c-Kit (IC50 = 10 nM); cFMS (IC50 = 20 nM); FLT3 (IC50 = 160 nM); KDR (IC50 = 350 nM); LCK (IC50 = 860 nM); FLT1 (IC50 = 880 nM); NTRK3 (IC50 = 890 nM)

Pexidartinib hydrochloride (0.25, 1 mg/kg, i.p., twice daily for 8 days) inhibits the proliferation of microglia and BrdU-positive cells in neonatal mice[2].
Pexidartinib hydrochloride (1 mg/kg, twice daily for 8 day) does not appear to have any discernible effects on mice's cleaved caspase-3-positive cells[2].
Pexidartinib hydrochloride (50 mg/kg; p.o.; every other day for three weeks) decreases tissue macrophage counts without changing glucose homeostasis in mice[4].

---

---

Blockade of Chemotherapy-Induced TAM Recruitment [1]
To determine whether tumor-infiltrating TAMs also regulate sensitivity of MECs to cytotoxic therapy, we blocked TAM infiltration in vivo with immunologic and pharmacologic agents (Supplementary Fig. S4) and evaluated myeloid cell infiltration of tumors from treated mice (Supplementary Fig. S5). Mice bearing orthotopic mammary tumors were treated with neutralizing monoclonal antibodies (mAb) CSF1 (clone 5S1) or CD11b (clone M1/70), or a competitive ATP inhibitor with potent (nM) specificity for CSF1 and cKIT receptor tyrosine kinases (Pexidartinib), either as a monotherapy or in combination with PTX. CD11b is an integrin cell adhesion molecule expressed on granulocytes, macrophages, monocytes, dendritic cells (DC), and natural killer cells that in part regulates transendothelial migration of cells into tissue and tumor parenchyma. PLX3397 has 10- to 100-fold selectivity for cKIT and CSF1R, as opposed to other related kinases, such as KDR (see Supplementary Fig. S6A and Methods; ref. 20).

Fluorescence-activated cell sorting analysis of the predominant myeloid subtypes infiltrating mammary tumors revealed that either as monotherapy, or in combination with PTX, CD45+CD11b+Ly6C−Ly6G−F4/80+ TAM recruitment was significantly diminished following treatment with either αCSF1 mAb or Pexidartinib, with no effect on infiltration of CD45+CD11b+LY6Ghigh iMCs or CD45+CD11blow/−Ly6C−CD22−Ly6G−CD11chighMHCIIhigh DCs (Fig. 3A and B; Supplementary Fig. S5A and B). Treatment with aCD11b mAb decreased both TAM and iMC infiltration (Fig. 3A). Analysis of the maturation and differentiation status of TAMs remaining in mammary tumor tissue following αCSF1 or PLX3397 treatment revealed no significant change in CD11b, CD11c, F4/80, CD45, or MHCII expression (Supplementary Fig. S5B). However, examination of mammary tumor sections revealed a population of perivascular CSF1-independent F4/80+ TAMs remaining (Fig. 3C). Blockade of TAM recruitment was a direct effect of CSF1/CSF1R blockade: In vitro CSF1R inhibition efficiently blocked CD11b+ monocyte chemotaxis in response to control or PTX-treated pMEC–conditioned medium, with no effect on chemotaxis of CD3+ T lymphocytes (Fig. 3D; Supplementary Fig. S3G). These results were mirrored in vivo; treatment of late-stage MMTV-PyMT mice with PLX3397 significantly inhibited both steady-state and PTX-induced tumor infiltration by CD45+CD11b+Ly6C−Ly6G−F4/80+ TAMs (Fig. 3E; Supplementary Fig. S5) without altering TAM maturation/differentiation (Supplementary Fig. S6B).

We next treated 80-day-old MMTV-PyMT mice, or mice bearing syngeneic orthotopic PyMT-derived tumors (~1.0 cm) with αCSF1, αCD11b, or Pexidartinib (vs controls) for 5 days, followed by 4 cycles of PTX (10 mg/kg, i.v.; Supplementary Fig. S4). Primary tumor burden at study endpoints (2.0 cm primary tumors or 100 days of age) was significantly reduced in mice treated with combined αCSF1/PTX, αCD11b/PTX, or PLX3397/PTX therapy, compared to mice treated with these as single agents (Fig. 4A and B; Supplementary Fig. S7A). Similar results were observed in syngeneic mice bearing orthotopic PyMT-derived mammary tumors receiving combined PLX3397/carboplatin (CBDCA) therapy (Fig. 4B).

Mammary tumors in MMTV-PyMT mice progress through well-defined stages of cancer development, similar to progression of breast cancer in women, including tissue with florid ductal hyperplasia, ductal carcinoma in situ with early stromal invasion, and poorly differentiated invasive ductal carcinoma (15, 21). Using this staging criterion, we observed that mammary tumors arising in MMTV-PyMT mice treated with combined Pexidartinib/PTX therapy exhibited decreased development of late-stage carcinoma, compared with tumors in age-matched mice treated with either PTX or Pexidartinib as monotherapy (Fig. 4C; Supplementary Fig. S7B). Moreover, the late-stage carcinomas that did develop in PLX3397/PTX-treated mice contained large areas of necrosis (Supplementary Fig. S7C) characterized by increased presence of apoptotic cells, as measured by cleaved caspase 3-positivity (Fig. 4D) with no accompanying change in epithelial proliferation (Supplementary Fig. S7D).

---

Decreased Vascular Density Accompanies Improved Chemosensitivity [1]
It is known that TAMs provide VEGF to developing mammary tumors and thereby regulate angiogenic programming of tissue (22–24). Chemosensitivity to CDDP in MMTV-PyMT mice is in part regulated by myeloid-derived VEGF (25); thus, we sought to determine if TAM depletion altered VEGF expression and/or density of CD31+ vessels in MMTV-PyMT mice treated with PTX. Whereas total VEGF mRNA expression was significantly reduced by Pexidartinib (Fig. 4E), this 70% reduction did not correlate with a change in vascular density (Fig. 4F). In contrast, combined PLX3397/PTX therapy resulted in a significant reduction in CD31+ vessel density within mammary tumors, paralleling induction of apoptosis and necrosis (Fig. 4F).

