RIP1 kinase (IC50 = 29 nM)
GSK-963 targets receptor-interacting protein 1 (RIP1) kinase (IC₅₀ = 1–4 nM for inhibiting RIP1-dependent cell death in human and murine cells; >10,000-fold selective for RIP1 over 339 other human kinases at 10 μM; no measurable activity against indoleamine-2,3-dioxygenase (IDO)) [1]

GSK-963 inhibits RIP1-dependent cell death in human and murine cells with an IC50 range of 1 to 4 nM[1].
GSK′963 is a potent and selective inhibitor of RIP1 kinase in biochemical assays. GSK′963 is a selective and potent inhibitor of necroptosis in murine and human cells in vitro.

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1. GSK-963 exhibits potent binding affinity to the ATP-binding pocket of RIP1 in a fluorescence polarization (FP) binding assay, with significantly higher affinity than Necrostatin-1 (Nec-1); in an ADP-Glo kinase assay, it potently inhibits the autophosphorylation of the RIP1 kinase domain in vitro, and its efficacy is far superior to Nec-1 [1]
2. In cell-based necroptosis assays, GSK-963 inhibits TNF + zVAD-induced necroptosis with an IC₅₀ of 1–4 nM in mouse fibrosarcoma L929 cells, human monocytic U937 cells, primary murine bone marrow-derived macrophages (BMDM), and primary human neutrophils (stimulated with TNF + zVAD + SMAC mimetic); its inactive enantiomer GSK-962 shows no inhibitory effect at the same concentrations [1]
3. GSK-963 (100 nM) has no effect on caspase 3/7 activity in BMDM treated with TNF + cycloheximide (a model of apoptosis), nor does it inhibit TNF-induced IκB phosphorylation (at 5 min) or degradation (at 15 min) in BMDM, indicating that it does not interfere with apoptotic signaling or NF-κB activation [1]
4. GSK-963 shows no inhibitory activity against IDO in an in vitro enzymatic assay (menadione was used as a positive control for IDO inhibition), and it does not block the activity of any of the 339 tested human kinases at a concentration of 10 μM [1]

GSK-963 is a powerful inhibitor of a lethal shock caused by TNF+zVAD. In comparison to Nec-1, GSK-963 would maintain blood concentrations above the level necessary for 90% inhibition of RIP1 activity for a longer period of time at 2 mg/kg.

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GSK′963 is a potent inhibitor of a TNF+zVAD-mediated lethal shock: researchers assessed the pharmacokinetic profiles of GSK′963 and Nec-1 following intraperitoneal (i.p.) administration in vivo. Nec-1 demonstrated an ~10-fold higher exposure compared with GSK′963 at 10 mg/kg, although the apparent half-life of GSK′963 was greater than that for Nec-1 (Figure 3a; Supplementary Figure 1a). However, pharmacodynamic modeling of both compounds based on the mouse pharmacokinetic profiles (Figure 3a; Supplementary Figure 1a) and compound potencies in TNF+zVAD-treated L929 cells (Figure 2a) indicated that at 2 mg/kg, GSK′963 would maintain blood concentrations above the concentration required for 90% inhibition of RIP1 activity for an extended period of time compared with Nec-1 (Figure 3b; Supplementary Figure 1b). To directly test the efficacy of GSK′963 in vivo, we made use an acute model of sterile shock. Administration of TNF+zVAD results in a lethal hypothermia that has previously been shown to be dependent on RIP1 kinase activity.5,18 Treatment of animals with 2 mg/kg of GSK′963 resulted in a complete protection from TNF+zVAD-induced temperature loss, with the 0.2 mg/kg dose also showing a significant response (Figure 3c). As expected, GSK′962 had no effect on the TNF+zVAD-induced shock, confirming that GSK′963 was acting selectively through RIP1 kinase inhibition (Figure 3d). Interestingly, Nec-1 had no effect in the model at 0.2 mg/kg, a dose that is commonly used to inhibit RIP1 in vivo in the literature, and showed a minimal level of protection at a 10-fold higher dose (Figure 3e). Together these results demonstrate that compared with Nec-1, GSK′963 represents a better tool molecule to explore acute RIP1 kinase biology in vivo[1].

