PARP1 ( IC50 = 3 nM ); PARP2 ( IC50 = 1400 nM )

AZD5305 has excellent pharmacokinetics in preclinical species, good secondary pharmacology, good physicochemical properties, and is a highly selective inhibitor for PARP1 over other PARP family members. In vitro, AZD5305 lessens the anti-proliferation effects on human bone marrow progenitor cells.

AZD5305 exhibits excellent in vivo efficacy as a potent and selective PARP1 inhibitor and PARP1-DNA trapper in a BRCA mutant HBCx-17 PDX model.[2]

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AZD5305 demonstrates sustained antitumor activity in BRCAm xenograft and PDX models in vivo. Antitumor efficacy of AZD5305 correlates with PK and PD in BRCA1m tumor models. AZD5305 and carboplatin show combination benefit in HBCx-9 PDX and SUM149PT xenograft model. AZD5305 demonstrates reduced hematologic toxicity when compared with dual PARP1/2 inhibitors in rat preclinical models. [3]

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AZD5305 demonstrates sustained antitumor activity in BRCAm xenograft and PDX models in vivo[3]
The antitumor efficacy of Saruparib (AZD5305) monotherapy was evaluated in vivo in BRCAm xenografts and PDX models. In the MDA-MB-436 BRCA1m TNBC model, daily treatment with ≥ 0.1 mg/kg of Saruparib (AZD5305) resulted in profound regressions (≥90%). Further reductions of AZD5305 dose levels to 0.03 mg/kg and 0.01 mg/kg resulted in diminished antitumor efficacy (40% regression and not efficacious, respectively; Fig. 3A). In Capan-1 xenografts, a BRCA2m pancreatic model, daily treatment with 1 or 10 mg/kg of Saruparib (AZD5305) resulted in tumor stasis, while a 0.1 mg/kg oral daily dose led to 52% tumor growth inhibition[3].

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With suitable oral exposure in the mouse, compound 25/Saruparib (AZD5305) was tested for efficacy in vivo in a BRCA1m triple-negative breast cancer (TNBC) patient-derived explant (PDX) model, HBCx-17 (Xentech France) (Figure 4). Tumor fragments were implanted subcutaneously into mice, and the mice were randomized into treatment groups when the mean tumor volume reached approximately 0.1 cm3. Animals were treated daily for 5 weeks from the day after the randomization with the vehicle or compound 1 at 100 mg/kg or compound 25 at 10, 1, 0.1, and 0.03 mg/kg. Compound 25 dosed daily at 10 mg/kg and 1 mg/kg delivered over 80% tumor regression, which was comparable to the effect of compound 1 at 100 mg/kg, while 0.1 mg/kg of compound 25 resulted in 73% regression. Compound 25 dosed as low as 0.03 mg/kg also resulted in tumor regressions (25%)[2].

Compound coupling and competition affinity pulldowns [2]
PARP affinity probe was immobilized on sepharose beads through covalent linkage using primary amino compound 101 (see synthetic experimental section for preparation) and carboxyl groups as described previously.10 One mL of NHS-activated sepharose and compound 101 (2μmol/mL) were equilibrated in DMSO. 15 μL of triethylamine was added to start the coupling reaction and the mixture was incubated on an end-over-end shaker for 20 h in the dark. Free NHS-groups on the beads were blocked by adding 50 μL amino ethanol and incubation on an end-over-end shaker for 16-20 h in the dark. Coupled beads were washed and stored in isopropanol at 4 °C in the dark. Competition affinity pulldowns were performed as described previously10. Briefly, MDA-MB-436 cells were lysed in 0.8 % NP40, 50 mM Tris-HCl pH 7.5, 5 % glycerol, 1.5 mM MgCl2,150 mM NaCl, 1 mM Na3VO4, 25 mM NaF, 1 mM DTT, HALT protease inhibitor cocktail. Lysate (5 mg total protein each) was pre-incubated with increasing competitor (test compounds 1-5 or Saruparib (AZD5305)) concentrations (DMSO vehicle, 0.64 nM, 3.2 nM, 16 nM, 80 nM, 400 nM, 2 µM, 10 µM and 50 µM) on an end-over-end shaker for 45 min at 4°C. Subsequently, lysates were incubated with beads for 30 min at 4°C. The beads were washed and collected by centrifugation. To assess the degree of protein depletion from the lysates, a second pulldown was performed with fresh beads and the flow through of the DMSO control.10 Bound proteins were subjected to on-bead digestion for 12h at 37°C with trypsin (2ug) in the presence of 50mM tetraethylammonium tetrahydroborate (TEAB). Peptides were labeled with TMT 10-plex, combined into a single sample and then subjected to offline fractionation by high pH reverse-phase HPLC (Agilent 1200) on an Gemini C18 column (5 uM, 110 Å, 150 x 2 mm; Phenomenex) with 20mM ammonium hydroxide in water as mobile phase A and 20 mM ammonium hydroxide in acetonitrile. The 96 resulting fractions were pooled in a non-continuous manner into 8 fractions for subsequent mass spectrometry analysis.