---

CSF1-Signaling Blockade Enhances Antitumor Immunity and CTL Infiltration in Response to Chemotherapy [1]
Because analysis of human breast cancer tissues revealed that high stromal TAM density inversely correlated with CD8+ T-cell infiltration (Supplementary Table S1), we predicted that depletion of TAMs would enhance CD8+ CTL infiltration and thereby foster an antitumor immune microenvironment. Analyses of tumor-infiltrating T lymphocytes in mice treated with αCSF1/PTX or Pexidartinib/PTX by flow cytometry or IHC revealed significantly increased presence of CD4+ and CD8+ T cells in mammary tumors (Fig. 5A and B; Supplementary Fig. S8A). Consistent with these findings, cytokine mRNA expression in mammary tissue derived from PLX3397/PTX-treated MMTV-PyMT mice revealed increased mRNA expression of cytotoxic effector molecules, including IFN-γ, granzyme A, granzyme B, perforin-1, and the type 1 DC effector molecules IL12p35 and IFN-α (Fig. 5C). In contrast, expression of the immunosuppressive molecule arginase-1 was decreased by PLX3397/PTX therapy (Fig. 5C). This reprogramming of the immune microenvironment was accompanied by increased tumor infiltration of CD45+CD11blow/−CD19−Ly6G−Ly6ClowCD11chighMHCIIhigh DCs (Fig. 5D), indicating that combined treatment of MMTV-PyMT mice with PLX3397/PTX fostered an antitumor immune response by T lymphocytes expressing high levels of cytotoxic effector molecules.

---

Macrophage Depletion Enhances Chemotherapeutic Response in a CD8∙ CTL-Dependent Manner [1]
To determine whether increased chemosensitivity of mammary tumors in Pexidartinib/PTX-treated mice was dependent on enhanced CD8+ T-cell response, we depleted CD8+ T cells from late-stage MMTV-PyMT mice treated with PTX or PLX3397 or both. Findings from this study revealed that the improved outcome with enhanced chemosensitivity resulting from combined PLX3397/PTX therapy was indeed a CD8+ T-cell–dependent response (Fig. 6A and B; Supplementary Fig. S8B). We found that CD8 depletion also resulted in increased tumor grade and decreased presence of cleaved caspase-3–positive cells in mice that had received combined PLX3397/PTX therapy (Fig. 6C and D). Taken together, these data indicate that the enhanced cytotoxic response elicited by CSF1R-signaling blockade was CD8+ T-cell–dependent.

---

Combined Macrophage Depletion and Chemotherapy Blocks Metastasis in a CD8-Dependent Manner [1]
Long-term survival of breast cancer patients is often limited by disseminated metastases following surgical resection of primary tumors. Analysis of leukocyte profiles in human breast cancers demonstrated that OS, and thus presumably metastatic spread, were regulated by the spectrum of tumor-infiltrating T lymphocytes and macrophages present. In MMTV-PyMT mice, although neither CSF1R-signaling blockade nor PTX therapy alone inhibited development of pulmonary metastasis, mice receiving combined Pexidartinib/PTX exhibited >85% reduction in pulmonary metastases that was in part CD8+ T-cell–dependent (Fig. 6E).

---

Pexidartinibtreatment of neonatal mice decreased the number of microglia and BrdU-positive proliferative cells [2]
PLX3397, a colony-stimulating factor 1 receptor (CSF1R) inhibitor, is generally used for microglial depletion. In this study, neonatal mice were treated with PLX3397 with an intraperitoneal injection (Figure 3A). The treatment with PLX3397 twice daily from P0 to P7 decreased the number of microglia (p=0.015, Figure 3B,C). PLX3397 also decreased the number of BrdU-positive proliferative cells in the retina at 500 μm from the optic nerve (p=0.021, Figure 3D,E). To determine that the proliferative cells were retinal precursor cells, we costained BrdU and the retinal precursor cell markers, Pax6 and Chx10, which is also known as visual system homeobox 2 (Vsx2). PLX3397 statistically significantly decreased the BrdU and Chx10 double-positive cells and tended to decrease BrdU and Pax6 double-positive cells (p=0.038, Figure 3F,G). PLX3397 did not alter the number of cleaved caspase-3-positive cells (Figure 3H).

---

Pexidartinib/PLX3397 treatment substantially reduced macrophage numbers in adipose tissue of both chow and high-fat diet fed mice without affecting total myeloid cell levels. Despite this, PLX3397 did not greatly alter glucose homeostasis, did not affect high-fat diet-induced increases in visceral fat cytokine expression (Il-6 and Tnfa) and had limited effect on the phosphorylation of the stress kinases JNK and ERK and macrophage polarization. Conclusions: Our results indicate that macrophage infiltration of adipose tissue induced by a high-fat diet may not be the trigger for impairments in whole body glucose homeostasis, and that anti-CSF1 therapies are not likely to be useful as treatments for insulin resistance [4].

---

Activation of CSF1R promotes PTCL growth in-vivo [5]
In order to further address the therapeutic relevance of these findings, the growth of T-cell lymphoma xenografts was evaluated upon CSF1R inhibition with Pexidartinib. Murine CSF1 does not bind human CSF1R(48,59); therefore, the autocrine-dependent CSF1R activation was evaluated in Karpas 299 xenografts generated in NSG mice. Tumor-bearing mice were treated with sham- or pexidartinib-containing chow, and no treatment-related toxicity was appreciated. An approximately 50% reduction in tumor growth was observed in pexidartinib-treated mice (n=24, p<0.05; Figure 6A and B), and increased apoptosis was observed from protein extracts of tumors treated with pexidartinib (Figure 6C). Phosphorylation of CSF1R (Y699) and p70S6K (T389) were examined as pharmacodynamic biomarkers using protein extracts from these xenografts, and a significant reduction in phosphorylation was observed in pexidartinib-treated mice (Figure 6D). In similarly designed experiments, SUP-M2 cells (that require exogenous CSF1) were utilized, and xenografts generated in immunodeficient mice that either transgenically express human CSF1(48) or in non-CSF1 expressing controls. An approximately 3-fold increase in tumor volume was observed in CSF1 producing mice compared with control mice (n=32, p<0.001; Figure 6E and F). Importantly, tumor growth was inhibited in pexidartinib-treated CSF1 transgenic mice (n=15, p<0.001; Figure 6E and F). However, no significant change in tumor growth was observed in the control mice treated with pexidartinib (Figure 6E and F). Overall, these findings demonstrate that activation of CSF1R, in either an autocrine- or paracrine-dependent manner, promotes T-cell lymphoma growth, and further supports CSF1R as a rational therapeutic target in these lymphomas (supplementary figure 7).