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1. In a mouse model of TNF + zVAD-induced sterile shock, intraperitoneal (i.p.) pretreatment with GSK-963 (0.2 mg/kg and 2 mg/kg) 15 minutes prior to intravenous (i.v.) injection of TNF + zVAD significantly protects C57BL/6 mice from hypothermia over a 3-hour monitoring period; this protective effect is dose-dependent and far more potent than that of Nec-1 at matched doses (0.2 mg/kg and 2 mg/kg) [1]
2. The inactive enantiomer of GSK-963, GSK-962, has no protective effect against TNF + zVAD-induced hypothermia even at a high dose of 20 mg/kg (i.p.), confirming the on-target effect of GSK-963 on RIP1 [1]
3. Pharmacokinetic modeling shows that the plasma exposure of GSK-963 after i.p. administration at 10 mg/kg in C57BL/6 mice is sufficient to achieve predicted RIP1 inhibition (based on its potency in L929 cell assays) at doses of 0.2 mg/kg and 2 mg/kg [1]

Compound potency against RIP1 kinase activity was determined using an ADP-Glo luminescence assay, which measures the conversion of ATP to ADP as previously described. In brief, the primary reaction consisted of 10 nM GST-RIPK1 (1–375) and 50 μM ATP in 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.5 mg/ml BSA, and 0.02% CHAPS. Five microliter of enzyme and 5 μl of ATP were added to the plate at twice the final assay concentration and incubated at room temperature for 4 h. The luminescence was measured on a plate reader. Test compound inhibition was expressed as percent inhibition of internal assay controls.

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Fluorescent polarization (FP) binding assay[1]
A FP-based binding assay was developed to quantitate interaction of novel test compounds at the ATP-binding pocket of RIP1, by competition with a fluorescently labeled ATP-competitive ligand, as previously described.18 In brief, GST-RipK1 (1–375) was purified and was used at a final assay concentration of 10 nM. A fluorescent-labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo [2“,3“]indolizino[8“,7“:5ʹ,6ʹ]pyrano[3ʹ,2ʹ:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate was used at a final assay concentration of 5 nM. Samples were read on an Analyst multimode reader. Test compound inhibition was expressed as percent (%) inhibition of internal assay controls.

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ADP-Glo kinase assay[1]
Compound potency against RIP1 kinase activity was determined using an ADP-Glo luminescence assay, which measures the conversion of ATP to ADP as previously described.18 In brief, the primary reaction consisted of 10 nM GST-RIPK1 (1–375) and 50 μM ATP in 50 mM HEPES pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.5 mg/ml BSA, and 0.02% CHAPS. Five microliter of enzyme and 5 μl of ATP were added to the plate at twice the final assay concentration and incubated at room temperature for 4 h. The luminescence was measured on a plate reader. Test compound inhibition was expressed as percent inhibition of internal assay controls.

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Kinase selectivity[1]
GSK′963 was tested against 339 kinases using a P33-radiolabeled assay at Reaction Corp Biology (http://www.reactionbiology.com). The compound was tested at a single dose in duplicate at 10 μM. Reactions were carried out at 10 μM ATP. Data are reported as % enzyme activity (relative to DMSO controls). The full data set is shown in the Supplementary Table I.

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IDO enzymatic assay[1]
IDO activity was determined as described by Takahashi et al.25 using recombinant human IDO purchased from R&D Systems. The yellow pigment derived from kynurenine was measured at 490 nm using the Spectramax microplate reader.

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Caspase 3/7 assay[1]
To induce apoptosis, BMDM pretreated with GSK′963 (100 nM), GSK′962 (100 nM) or Nec-1 (10 μM) for 30 min were stimulated with TNF (50 ng/ml) and CHX (12 μg/ml). Caspase 3/7 activity was measured at 6 h using the Caspase-Glo 3/7 assay.