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Binding assay using TIRF microscopy (M&M) [2]
For surface-immobilization, full-length PARP1 and PARP2 was modified by chemical biotinylation. In brief, protein was incubated with NHS-PEG12-biotin in a 1:4 ratio for 4h. Residual reagent was removed by SEC. Microscopy-compatible 384-wells glass plates was cleaned using a S21 1:1:5 mixture of NH4:H2O2:H2O and heated to ~80 °C, before extensively washed with MQ. The surface was functionalized with PLL-g-PEG-biotin (SuSoS) at 10 ug/mL (overnight incubation) to eliminate unspecific binding of protein and probe before subsequent addition of pre-mixed biotinylated PARP1/2 and Neutravidin in a 1:1 ratio at 100 nM (>2h incubation). For affinity determination, dilution series (3-fold) of all compounds were mixed with Alexa647-probe (in-house custom synthesis) at 2.5 nM (final concentration) and added to the wells. Due to the slow binding kinetics and high potency of compounds, all samples were incubated for >4h to reach equilibrium. Dilution series (3-fold) of probe only was used to determine Kd of probe to enable Ki calculations. For determination of residence time, 500 nM of compound was added to each well and allowed to equilibrate before extensive washing using plate washer (BioTek) and addition of probe. The plate was rapidly transferred to the TIRF microscope for time-lapse imaging with an acquisition rate of 5 min/frame to minimize bleaching. Samples were imaged using a 60x oil immersion objective (NA= 1.49) mounted on a Ti Eclipse inverted microscope, equipped with a Cy5 filter cube, CoolLED pE4000 illumination system (635 nm illumination) and Orca Flash 4.0 CMOS camera. Multiple images (200×200 µm2 ) were acquired per sample using identical illumination (100 ms exposure time) and focus (via perfect focus system). Quantification of image intensity was used to determine steady state binding levels, which were fitted to a 1:1 binding model to determine IC50.

BRCA2 and DLD-1(^-/-) Using a Multidrop Combi, 40 μL of DLD-1 cells per well are seeded into 384-well plates at a density of 5000 cells/mL and 2.5 × 104 cells/mL, respectively, in complete media. The plates are then incubated for an overnight period at 37 °C with 5% CO2. Day 0 plate was incubated for more than three hours at room temperature (RT) after sytox green (5 μL, 2 μM) and saponin (10 μL, 0.25% stock) were added using a Multidrop Combi. The plate was then sealed with a black adhesive lid. Cell Insight Focused with a 4× objective is used to image cells. Using an Echo 555, AZD5305 is added, and the mixture is then incubated for seven days at 37 °C with 5% CO2. Day 8: Plates are filled with sytox green (5 μL, 2 μM) and saponin (10 μL, 0.25% stock). A black adhesive lid is used to seal the plate, and it is incubated for more than three hours at room temperature. On a 4× Objective Cell Insight, every cell is read.

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Hematotoxicity Assay[1]
All inhibitors were diluted in DMSO to a concentration of 3 mM (talazoparib) or 10 mM (compound 25/Saruparib (AZD5305)) and stored under nitrogen. CD34+ hematopoietic stem and progenitor cells were cultured overnight in StemSpan SFEM II media supplemented with 25 ng/mL SCF, 50 ng/mL TPO, and 50 ng/ml Flt3-L at 37 °C, 5% CO2. The following day, cells were resuspended in CellExpand Suspension Expansion Culture BFU, plated at 500 cells per well in 96-well plates, and technical replicates were treated with a vehicle or a concentration range of the compound. After 5 days of incubation, the number of viable cells per well was determined using CellTiter-Glo 2.0 with the luminescence readout being performed on an Envision plate reader. Data were normalized to vehicle controls and dose–response curves generated using non-linear regression (curve fit) analysis using GraphPad Prism (v9).