Biochemical selectivity and potency of Pexidartinib (PLX3397): [1]
Pexidartinib (PLX3397) selectively inhibits the c-Fms and the c-Kit receptor tyrosine kinases, with biochemical IC50 values of 0.02 µM and 0.01 µM respectively (Figure S6A). Pexidartinib (PLX3397) was identified as a potent CSF-1R and c-KIT kinase inhibitor by using a Scaffold- and X-ray structure-based discovery approach. In a comprehensive screen of 226 different kinases, including representatives of all protein kinase subfamilies and several lipid kinases, Pexidartinib (PLX3397) at 0.03 µM and 1.0 µM only inhibited five other kinases significantly. Pexidartinib (PLX3397) was selected based on inhibition of the CSF1-dependent proliferation of the murine myelogenous leukemia cell line M-NFS-60, with an IC50 of 0.44 µM the murine macrophage cell line Bac1.2F5, with an IC50 of 0.22 µM. The human acute megakaryoblastic leukemia cell line M-07e, which depends on the addition of SCF for growth, was inhibited by Pexidartinib (PLX3397) with an IC50 of 0.1 µM. These sub-micromolar potencies confirm that Pexidartinib (PLX3397) can enter cells and inhibit Fms-driven cell growth.[1]

---

 Pexidartinib (PLX-3397) is an ATP-competitive, potent, and selective inhibitor of CSF1R (cFMS) and c-Kit that exhibits selectivity for c-Kit and CSF1R over other related kinases, FLT3, KDR (VEGFR2), LCK, FLT1 (VEGFR1), and NTRK3 (TRKC), with IC50 values of 160, 350, 860, 880, and 890 nM, respectively.

Proliferation, apoptosis and colony formation assays[5]
Colony formation assays were performed with Methocult H4230 per manufacturer’s instructions. Briefly, approximately 5000 cells were seeded with or without doxycycline (10ng/ml), colonies were grown for 7 days before staining with p-iodonitrotetrazolium chloride. Proliferation was evaluated with CellTiter Glo assays over 72 hours. Multiparametric evaluation of cellular apoptosis was performed by flow cytometry analysis of Annexin V incorporation, coupled with either Propidium Iodide or DilC1(5) incorporation. Staining and detection procedures for flow cytometry were performed according to manufacturer instructions.

---

CSF1R activity promote the growth of T-cell lymphomas in-vitro[5]
Having established the expression and activation of CSF1R in TCL, we adopted a loss-of-function strategy to address its potential oncogenic role in these TCL using complementary molecular and pharmacologic approaches. We first used a clinically available and rationally designed tyrosine kinase inhibitor that is selective for CSF1R (Pexidartinib, PLX3397). In order to confirm CSF1R inhibition upon pexidartinib treatment, TCL cells with autocrine-activation of CSF1R were treated with pexidartinib. A marked decrease in CSF1R phosphorylation was observed upon treatment with pexidartinib (Figure 2A, supplementary figure 4A). Importantly, pexidartinib did not show any effect on the phosphorylation levels of the oncogenic kinase NPM-ALK which is expressed in a portion of the TCL cells evaluated (supplementary figure 4B). In addition, a dose-dependent decrease in proliferation was observed with exposure to pexidartinib (Figure 2B and supplementary figure 4D–E), however these effects were not observed in TCL cells that do not express CSF1R, supporting the relative selectivity of this FDA-approved agent (supplementary figure 4C). Consistent with these findings, treatment with pexidartinib was associated with increased apoptosis of TCL cells, as demonstrated by phosphatidylserine exposure (Figure 2C–E), PARP cleavage and Caspase 3 cleavage[5].

---

PLX3397/Pexidartinib is found to be a strong inhibitor of both CSF-1R and c-KIT kinase through the application of scaffold- and X-ray structure-based discovery methodology. The SelectScreen^TM profiling service provided the IC50 data.

---

Leukocyte chemotaxis assay: For cell migration, PBLs were collected from peripheral blood of FVB/n mice following cardiac puncture, and seeded (105 cells/ 100 µl DMEM containing 0.1% BSA) onto the top chamber of transwell filters (3-µm). Filters were placed in a 24-well plate that contains conditioned medium isolated from vehicle or PTX (20 nM) pretreated MMTV-PyMT-derived MECs. For some conditions, Pexidartinib/PLX3397 (50 nM) was added to the upper chamber. 6-hr following incubation, the media in the lower chamber was isolated and analyzed by flow cytometry for CD11b, CD3 and 7AAD as described above. Samples were run in triplicates for each experimental group and experiments were repeated twice. [1]

Neonatal mice
0.25, 1 mg/kg
I.P. twice daily for 8 days

---

Two murine models of mammary tumor development were used to analyze response to chemotherapy (Supplementary Fig. S3). The first model used MMTV-PyMT mice (Supplementary Fig. S3A). The 80-day-old MMTV-PyMT female littermates were randomized by initial tumor volume and fed either PLX3397 (20, 61, 62) formulated in mouse chow or control chow (provided by Plexxikon Inc). PLX3397 was formulated in mouse chow so that the average dose per animal per day was 40 mg/kg. When PLX3397-treated MMTV-PyMT mice reached 85 days of age, they were then administered PTX (Hospira) every 5 days by i.v. injection into the retroorbital plexus. PTX was given at 10 mg/kg of the animal per injection, diluted in PBS. Tumor burden was evaluated by caliper measurement every 5 days following the start of PLX3397 treatment. Prior to tissue collection, mice were cardiac-perfused with PBS to clear peripheral blood. Mammary tumor tissue from PBS-perfused MMTV-PyMT mice was analyzed by flow cytometry and qRT-PCR 2 days after the second dose of PTX, when metastatic burden and tumor grade were determined. Primary tumor burden was determined by caliper measurements on live sedated mice. Metastatic burden was assessed by serial sectioning of formalin-fixed paraffin-embedded lung tissue whereby the entire lung was sectioned and the number of metastatic foci (>5 cells) was determined on 6 sections taken every 100 µm following H&E staining. Lungs from >10 mice/group were analyzed[1].