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1. Fluorescence Polarization (FP) Binding Assay: Recombinant RIP1 protein was incubated with serial concentrations of GSK-963, GSK-962, and Nec-1 to evaluate their binding affinity to the ATP-binding pocket of RIP1. Fluorescence polarization signals were measured, dose-response curves were generated (n=7 for GSK-963, n=4 for GSK-962, n=52 for Nec-1), and the binding potency of each compound was compared [1]
2. ADP-Glo Kinase Assay: The recombinant RIP1 kinase domain was incubated with different concentrations of GSK-963, GSK-962, or Nec-1 in the presence of ATP to assess the inhibition of RIP1 autophosphorylation. After incubation, ADP-Glo reagent was added to detect the amount of ADP produced (a marker of kinase activity), dose-response curves were plotted (n=2 for GSK-963, n=2 for GSK-962, n=20 for Nec-1), and the IC₅₀ values for RIP1 kinase inhibition were calculated [1]
3. Kinase Selectivity Assay: GSK-963 was tested at a concentration of 10 μM against a panel of 339 human kinases. The activity of each kinase was measured, and the inhibition rate was calculated; the results showed that GSK-963 did not inhibit the activity of any of these kinases [1]
4. IDO Enzymatic Activity Assay: GSK-963, GSK-962, Nec-1, and menadione (positive control) were incubated with IDO enzyme and its substrate in an in vitro system. The enzymatic activity of IDO was measured, and the results confirmed that GSK-963 had no measurable IDO inhibitory activity (data from three independent experiments) [1]

For immunoblot analysis, BMDM are stimulated with 50 ng/ml TNF for 5 and 15 minutes after being pretreated for 30 minutes with GSK'963 (100 nM), GSK'962 (100 nM), or Nec-1 (10 μM). Lysates made with protease and phosphatase inhibitors in 1× Cell Lysis Buffer are separated on 4-12% SDS-PAGE and blotted onto nitrocellulose membrane. IκB, phospho-IκB, and tubulin are probed as loading controls on blots.

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Cell culture[1]
Mouse fibrosarcoma L929 cells (ATCC# CCL-1) and human monocytic U937 cells were cultured in RPMI media supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 U/ml streptomycin. BMDM were prepared from C57BL/6 mice by differentiation with 10 ng/ml M-CSF for 7 days and cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin. Primary human neutrophils were isolated from human blood following the standard method comprised of sequential sedimentation in dextran, density centrifugation in Ficoll–Hypaque and lysis of contaminating red blood cells.

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Cell-based assays[1]
Necroptotic cell death was induced in BMDM, L929 and U937 cells with TNF in the presence of caspase inhibitor zVAD-FMK or QVD-Oph (BMDM: 50 ng/ml TNF+50 μM zVAD; L929: 100 ng/ml TNF+50 μM QVD; U937: 100 ng/ml TNF+25 μM QVD). To evaluate the effect of RIP1 inhibitors, cells were pretreated with compound (dose–response) for 30 min. Induced cell death was evaluated 19–21 h later by measuring cellular ATP levels using CellTiter-Glo Luminescent Cell Viability assay. To induce necroptosis in neutrophils, freshly isolated human neutrophils were stimulated with TNF (10 ng/ml), QVD-Oph (50 μM) and SMAC mimetic (100 nM). Induced cell death was evaluated as above.

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1. Necroptosis Cell Viability Assay (CellTiter-Glo): Mouse fibrosarcoma L929 cells, human monocytic U937 cells, primary murine BMDM, and primary human neutrophils were seeded in plates and treated with serial concentrations of GSK-963, GSK-962, or Nec-1. Necroptosis was induced by TNF + zVAD (TNF + zVAD + SMAC mimetic for human neutrophils). After incubation, CellTiter-Glo reagent was added to measure cell viability, dose-response curves were generated (combined data from at least three independent experiments), and the IC₅₀ values for necroptosis inhibition were calculated [1]
2. Caspase 3/7 Activity Assay: Primary murine BMDM were treated with TNF + cycloheximide to induce apoptosis, and GSK-963 (100 nM), GSK-962 (100 nM), or Nec-1 (10 μM) was added simultaneously. At 3 hours post-treatment, Caspase-Glo 3/7 reagent was used to detect caspase 3/7 activity; cell viability was measured by CellTiter-Glo assay at 20 hours. The experiment was repeated four times independently, and the results showed no effect of GSK-963 on apoptotic signaling [1]
3. Western Blot for IκB Phosphorylation and Degradation: Primary murine BMDM were pretreated with GSK-963 (100 nM), GSK-962 (100 nM), or Nec-1 (10 μM) and then stimulated with TNF. Cell lysates were collected at 5 min (for IκB phosphorylation) and 15 min (for IκB degradation). Western blot was performed using antibodies against phosphorylated IκB, total IκB, and tubulin (loading control). The experiment was repeated with cells from four different animals, and the results confirmed that GSK-963 did not affect TNF-induced NF-κB signaling [1]