Animals were treated from the day after the randomization. Saruparib (AZD5305) and olaparib (AZD2281) were administered by oral gavage once daily (QD) at 10 mL/kg final dose volume. Olaparib was formulated in 10% DMSO (Sigma), 30% Kleptose. Saruparib (AZD5305) was formulated in water/HCl pH 3.5–4. Carboplatin was prepared fresh on the day of dosing and administered intraperitoneally at 10 mL/kg final dose volume. Carboplatin was formulated in 0.85% physiologic saline. For Xentech studies carboplatin stock (Teva or Sandoz) was diluted to the final concentration in 0.9% NaCl.[3]

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During monotherapy studies, rats received either 1 mg/kg Saruparib (AZD5305) or vehicle [0.5% w/v hydroxypropyl methylcellulose (HPMC)/0.1% Tween80 in water, adjusted for pH 3–3.2], 57 mg/kg niraparib or vehicle (0.5% w/v HPMC) or 100 mg/kg olaparib (co-administered with 0.5% w/v HPMC, 0.1% Tween 80 because this was a monotherapy arm from a combination study), or vehicle (0.5% w/v HPMC/0.1% Tween 80 in water) orally, daily for 14 days.
In combination studies, rats were dosed with 1 mg/kg Saruparib (AZD5305), 100 mg/kg olaparib, or vehicle daily. In the 14-day study, rats were dosed in combination with a single administration of 30 mg/kg carboplatin or vehicle (0.9% w/v physiologic saline) on day 1. In the 42-day two-cycle study, rats were dosed in combination with 40 mg/kg carboplatin or vehicle on day 1 and day 22. Carboplatin or vehicle control were delivered via a single slow (over 1 min) intravenous bolus injection into the lateral tail vein followed immediately with either vehicle control or test article.[3]

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For toxicokinetic analysis of Saruparib (AZD5305), niraparib, and olaparib, and hematology analyses, blood samples were collected via tail vein prick at various time points. All animals were monitored throughout the study and sacrificed on day 15 or day 42, approximately 24 hours after administration of the final dose. Bone marrow (left femurs) was fixed in 10% neutral buffered formalin prior to processing, then sectioned and stained with hematoxylin and eosin (H&E) for histopathologic evaluation. Right femurs were processed for bone marrow progenitor cell population analysis by flow cytometry.[3]

To correlate the antitumor efficacy of Saruparib (AZD5305) with its steady-state pharmacokinetics (PK) in the MDA-MB-436 model (Figure 3A), plasma samples were collected at different time points after administration on day 7. In the maximum efficacy dose group (≥0.1 mg/kg, tumor regression rate >90%; Figure 3A), the free plasma concentration of Saruparib (AZD5305) was higher than the IC95 (0.0064 μmol/L) of DLD-1 BRCA2−/− cell colony formation assay over the 24-hour dosing interval (Figure 4B). In contrast, in the 0.03 mg/kg group where a 40% regression rate was achieved, the concentration of Saruparib (AZD5305) in free plasma was above IC95 only for about 17 hours; while in the ineffective group (0.01 mg/kg), the concentration of Saruparib (AZD5305) in free plasma did not reach IC95. These data are consistent with the requirement of maintaining high and sustained target binding to achieve maximum efficacy. [2]

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The in vivo preclinical pharmacokinetic characteristics of compound 25 [Saruparib (AZD5305)] have been determined in mice, rats, dogs and cynomolgus monkeys (Table 7). Compound 25 exhibited extremely low plasma clearance (CLp) in mice, rats, dogs, and monkeys, at 0.23, 1.1, 0.33, and 0.84 mL/min/kg, respectively, and generally low steady-state distribution volume (Vss), at 0.17, 0.38, 0.30, and 0.38 L/kg, respectively. The corresponding plasma half-lives were 8.0, 4.6, 10, and 7.1 hours, respectively. Compound 25 showed high oral bioavailability in all species, consistent with its low hepatic clearance and high absorption fraction. With a free clearance of ≤34 mL/min/kg, combined with its high activity, a low effective dose in vivo is expected, while based on the similarly low in vitro CLint value, the drug is predicted to be effective in humans as well. [1]
The metabolic clearance pathway of compound 25 was studied in vitro in hepatocytes of preclinical animals and humans. Consistent with the low hepatocyte CLint values, the production of metabolites was limited; however, several metabolites were observed, primarily from the successive oxidation of methylcarboxamides, leading to the loss of the methyl group and subsequent further metabolism to carboxylic acids. In addition, other unlocalized oxidation reactions were observed (see Supplementary Information for details). [1]