---

A Ten-week-old mice were fed a chow or high-fat diet for 10 weeks and then treated with PLX3397 via oral gavage (50 mg/kg) every second day for 3 weeks, with subsequent monitoring of glucose tolerance, insulin sensitivity and assessment of adipose tissue immune cells.PLX3397 treatment substantially reduced macrophage numbers in adipose tissue of both chow and high-fat diet fed mice without affecting total myeloid cell levels. Despite this, PLX3397 did not greatly alter glucose homeostasis, did not affect high-fat diet-induced increases in visceral fat cytokine expression (Il-6 and Tnfa) and had limited effect on the phosphorylation of the stress kinases JNK and ERK and macrophage polarization.[4]

---

Preclinical Mouse Models and Animal Husbandry [1]
Mice harboring the PyMT transgene under the control of the MMTV promoter in the FVB/n strain were used. Two murine models of mammary tumor development were used to analyze response to chemotherapy (Supplementary Fig. S3). The first model used MMTV-PyMT mice (Supplementary Fig. S3A). The 80-day-old MMTV-PyMT female littermates were randomized by initial tumor volume and fed either Pexidartinib/PLX3397 formulated in mouse chow or control chow. PLX3397 was formulated in mouse chow so that the average dose per animal per day was 40 mg/kg. When PLX3397-treated MMTV-PyMT mice reached 85 days of age, they were then administered PTX every 5 days by i.v. injection into the retroorbital plexus. PTX was given at 10 mg/kg of the animal per injection, diluted in PBS. Tumor burden was evaluated by caliper measurement every 5 days following the start of PLX3397 treatment. Prior to tissue collection, mice were cardiac-perfused with PBS to clear peripheral blood. Mammary tumor tissue from PBS-perfused MMTV-PyMT mice was analyzed by flow cytometry and qRT-PCR 2 days after the second dose of PTX, when metastatic burden and tumor grade were determined. Primary tumor burden was determined by caliper measurements on live sedated mice. Metastatic burden was assessed by serial sectioning of formalin-fixed paraffin-embedded lung tissue whereby the entire lung was sectioned and the number of metastatic foci (>5 cells) was determined on 6 sections taken every 100 µm following H&E staining. Lungs from >10 mice/group were analyzed.

To assess tumor grade, the stage characterization technique classified tumor tissue into 3 levels of histologic progression by quantifying the area of transformed glands occupied by each stage. Progression follows from a “precancerous stage” characterized by premalignant hyperplasia and adenoma/mouse intestinal epithelium but with the retention of some normal ductal and acinar mammary gland morphology, to a more epithelial cell–dense “early carcinoma” with some stromal invasion, and finally to an invasive, high–mitotic index “late-stage carcinoma.”

The IHC analysis was conducted on tissue sections following the end of studies on 100-day-old MMTV-PyMT mice (detailed in Supplementary Fig. S4A). Vehicle-treated mice received PBS-only injections. We also used a syngeneic orthotopic implantable tumor model (referred to as PyMT-implantable in all figures and detailed in Supplementary Fig. S4B). For this model, single-cell suspensions of tumor cell pools isolated from mammary tumors of 3 or 4 100-day-old MMTV-PyMT mice were generated following collagenase A digestion (see discussion of flow cytometry analysis earlier). A total of 1.0 million tumor cells from pools were diluted in medium and basement membrane extract and injected orthotopically into uncleared mammary fat pads (4th gland) of 10-week-old virgin FVB/n female mice. Following implantation, tumors were allowed to grow to a mean diameter of 1.0 cm before enrollment into studies. Mice were randomized into treatment groups based on tumor size and treated with Pexidartinib/PLX3397 and PTX, as described above. For some studies, CBDCA was used and administered at 10 mg/kg of mouse per injection, in a similar manner to administration of PTX (see above). For mice with implantable tumors, tumor burden was evaluated by caliper measurement every 2 to 3 days following the start of PLX3397 treatment, and mammary tissue was analyzed by flow cytometry, IHC, and qRT-PCR at the end of the study (Supplementary Fig. S3B). Immune-depleted mice were injected i.p. every 5 days with either 1.0 mg anti-CD8 immunoglobulin G (YTS169.4) or isotype control rat immunoglobulin on day 1 followed by 500 µg every 5 days.

---

PLX3397/Pexidartinib treatment [2]
PLX3397 (Pexidartinib) was treated at a dose of 0.25 and 1 mg/kg (i.p., twice daily) to neonatal mice from P0 to P7. PLX3397 250 mg/ml in 100% dimethyl sulfoxide (DMSO) was prepared as the stock solution. The stock solution was diluted with PBS for the injected solution (0.25 mg/ml in PBS plus 0.1% DMSO). The control group was treated with an equal amount of 0.1% DMSO in PBS. BrdU was injected similarly as described above. At P7, the mice were euthanized, and the eyes were enucleated.

---

Mice were housed under specific-pathogen free conditions. SUPM2 or Karpas 299 cells (2 × 106) were injected subcutaneously in immunodeficient NSG mice or Rag2/Il2rg immunodeficient mice with transgenic expression of humanized CSF1 ligand(48). Upon injections, mice were fed with either nutritionally complete pexidartinib-containing (275mg PLX3397/kg) or control chow, provided under MTA by Plexxikon. Toxicity secondary to drug delivery was assessed by daily monitoring of clinical condition (appearance, activity and body condition). Tumors were measured and mice humanely euthanized approximately 14 days after pexidartinib- or control-treatment.