C57BL/6 mice were pretreated i.p. with Nec-1 (0.2 and 2 mg/kg), GSK′963 (0.2 and 2 mg/kg) or GSK′962 (20 mg/kg) 15 min prior to i.v. injection of TNF (1.25 mg/kg) and zVAD-FMK (16.7 mg/kg).
C57BL/6 mice Pharmacokinetic experiments[1]
Briefly, pharmacokinetic studies were conducted using n=3 animals per group for each compound. Animals received either GSK′963 or 7-Cl-O-Nec-1 as an i.p. injection prepared as aqueous solutions in 6% 11-b-hydroxypropyl cyclodextrin and contained 5% DMSO. Blood samples were obtained at various time points following intraperitoneal injection and diluted 1 : 1 with water prior to storage at −80 °C.
Prior to bioanalysis, samples were thawed and each analyte was isolated using a method based on protein precipitation. The resulting supernatant was injected into an LC/MS/MS system optimized for detection of the compound of interest. Data were reported as quantitative drug concentrations as determined by standard calibration curve analysis. Using these optimized conditions, the typical lower limit of quantitation achieved was 1.00 ng/ml for both compounds.

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TNF+zVAD-induced shock model[1]
C57BL/6 mice were pretreated i.p. with Nec-1 (0.2 and 2 mg/kg), GSK′963 (0.2 and 2 mg/kg) or GSK′962 (20 mg/kg) 15 min prior to i.v. injection of TNF (1.25 mg/kg) and zVAD-FMK (16.7 mg/kg). Temperature was monitored over the course of 3 h by rectal probe.

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1. Pharmacokinetic (PK) Study: C57BL/6 mice were administered GSK-963 via intraperitoneal injection at a dose of 10 mg/kg. Blood samples were collected at predetermined time points, plasma was isolated, and the concentration of GSK-963 in plasma was measured. The PK profile was generated from the combined results of three independent animals, and the predicted % inhibition of RIP1 was modeled based on its potency in L929 cell assays [1]
2. TNF + zVAD-Induced Sterile Shock Model: C57BL/6 mice were randomly divided into groups (seven animals per group). Mice were pretreated with GSK-963 (0.2 mg/kg or 2 mg/kg, i.p.), GSK-962 (20 mg/kg, i.p.), Nec-1 (0.2 mg/kg or 2 mg/kg, i.p.), or vehicle (control) 15 minutes before intravenous injection of TNF + zVAD. Rectal temperature was monitored every 30 minutes for 3 hours using a rectal probe. The experiment was repeated three times independently, and the temperature change curves for each group were plotted [1]

Next, the researchers evaluated the pharmacokinetic characteristics of GSK′963 and Nec-1 in vivo by intraperitoneal injection (ip). At a dose of 10 mg/kg, the exposure of Nec-1 was approximately 10-fold higher than that of GSK′963, despite the longer apparent half-life of GSK′963 than that of Nec-1 (Fig. 3a; Supplementary Fig. 1a). However, a pharmacodynamic model based on mouse pharmacokinetic characteristics (Fig. 3a; Supplementary Fig. 1a) and the efficacy of the compound in TNF+zVAD-treated L929 cells (Fig. 2a) showed that at a dose of 2 mg/kg, GSK′963 was able to maintain plasma concentrations above the concentration required to inhibit 90% of RIP1 activity for a longer period of time compared to Nec-1 (Fig. 3b; Supplementary Fig. 1b). [1]

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1. GSK-963 was administered intraperitoneally at a dose of 10 mg/kg to C57BL/6 mice, and plasma concentrations of the compound were measured at different time points to generate pharmacokinetic curves. [1]
2. Based on the observed pharmacokinetic curves of GSK-963 and its efficacy in inhibiting TNF+zVAD-induced necrosis and apoptosis in L929 cells, the predicted inhibition rates of RIP1 at doses of 0.2 mg/kg and 2 mg/kg were calculated, confirming that these doses can effectively inhibit RIP1 in vivo. [1]

[1]. Characterization of GSK'963: a structurally distinct, potent and selective inhibitor of RIP1 kinase. Cell Death Discov. 2015 Jul 27;1:15009.