Since myelotoxicity and peripheral blood adverse reactions are common clinical side effects of PARP1/2 dual inhibitors, we sought to understand the potential hematologic toxicity of compound 25 [Saruparib (AZD5305)] as early as possible. To assess this, we employed an in vitro hematologic toxicity assay to compare the effects of compound 25 [Saruparib (AZD5305)] and compound 4 on the viability of proliferating and differentiating hematopoietic stem/progenitor cells (HSPCs) after 5 days of treatment. Compound 4 showed a dose-dependent decrease in HSPC viability, with an initial concentration of 14 nM, an IC50 of 27 nM, and a maximum effect of 1% viability reduction (Figure 5, red data). In contrast, although compound 25 caused a decrease in cell viability at concentrations as low as 3 nM, no dose-dependent effect was observed, and therefore the IC50 value could not be determined. At a concentration of 100 nM, 46% of cells remained viable (compared to only 10% for compound 4), and at concentrations up to 10 μM, cell viability showed almost no further decrease, with a maximum viability of 38%. Therefore, these data suggest that compound 25 is less toxic to cultured human hematopoietic stem cells than compound 4. [2]

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Saruparib (AZD5305) showed lower hematologic toxicity compared to dual PARP1/2 inhibitors in a rat preclinical model. [3]
To determine whether the PARP1 selectivity of Saruparib (AZD5305) could alleviate the hematologic toxicity of dual PARP1/2 inhibitors observed in a rat preclinical model, we conducted a rat comparative study (16, 17). Saruparib (AZD5305) and clinical non-PARP1 selective PARP inhibitors were administered once daily for 14 days: AZD5305 1 mg/kg once daily; olaparib 100 mg/kg once daily; niraparib 57 mg/kg once daily. Exposure to AZD5305 and olaparib was designed to cover the IC95 values of their respective cells for approximately 24 hours (Fig. 5A; dashed lines indicate IC95). The free AUC (0–24) generated by the 57 mg/kg niraparib dose was 13.7 μmol/L/hour, which is generally consistent with the reported clinical free AUC of 11.4 μmol/L/hour (18) generated by the 300 mg clinical dose. Serial peripheral blood hematologic analysis was subsequently performed (Fig. 5B; Supplementary Figs. S5A–S5C), and bone marrow lineage precursor cells at the end of the experiment were directly assessed by flow cytometry (Fig. 5C; Supplementary Fig. S5D). Consistent with reported clinical anemia outcomes following olaparib treatment (14, 19, 20), we observed a persistent decrease in the number of reticulocytes (immature erythrocytes) in peripheral blood compared to the control group, although changes in total erythrocyte count and hemoglobin levels only began to appear in this 14-day study (Fig. 5B; Supplementary Fig. S5A). No significant changes were observed in other blood cell lineages (Supplementary Fig. S5A). Consistent with the changes observed in peripheral blood, flow cytometry analysis of bone marrow lineage precursor cells showed a decrease in the number of erythroid precursor cells, while myeloid and platelet precursor cells were unaffected (Fig. 5C; Supplementary Fig. S5D).

[1]. Cancer J . 2021 Nov-Dec;27(6):521-528.

[2]. J Med Chem . 2021 Oct 14;64(19):14498-14512.

[3]. Clin Cancer Res . 2022 Nov 1;28(21):4724-4736.

Saluparib is an orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor with potential chemosensitizing/radiosensitizing and antitumor activity. After administration, saruparib selectively targets and binds to PARP, blocking PARP-mediated single-strand DNA break repair (via the base excision repair pathway). This enhances the accumulation of DNA strand breaks, promotes genomic instability, and ultimately leads to apoptosis. This may enhance the cytotoxicity of DNA-damaging drugs. PARP catalyzes post-translational ADP-ribosylation modification of nucleoproteins, which signal and recruit other proteins to repair damaged DNA; single-strand DNA breaks activate PARP. The PARP-mediated repair pathway is aberrantly regulated in multiple cancer cell types. Poly(ADP-ribose) polymerase (PARP) inhibitors have revolutionized the treatment landscape for advanced ovarian cancer and expanded treatment options for other tumor types, including breast, pancreatic, and prostate cancer. However, despite the success of PARP inhibitors in current treatment modalities, not all patients benefit from them due to primary resistance and the potential for disease progression through various acquired resistance mechanisms. Furthermore, the toxicity of PARP inhibitors, primarily myelosuppression, leads to adverse reactions in some patients undergoing monotherapy and limits their use in certain appropriate combination therapies, such as chemotherapy and targeted therapy. Currently approved PARP inhibitors have roughly equivalent efficacy against PARP1 and PARP2 enzymes. This article reviews the development progress of next-generation selective PARP1 inhibitors that have entered Phase I clinical trials. These inhibitors have demonstrated higher PARP1 inhibitory efficacy and extremely high PARP1 selectivity in preclinical studies—characteristics that promise to improve clinical efficacy and broaden the therapeutic window. First-in-human clinical trials have been initiated to determine the safety, tolerability, recommended Phase II dose, and antitumor activity of these novel drugs. If successful, next-generation selective PARP1 drugs are expected to further expand the efficacy of cancer treatment beyond existing PARP inhibitor modalities. [1]