Absorption, Distribution and Excretion
Following a single dose in healthy subjects and multiple doses in patients, the mean Cmax was 8625 ng/mL, and the mean AUC was 77465 ng·h/mL. The median Tmax was 2.5 hours, and the time to reach steady-state plasma concentrations was approximately 7 days. Co-administration with a high-fat meal increased pexidartinib's Cmax and AUC by 100% and delayed Tmax by 2.5 hours. Pexidartinib is primarily excreted in feces, accounting for 65% of total pexidartinib clearance. Approximately 44% of the compound is recovered in its original form in feces via this route. Renal excretion accounts for 27% of total pexidartinib excretion, with over 10% of the compound remaining as N-glucuronide metabolites. The apparent volume of distribution of pexidartinib is approximately 187 liters. In rats, pexidartinib has been shown to penetrate the central nervous system.
The apparent clearance is approximately 5.1 L/h.

---

Metabolism/Metabolites
Pecilidatinib is primarily metabolized in the liver via CYP3A4-mediated oxidation and UGT1A4-mediated glucuronidation. UGT1A4-mediated glucuronidation results in a major inactive N-glucuronide metabolite, and the exposure to this metabolite after a single dose of percilidatinib is approximately 10% higher than that of the parent drug. Based on in vitro studies, CYP1A2 and CYP2C9 may also play minor roles in drug metabolism.

---

Biological Half-Life
The elimination half-life is approximately 26.6 hours.

Hepatotoxicity
Elevated serum transaminase levels are common during pexidartinib treatment, occurring in 50% to 90% of patients, with 12% to 20% of patients experiencing transaminase levels exceeding five times the upper limit of normal. Additionally, up to 20% of treated patients experience elevated alkaline phosphatase levels. In the registration trial, 5% of patients developed clinically significant liver injury with jaundice. Liver injury typically occurs within 2 to 6 weeks, with liver enzyme elevations exhibiting a mixed or cholestatic pattern. Autoimmune and immunohypersensitive features are not prominent. Liver biopsies revealed bile duct damage and loss; at least three patients in non-TGCT disease studies presented with features of bile duct reduction and disappearance syndrome, one of whom ultimately underwent liver transplantation. The clinical use of pexidartinib is limited, and the frequency and type of acute liver injury it causes remain unclear. Probability Score: B (likely to cause clinically significant liver injury).

---

Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of pexidartinib during lactation. Because pexidartinib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk may be very low. However, the manufacturer recommends discontinuing breastfeeding during pexidartinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

Protein binding
Based on in vitro plasma protein binding studies, pexidartinib binds to approximately 99% of serum proteins, including 99.9% to human serum albumin and 89.9% to α1-acid glycoprotein.

[1]. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov. 2011 Jun;1(1):54-67.

[2]. Microglia increases the proliferation of retinal precursor cells during postnatal development. Mol Vis. 2018 Jul 30;24:536-545.

[3]. A phase I study of pexidartinib, a colony-stimulating factor 1 receptor inhibitor, in Asian patients with advanced solid tumors. Invest New Drugs. 2020 Feb;38(1):99-110.

[4]. The CSF1 receptor inhibitor pexidartinib (PLX3397) reduces tissue macrophage levels without affecting glucose homeostasis in mice. Int J Obes (Lond). 2020;44(1):245-253.

[5]. Colony-stimulating Factor 1 Receptor (CSF1R) Activates AKT/mTOR Signaling and Promotes T-cell Lymphoma Viability. Clin Cancer Res. 2020 Feb 1; 26(3): 690–703.

Pexidartinib hydrochloride is the hydrochloride salt form of Pexidartinib, a small molecule receptor tyrosine kinase (RTK) inhibitor that inhibits proto-oncogene receptor tyrosine kinase (KIT), colony-stimulating factor-1 receptor (CSF1R), and FMS-like tyrosine kinase 3 (FLT3), exhibiting antitumor activity. After oral administration, Pexidartinib targets and binds to KIT, CSF1R, and FLT3 carrying internal tandem repeat (ITD) mutations, inhibiting their phosphorylation and thereby suppressing tumor cell proliferation. FLT3, CSF1R, and FLT3 are overexpressed or mutated in various cancer cell types and play important roles in tumor cell proliferation and metastasis.
See also: Pexidartinib (active fraction). Pexidartinib is a pyrrolopyridine compound with the chemical name 5-chloro-1H-pyrrolo[2,3-b]pyridine, substituted at the 3-position with [6-({[6-(trifluoromethyl)pyridin-3-yl]methyl}amino)pyridin-3-yl]methyl]. It is a potent multi-target receptor tyrosine kinase inhibitor that inhibits CSF-1R, KIT, and FLT3 (IC50 values of 20 nM, 10 nM, and 160 nM, respectively). It is FDA-approved for the treatment of adult patients with symptomatic tenosynovial giant cell tumor (TGCT). It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor and an anti-tumor drug. It is a pyrrolopyridine compound, organochlorine compound, aminopyridine compound, organofluorine compound, and secondary amino compound. Pexidartinib is a selective tyrosine kinase inhibitor whose mechanism of action is through inhibition of the colony-stimulating factor (CSF1)/CSF1 receptor pathway. Pexidartinib was originally developed by Daiichi Sankyo Co., Ltd., and in August 2019, it was approved by the U.S. Food and Drug Administration (FDA) as the first systemic therapy for the treatment of symptomatic tenosynovitis in adults. Tenosynovitis is a rare, non-malignant tumor that causes thickening and overgrowth of the synovium and tendon sheath, leading to damage to surrounding joint tissues. Tenosynovitis is often accompanied by severe symptoms and can cause significant functional limitations and a decreased quality of life. While surgical resection is currently the standard treatment for tenosynovitis, surgery is considered clinically ineffective for certain types of tumors and carries a high risk of lifelong recurrence. Pexidartinib's mechanism of action is to block the activated immune response in tenosynovitis. Clinical trials have shown that pexidartinib can improve symptoms and functional outcomes in patients with tenosynovitis. Pexidartinib is an oral formulation, commonly marketed under the brand name Turalio. Pexidartinib is a kinase inhibitor. Pexidartinib's mechanism of action involves its role as a kinase inhibitor, tyrosine kinase inhibitor, colony-stimulating factor receptor type 1 (CSF-1R) inhibitor, cytochrome P450 3A inducer, cytochrome P450 2B6 inhibitor, and UGT1A1 inhibitor. Pexidartinib is an oral small-molecule multi-kinase inhibitor used to treat tenosynovial giant cell tumor and is an anti-tumor drug. Elevated serum transaminases and alkaline phosphatase levels are frequently observed during pexidartinib treatment and have been associated with numerous clinically significant cases of liver injury, characterized by progressive intrahepatic bile duct damage, some of which ultimately led to liver transplantation or death. Pexidartinib is a small-molecule receptor tyrosine kinase (RTK) inhibitor that inhibits proto-oncogene receptor tyrosine kinase (KIT), colony-stimulating factor-1 receptor (CSF-1R), and FMS-like tyrosine kinase 3 (FLT3), exhibiting anti-tumor activity. After oral administration, Pexidartinib targets and binds to KIT, CSF1R, and FLT3 carrying internal tandem repeat (ITD) mutations, inhibiting their phosphorylation. This leads to suppression of tumor cell proliferation. FLT3, CSF1R, and FLT3 are overexpressed or mutated in a variety of cancer cell types and play important roles in tumor cell proliferation and metastasis.
See also: Pexidartinib hydrochloride (salt form).
Drug Indications

