studies in dogs[2]
Adult male beagle dogs (originating from Marshall Farms at Montichiari, Italy) from the permanent MAP/DMPK stock were kept under standard conditions in dog pens according to Swiss Animal Welfare guidelines (standard dog chow once daily, free access to tap water throughout the study). The dogs were fasted 12 h before dosing and then fed 2 h post dose. Compound administration was in the morning (7-8 AM). Intravenous and oral dosing was performed in the same animals. Washout time was one week between applications. The test substance were administered intravenously as a solution in 1-methyl-2- pyrrolidone and polyethylene glycol 200 (15:85, v/v) at a dose of 0.1 mg/kg and orally as a homogenous aqueous suspension in Tween 80 and carboxy methyl cellulose sodium 0.5/0.5/99 (w/w) at a dose of 0.3 mg/kg.
In vivo pharmacokinetic studies in cynomolgus monkeys [2]
Two male and one female adult cynomolgus monkey from the permanent MAP/DMPK stock were kept under standard conditions according to Swiss Animal Welfare guidelines (standard pelleted monkey chow plus fruits in the morning and vegetables in the afternoon, free access to tap water throughout the study). During the single dose experiments up to the 7 h sampling time, the animals were kept singly in experimental cages. Before and after that, the animals were group-housed in large cages, and singled out short-term to take the remaining blood samples. The monkeys were not fasted before dosing and fed 2 h post dose. Compound administration was in a cassette format with application of a mixture of 2 compounds in the morning. Intravenous and oral dosing was performed in the same animals. Washout time was one week between applications. The test substance were administered intravenously as a solution in 1-methyl-2-pyrrolidone and polyethylene glycol 200 (15:85, v/v) at a dose of 0.1 mg/kg and orally as a homogenous aqueous suspension in Tween 80 and carboxy methyl cellulose sodium 0.5/0.5/99.5 (w/w) at a dose of 0.3 mg/kg.

[1]. Effective 'Activated PI3Kd Syndrome' -targeted therapy with PI3Kd inhibitor leniolisib. The New England journal of medicine: NEJM. ISSN 0028-4793; 1533-4406

[2]. Hoegenauer K, et al. Discovery of CDZ173 (Leniolisib), Representing a Structurally Novel Class of PI3K Delta-Selective Inhibitors. ACS Med Chem Lett. 2017 Aug 25;8(9):975-980.

C21H25F3N6O2

450.47

450.2

C, 55.99; H, 5.59; F, 12.65; N, 18.66; O, 7.10

1354690-24-6

Leniolisib phosphate;1354691-97-6

White to off-white solid powder

2.96

83.48

FC(C1=C(OC)N=CC(N(CCC2=NC=N3)CC2=C3N[C@@H]4CN(C(CC)=O)CC4)=C1)(F)F

MWKYMZXCGYXLPL-ZDUSSCGKSA-N

InChI=1S/C21H25F3N6O2/c1-3-18(31)30-6-4-13(10-30)28-19-15-11-29(7-5-17(15)26-12-27-19)14-8-16(21(22,23)24)20(32-2)25-9-14/h8-9,12-13H,3-7,10-11H2,1-2H3,(H,26,27,28)/t13-/m0/s1

(S)-1-(3-((6-(6-methoxy-5-(trifluoromethyl)pyridin-3-yl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)propan-1-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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.55 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 25.0 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: ≥ 2.5 mg/mL (5.55 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 25.0 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: ≥ 2.5 mg/mL (5.55 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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