Necrostatin-1 (Nec-1), apoptosis, and its signaling pathway regulated by RIP1 kinase activity are increasingly becoming key drivers of inflammation in various diseases. Through the use of the RIP1 kinase inhibitor Necrostatin-1 (Nec-1), a deeper understanding has been gained of how RIP1 regulates necrotic cell death. Nec-1 has been a transformative tool for exploring the function of RIP1 kinase activity; however, its application is limited by factors such as moderate potency, off-target activity against indoleamine-2,3-dioxygenase (IDO), and poor pharmacokinetic properties. These limitations of Nec-1 have spurred efforts to find next-generation tools for studying RIP1 function, ultimately leading to the discovery of 7-Cl-O-Nec-1 (Nec-1s). Nec-1s exhibit superior pharmacokinetic properties and do not inhibit IDO. This article describes the properties of GSK′963, a chiral small-molecule RIP1 kinase inhibitor with a chemical structure distinct from both Nec-1 and Nec-1s. In biochemical and cellular experiments, GSK′963 demonstrated significantly higher potency than Nec-1, with IC50 values for inhibiting RIP1-dependent cell death in human and murine cells ranging from 1 to 4 nM. GSK′963 exhibited over 10,000-fold higher selectivity for RIP1 compared to 339 other kinases, showed no significant activity against IDO, and possesses an inactive enantiomer, GSK′962, which can be used to validate its targeting effects. The enhanced in vitro activity of GSK′963 was also demonstrated in vivo; in a TNF-induced aseptic shock model, the same dose of GSK′963 provided significantly better protection against hypothermia than Nec-1. We believe that GSK′963 represents a next-generation tool for studying RIP1 function in vitro and in vivo, and will contribute to elucidating our current understanding of the role of RIP1 in disease pathogenesis. [1]

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In this study, we characterized GSK′963, a novel small-molecule inhibitor of RIP1 kinase with a structure distinct from Nec-1 and Nec-1s. GSK′963 is more than 200 times more potent than Nec-1, exhibits extremely high selectivity for RIP1 kinase activity, and has no effect on IDO activity, TNF-mediated NFκB activation, or apoptosis. As a chiral molecule, GSK′963 can be used as a negative control with its chemically identical inactive enantiomer to validate the inhibitor's targeting activity. Furthermore, in a TNF-induced aseptic shock model in vivo, GSK′963 provided complete protection at doses where Nec-1 did not show significant protective effects in this model. In conclusion, we believe that GSK′963 represents another new generation of tools for exploring the biological effects mediated by RIP1 kinase. [1]

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In summary, we have discovered a novel, highly effective, and selective inhibitor of RIP1 kinase activity. We believe that GSK′963 represents a new generation of tool inhibitors for in vitro studies of RIP1 biology, with significant advantages over commercially available necrosis inhibitors.

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1. GSK-963 is a chiral small molecule RIP1 kinase inhibitor with a chemical structure different from Nec-1 and Nec-1s (7-Cl-O-Nec-1); it overcomes the limitations of Nec-1 (moderate potency, off-target IDO inhibition, poor pharmacokinetic properties)[1]
2. GSK-963 has an inactive enantiomer (GSK-962), which can be used as a negative control to verify the targeting effect of RIP1 inhibition in in vitro and in vivo experiments[1]
3. Necrotic apoptosis and RIP1 kinase-mediated signal transduction are key drivers of inflammation in various disease conditions; GSK-963 is a new generation of tool compound that, due to its high efficiency, high selectivity and lack of off-target activity, can be used to study the role of RIP1 in the pathogenesis of diseases[1]
4. GSK-963 specifically inhibits RIP1-dependent necrotizing apoptosis without affecting apoptosis signaling pathways (caspase 3/7 activity) or NF-κB activation (IκB phosphorylation/degradation), indicating that it has target specificity for RIP1 kinase [1].

C14H18N2O

230.31

230.141

C, 73.01; H, 7.88; N, 12.16; O, 6.95

2049868-46-2

GSK962;2049872-86-6

122703613

Light yellow to yellow solid powder

1.06±0.1 g/cm3

342.8±45.0 °C

2.1

0

2

2

17

311

1

O=C(C(C)(C)C)N1C(C2C=CC=CC=2)CC=N1

NJQVSLWJBLPTMD-LBPRGKRZSA-N

InChI=1S/C14H18N2O/c1-14(2,3)13(17)16-12(9-10-15-16)11-7-5-4-6-8-11/h4-8,10,12H,9H2,1-3H3/t12-/m0/s1

2,2-dimethyl-1-[(3S)-3-phenyl-3,4-dihydropyrazol-2-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 (10.85 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 2: ≥ 2.5 mg/mL (10.85 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.)