Pexidartinib is indicated for the treatment of adult symptomatic giant cell tumor of the tenosynovium (TGCT) with severe complications or functional impairment that cannot be improved by surgery.
Treatment of giant cell tumor of the tenosynovium.
Treatment of benign soft tissue tumors.
Mechanism of Action

Giant cell tumor of the tenosynovium is a rare non-malignant tumor that causes abnormal growth and damage to the synovium, bursa, or tendon sheath. The recruitment of immune cells, especially macrophages, is closely associated with tumorigenesis of giant cell tumor of the tenosynovium. Macrophages drive pro-tumor inflammation and play a central role in all stages of tumor progression. As the most abundant immune cells in the tumor microenvironment of solid tumors, macrophages promote a variety of processes that enhance tumor survival, such as angiogenesis, tumor cell invasion, and intravascular infiltration at the primary site. They also modulate the tumor immune response to inhibit tumor clearance and interact directly with tumor cells to activate pro-survival signaling pathways. Macrophage recruitment, proliferation, and irreversible differentiation are regulated by colony-stimulating factor-1 (CSF-1), a cytokine that frequently metastasizes and is highly expressed in tenosynovial giant cell tumors. High expression of CSF-1 and its receptor (CSF1R) is also associated with various malignant tumor models. Pexidartinib is a selective CSF1R inhibitor that targets the CSF1/CSF1R pathway. It activates the autoinhibitory state of CSF1R by interacting with the juxtamembranous region of CSF1R. This juxtamembranous region is responsible for the folding and inactivation of the kinase domain, thereby preventing the binding of CSF1 and ATP to this region. Because CSF1 cannot bind to its receptor, CSF1R cannot undergo ligand-induced autophosphorylation. By inhibiting the CSF1R signaling pathway, pecidatinib can suppress tumor cell proliferation and downregulate disease-involved cells, such as macrophages. Studies have also shown that this compound can inhibit CD117 or proto-oncogene receptor tyrosine kinase (cKIT), mutant fms-like tyrosine kinase 3 (FLT3), and platelet-derived growth factor receptor (PDGFR)-β, all of which are involved in regulating key cellular processes such as cell proliferation and survival. Immune regulatory pathways influence multiple aspects of cancer development. This article demonstrates that the abundance of macrophages and T cells in breast cancer can serve as prognostic indicators of recurrence-free survival and overall survival. Our evidence suggests that chemotherapy response is partially regulated by these leukocytes; cytotoxic therapy induces breast epithelial cells to produce monocyte/macrophage recruitment factors, including colony-stimulating factor 1 (CSF1) and interleukin-34, which collectively enhance CSF1 receptor (CSF1R)-dependent macrophage infiltration. Blocking macrophage recruitment with a CSF1R signaling pathway antagonist in combination with paclitaxel treatment improved survival in mice with breast cancer by slowing primary tumor development and reducing lung metastases. These improved breast cancer development processes were accompanied by reduced vascular density and the emergence of anti-tumor immune programs that suppressed tumors in a CD8+ T cell-dependent manner. These data provide a theoretical basis for targeting macrophage recruitment/response pathways (especially CSF1R) in combination with cytotoxic therapy and help identify breast cancer patients who may benefit from this novel treatment approach. [1]

---

Pharmacodynamics
Pexidartinib works by inhibiting the growth of tenosynovial giant cell tumors. In clinical trials involving patients with symptomatic tenosynovial giant cell tumors, pexidartinib had a higher overall response rate compared to placebo, characterized by improved patient symptoms and functional prognosis. Pexidartinib works by inhibiting the activation and signaling of pro-tumor cytokines and receptor tyrosine kinases that play a central role in tumor cell proliferation and survival. Taking pericidatinib with high-fat foods may increase the incidence and severity of adverse reactions, including hepatotoxicity.