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Poly(ADP-ribose) polymerase (PARP) inhibitors have been approved for the treatment of homologous recombination repair deficient tumors, including those with BRCA mutations. However, some PARP inhibitors are not effective when used in combination with first-line chemotherapy, usually due to overlapping hematologic toxicities. Currently approved PARP inhibitors have lower selectivity for PARP1 than PARP2 and 16 other members of the PARP family, which we speculate may be one of the reasons for toxicity. Recent literature suggests that PARP1 inhibition and PARP1-DNA capture are key to efficacy in the context of BRCA mutations. This article describes the structure and properties of compound 25 (AZD5305), a highly potent and selective PARP1 inhibitor and PARP1-DNA capture agent that has shown excellent in vivo efficacy in a BRCA-mutated HBCx-17 PDX model. Compound 25 has much higher selectivity for PARP1 than other members of the PARP family, good secondary pharmacology and physicochemical properties, excellent pharmacokinetic characteristics in preclinical animal models, and minimal impact on cultured human bone marrow progenitor cells in vitro. [2] Objective: We hypothesized that inhibition and capture of PARP1 alone would achieve antitumor activity. In particular, we aimed to achieve selectivity for PARP2, as PARP2 has been shown to play an important role in the survival of hematopoietic/stem cell progenitor cells in animal models. We developed AZD5305 with the aim of improving clinical efficacy and broadening the therapeutic window. This next-generation PARP inhibitor (PARPi) is expected to revolutionize the clinical efficacy of first-generation PARPPi, especially in combination therapy. Experimental Design: The PARylation inhibition, PARP-DNA capture, and antiproliferative capacity of AZD5305 were tested in vitro. Its in vivo efficacy was evaluated in mouse xenograft and PDX models. Its hematologic toxicity was evaluated in rat models, including monotherapy and combination therapy. Results: AZD5305 is a highly potent and selective PARP1 inhibitor, with 500-fold higher selectivity for PARP1 than for PARP2. AZD5305 inhibits the growth of DNA repair-deficient cells with minimal or no effect on other cells. Unlike first-generation PARP inhibitors, AZD5305 had minimal effect on hematological parameters in preclinical rat models at the predicted clinically effective dose. Animal models treated with ≥0.1 mg/kg of AZD5305 once daily showed greater tumor regression and longer duration of remission than animal models treated with 100 mg/kg of olaparib once daily. Conclusion: AZD5305 can efficiently and selectively inhibit PARP1, thereby producing excellent antiproliferative activity and unprecedented selectivity for DNA repair-deficient cells and cells with normal DNA repair function. These data confirm the hypothesis that targeting PARP1 alone can retain the therapeutic advantages of non-selective PARP inhibitors while reducing the risk of hematologic toxicity. AZD5305 is currently undergoing a phase I clinical trial [3].

C22H26N6O2

406.4808

406.21

C, 65.01; H, 6.45; N, 20.68; O, 7.87

2589531-76-8

155586901

Off-white to light yellow solid powder

1.1

2

6

5

30

660

0

WQAVGRAETZEADU-UHFFFAOYSA-N

InChI=1S/C22H26N6O2/c1-3-16-11-19-20(26-21(16)29)10-15(12-24-19)14-27-6-8-28(9-7-27)17-4-5-18(25-13-17)22(30)23-2/h4-5,10-13H,3,6-9,14H2,1-2H3,(H,23,30)(H,26,29)

5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl]-N-methylpyridine-2-carboxamide

AZD-5305; AZD5305; 2589531-76-8; Saruparib; 16MZ1V3RBT; AZD-5305 [WHO-DD]; example 4 [WO2021013735]; 5-(4-((7-Ethyl-6-oxo-5,6-dihydro-1,5-naphthyridin-3-yl)methyl)piperazin-1-yl)-N-methylpicolinamide; AZD 5305

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)

DMSO: 12.5~20 mg/mL (30.8~49.2 mM)
Ethanol: 2 mg/mL

Solubility in Formulation 1: ≥ 0.56 mg/mL (1.38 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 5.6 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: ≥ 0.56 mg/mL (1.38 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 5.6 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: ≥ 0.56 mg/mL (1.38 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 5.6 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 0.8mg/ml (1.97mM)

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