---

Pericidatinib is a pyrrolopyridine compound with the chemical name 5-chloro-1H-pyrrolo[2,3-b]pyridine, substituted at the 3-position with [6-({[6-(trifluoromethyl)pyridin-3-yl]methyl}amino)pyridin-3-yl]methyl. It is a potent multi-target receptor tyrosine kinase inhibitor that inhibits CSF-1R, KIT, and FLT3 (IC50 values of 20 nM, 10 nM, and 160 nM, respectively). It has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of symptomatic giant cell tumor of the tenosynovium sheath (TGCT) in adults. It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor and an antitumor drug. It is a pyrrolopyridine compound, organochlorine compound, aminopyridine compound, organofluorine compound, and secondary amino compound. Pexidartinib is a selective tyrosine kinase inhibitor that works by inhibiting the colony-stimulating factor (CSF1)/CSF1 receptor pathway. Originally developed by Daiichi Sankyo, Inc., pexidartinib received FDA approval in August 2019, becoming the first systemic therapy for the treatment of symptomatic tenosynovial giant cell tumors in adults. Tenosynovial giant cell tumors are rare, non-malignant tumors that cause thickening and overgrowth of the synovium and tendon sheath, leading to damage to surrounding joint tissues. Patients with tenosynovial giant cell tumors often experience severe symptoms and face a significant risk of functional impairment and decreased quality of life. While surgical resection is currently the standard treatment for tenosynovial giant cell tumors, surgery is considered clinically ineffective for certain types of tumors and carries a high risk of lifelong recurrence. Pexidartinib's mechanism of action is to block the activated immune response in tenosynovial giant cell tumors. Clinical trials have shown that pexidartinib can improve symptoms and functional prognosis in patients with tenosynovial giant cell tumors. Pexidartinib is available in oral formulations, typically marketed under the brand name Turalio. Pexidartinib is a kinase inhibitor. Its mechanism of action involves acting as a kinase inhibitor, a tyrosine kinase inhibitor, a colony-stimulating factor receptor type 1 (CSF-1R) inhibitor, a cytochrome P450 3A inducer, a cytochrome P450 2B6 inhibitor, and a UGT1A1 inhibitor. Pexidartinib is an oral small-molecule multi-kinase inhibitor used to treat tenosynovial giant cell tumor and is an anti-tumor drug. Elevated serum transaminases and alkaline phosphatase levels are frequently observed during pexidartinib treatment and have been associated with numerous clinically significant cases of liver injury, characterized by progressive intrahepatic bile duct damage, some of which ultimately led to liver transplantation or death.
Pecidatinib is a small molecule receptor tyrosine kinase (RTK) inhibitor that inhibits proto-oncogene receptor tyrosine kinase (KIT), colony-stimulating factor-1 receptor (CSF1R), and FMS-like tyrosine kinase 3 (FLT3), exhibiting antitumor activity. After oral administration, pecidatinib targets and binds to KIT, CSF1R, and FLT3 carrying internal tandem repeat (ITD) mutations, inhibiting their phosphorylation. This leads to suppression of tumor cell proliferation. FLT3, CSF1R, and FLT3 are overexpressed or mutated in various cancer cell types and play important roles in tumor cell proliferation and metastasis.
PEXIDARTINIB is a small molecule drug currently in Phase IV clinical trials (covering all indications). It was first approved in 2019 for the treatment of cancer and tenosynovial giant cell tumor, and has 16 investigational indications. This drug has been placed on the FDA's black box warning list.

---

Immune regulatory pathways influence multiple aspects of cancer development. This study demonstrates that the abundance of macrophages and T cells in breast cancer can serve as prognostic indicators of recurrence-free survival and overall survival. Our evidence suggests that chemotherapy response is partially regulated by these leukocytes; cytotoxic therapy induces the production of monocyte/macrophage recruitment factors, including colony-stimulating factor 1 (CSF1) and interleukin-34, by mammary epithelial cells, which collectively enhance CSF1 receptor (CSF1R)-dependent macrophage infiltration. Blocking macrophage recruitment with CSF1R signaling pathway antagonists, combined with paclitaxel treatment, improved survival in tumor-bearing mice by slowing primary tumor progression and reducing lung metastases. These improvements in breast cancer development were accompanied by decreased vascular density and the emergence of anti-tumor immune programs, thereby inhibiting tumor growth in a CD8+ T cell-dependent manner. These data provide a theoretical basis for targeting macrophage recruitment/response pathways (especially CSF1R) in combination with cytotoxic therapy and identify a potential patient population for breast cancer who may benefit from this novel treatment approach.
Significance: These findings suggest that the response to chemotherapy is partly regulated by the tumor immune microenvironment. Commonly used cytotoxic drugs can induce tumor cells to produce monocyte/macrophage recruitment factors, thereby enhancing macrophage infiltration into breast adenocarcinoma. Blocking the pathway mediating macrophage recruitment, combined with chemotherapy, can significantly reduce the progression of primary tumors, reduce metastasis, and improve survival through a CD8+ T cell-dependent mechanism. This suggests that the tumor immune microenvironment can be reprogrammed to promote anti-tumor immunity and improve the response to cytotoxic therapy. [1]

---

Objective: In mice, retinal development continues in the postnatal stage and is accompanied by the proliferation of retinal progenitor cells. Previous studies have shown that the number of microglia gradually increases from day 0 (P0) to day 7 (P7) in the postnatal stage. However, how microglia participate in retinal development remains unclear.
Methods: This study employed two methods to investigate the role of microglia in retinal development, namely microglia activation and loss, using lipopolysaccharide (LPS) and PLX3397 (Pexidartinib), respectively.
Results: Intraperitoneal injection (ip) of 1 mg/kg LPS in newborn mice increased the number of retinal microglia at P7. Compared with the control group, the LPS-treated group showed an increased number of 5-bromo-2'-deoxyuridine (BrdU)-positive proliferating cells. These proliferating cells primarily colocalized with the retinal progenitor marker pairing cassette 6 (Pax6). However, PLX3397 treatment-induced microglia depletion reduced the number of BrdU-positive proliferating cells. Furthermore, anterior granulocyte deficiency also reduced the number of microglia and retinal progenitor cells.
Conclusion: These findings suggest that microglia regulate the proliferation of immature retinal cells. [2]

---

Background: Pexidartinib is a novel oral small molecule tyrosine kinase inhibitor with strong selectivity for colony-stimulating factor 1 receptor. This phase I, non-randomized, open-label, multiple-dose study evaluated the safety and efficacy of pexidartinib in Asian patients with symptomatic advanced solid tumors. Materials and Methods: Patients were treated with pexidartinib: Group 1, 600 mg/day; Group 2, 1000 mg/day for 2 weeks, followed by 800 mg/day. The primary objective was to assess the safety and tolerability of pexidartinib and determine the recommended phase 2 dose; secondary objectives were to assess efficacy and pharmacokinetic characteristics. Results: All 11 patients (6 males, 5 females; median age 64 years, range 23–82 years; cohort 1 n=3; cohort 2 n=8) experienced at least one adverse event during treatment; of these, 5 experienced at least one grade ≥3 adverse event. The most common adverse events (18%) included elevated aspartate aminotransferase, elevated serum alkaline phosphatase, elevated gamma-glutamyl transferase, and anemia. The recommended phase II dose was 1000 mg/day for 2 weeks, followed by 800 mg/day. With increasing pexidartinib dose, pexidartinib exposure, area under the plasma concentration-time curve (AUC0–8h), and maximum plasma concentration (Cmax) increased on days 1 and 15, while the time to reach Cmax (Tmax) remained consistent across all dose groups. Repeated daily administration of pexidartinib increased pexidartinib exposure and plasma adiponectin and colony-stimulating factor 1 levels. One (13%) patient with tenosynovial giant cell tumor achieved objective tumor response. Conclusion: This is the first study to evaluate the efficacy of pexidartinib in Asian patients with advanced solid tumors. Pexidartinib was safe and well-tolerated in this population at the previously established Phase II recommended dose for Western patients (funded by Daiichi Sankyo Co., Ltd.; clinicaltrials.gov registration number: NCT02734433). [3]

---

Background and Objectives: Excessive accumulation of adipose tissue macrophages in obesity is associated with mediating inflammatory responses that impair glucose homeostasis and promote insulin resistance. Colony-stimulating factor 1 (CSF1) controls macrophage differentiation, and this study aimed to investigate the effect of the CSF1 receptor inhibitor Pexidartinib/PLX3397 on the level of macrophages in mouse adipose tissue and to understand its effect on glucose homeostasis.
Methods: Ten-week-old mice were fed either a normal diet or a high-fat diet for 10 weeks, followed by treatment with PLX3397 (50 mg/kg) via gavage every other day for 3 weeks. Glucose tolerance, insulin sensitivity, and adipose tissue immune cells were then monitored.
Results: PLX3397 treatment significantly reduced the number of macrophages in the adipose tissue of mice in both the normal and high-fat diet groups, but did not affect the total myeloid cell level. Nevertheless, PLX3397 did not significantly alter glucose homeostasis, nor did it affect the high-fat diet-induced increase in visceral adipocyte cytokines (IL-6 and TnFA), and had limited effects on the phosphorylation of stress kinases JNK and ERK, or macrophage polarization.
Conclusion: Our results suggest that high-fat diet-induced adipose tissue macrophage infiltration may not be a trigger for impaired systemic glucose homeostasis, and anti-CSF1 therapy is unlikely to be effective in treating insulin resistance. [4]

---

Objective: Peripheral T-cell lymphoma is clinically aggressive and usually has a poor prognosis because currently available therapies rarely achieve complete or durable remission. Recent evidence suggests that lymphoma-associated macrophages play a crucial role in the progression of T-cell lymphomas, but the specific signaling pathways involved in the interaction between malignant T cells and their microenvironment remain poorly understood. Colony-stimulating factor 1 receptor (CSF1R, CD115) is essential for the homeostasis of tissue-resident macrophages. Interestingly, its aberrant expression has been reported in some tumors. This study evaluated the expression of CSF1R in T-cell lymphomas and its oncogenic role.
Experimental Design: We conducted loss-of-function studies in various in vitro and in vivo models, including pharmacological inhibition using the clinically available tyrosine kinase inhibitor pexidartinib. Furthermore, we performed proteomics and genomics screening to identify downstream signaling pathways activated by CSF1R signaling.
Results: We observed aberrant expression of CSF1R in many T-cell lymphomas, including a significant number of peripheral and cutaneous T-cell lymphomas. Colony-stimulating factor 1 (CSF1) leads to autophosphorylation and activation of CSF1R in malignant T cells via autocrine or paracrine pathways. Furthermore, CSF1R signaling is associated with significant changes in gene expression and phosphorylated proteome, suggesting that the PI3K/AKT/mTOR pathway is involved in CSF1R-mediated T-cell lymphoma growth. We also demonstrated that inhibition of CSF1R was associated with reduced T-cell lymphoma growth in in vivo and in vitro models. Conclusion: In summary, these findings suggest that CSF1R is associated with the development of T-cell lymphoma and has important therapeutic implications. [5]

C20H16CL2F3N5

454.275752067566

453.073

C, 52.88; H, 3.55; Cl, 15.61; F, 12.55; N, 15.42

2040295-03-0

Pexidartinib;1029044-16-3; 2040295-03-0 (HCl); 2169310-71-6 (2HCl)

73053710

White to off-white solid powder

3

7

5

30

537

0

ClC1=CN=C2C(=C1)C(=CN2)CC1C=NC(=CC=1)NCC1C=NC(C(F)(F)F)=CC=1.Cl

CJLUYLRKLUYCEK-UHFFFAOYSA-N

InChI=1S/C20H15ClF3N5.ClH/c21-15-6-16-14(10-28-19(16)29-11-15)5-12-2-4-18(26-7-12)27-9-13-1-3-17(25-8-13)20(22,23)24;/h1-4,6-8,10-11H,5,9H2,(H,26,27)(H,28,29);1H

5-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]-N-[[6-(trifluoromethyl)pyridin-3-yl]methyl]pyridin-2-amine;hydrochloride

PLX3397; PLX-3397; PLX 3397; YS6WAI3XN7; UNII-YS6WAI3XN7; Pexidartinib (hydrochloride); CML-261; Pexidartinib HCl; PLX3397 HCl; YS6WAI3XN7; CML 261; CML261; FP-113; FP 113; FP113; Pexidartinib HCl; Pexidartinib hydrochloride; Turalio; PLX3397 HCl;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~60 mg/mL (~132.1 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.50 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.50 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.

---

Solubility in Formulation 4: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 5: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

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