HIF-1α

PX-478 was applied to HIF-1α protein-expressing PC3 and DU 145 cells for 20 hours at normoxic conditions. PX-478 had a greater effect on PC3 cells than DU 145 cells. According to densitometric analysis, PC3 cells suppressed HIF-1α with an IC50 of 20–25 μM under normoxic circumstances, whereas DU inhibited HIF-1α with an IC50 of 40–50 μM. Different concentrations of PX-478 (10, 20, 30, 40, 50, and 60 μM) were applied to PC3 and DU 145 cells under normoxic or hypoxic conditions for a duration of 18 to 20 hours. PC3 cells were more sensitive to PX-478 than DU 145 cells in normoxic conditions. Clonogenic viability for PC3 cells (n=3) and DU 145 cells with IC50 values of 17 μM and 35 μM, respectively. The drug's half-life (IC50) for PC3 cells was 16 μM and for DU 145 cells it was 22 μM when the cells were treated for eighteen hours under hypoxic circumstances. Consequently, in hypoxic settings, DU 145 cells are more susceptible to PX-478 [1].

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Overexpression of hypoxia-inducible factor-1alpha (HIF-1alpha) in human tumors is associated with poor prognosis and poor outcome to radiation therapy. Inhibition of HIF-1alpha is considered as a promising approach in cancer therapy. The purpose of this study was to test the efficacy of a novel HIF-1alpha inhibitor PX-478 as a radiosensitizer under normoxic and hypoxic conditions in vitro. PC3 and DU 145 prostate carcinoma cells were treated with PX-478 for 20 hr, and HIF-1alpha protein level and clonogenic cell survival were determined under normoxia and hypoxia. Effects of PX-478 on cell cycle distribution and phosphorylation of H2AX histone were evaluated. PX-478 decreased HIF-1alpha protein in PC3 and DU 145 cells. PX-478 produced cytotoxicity in both cell lines with enhanced toxicity under hypoxia for DU-145. PX-478 (20 mumol/L) enhanced the radiosensitivity of PC3 cells irradiated under normoxic and hypoxic condition with enhancement factor (EF) 1.4 and 1.56, respectively. The drug was less effective in inhibiting HIF-1alpha and enhancing radiosensitivity of DU 145 cells compared to PC3 cells with EF 1.13 (normoxia) and 1.25 (hypoxia) at 50 mumol/L concentration. PX-478 induced S/G2M arrest in PC3 but not in DU 145 cells. Treatment of PC3 and DU 145 cells with the drug resulted in phosphorylation of H2AX histone and prolongation of gammaH2AX expression in the irradiated cells. PX-478 is now undergoing Phase I clinical trials as an oral agent. Although the precise mechanism of enhancement of radiosensitivity remains to be identified, this study suggests a potential role for PX-478 as a clinical radiation enhancer [1].

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Researchers tested the hypothesis that Hif1α inhibition can prevent HO. For this purpose, researchers used the drug PX-478, which has been shown to inhibit Hif1α transcription and translation. In vitro treatment of cells derived from the tenotomy site 3 weeks after injury (3WLST) and cultured in hypoxia showed diminished levels of the Hif1α transcript and of the chondrogenic gene transcripts Sox9 and Acan (aggrecan) upon treatment with PX-478 (Fig. 4A). Additionally, PX-478 and rapamycin, a previously described Hif1α inhibitor, both significantly diminished Hif1α produced by mesenchymal cells isolated from tendon, confirming again that these drugs affect Hif1α levels in cells local to the future HO site [2].

For two weeks after birth, (Nfatc1-Cre/caACVR1fl/fl) mice with congenital heart disease received PX-478 every other day. The amount of ectopic bone in treated mice's ankle joints was significantly less (6.8 mm3 vs. 2.2 mm3, P<0.01) when compared to mutant mice that received vehicle treatment[2].

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Researchers next tested whether treatment with PX-478 decreases Hif1α expression and cartilage formation in vivo, and consequently inhibits overall development of HO. Mice received burn/tenotomy and were subsequently treated with PX-478; histologic evaluation after 3 wk confirmed a substantial decrease in the cartilage anlagen, which is typically present after 3 wk (Fig. 4B). Furthermore, they found diminished Hif1α expression 3 wk after injury (Fig. 4C). Consistent with these data, expression of Sox9 was considerably diminished in the PX-478–treated group (Fig. 4C). Moreover, burn/tenotomy mice treated with PX-478 demonstrated a significant reduction in total HO volume at 5 wk (4.3 mm3 vs. 1.5 mm3, P < 0.05) and 9 wk (5.8 mm3 vs. 2.3 mm3, P < 0.05) after injury (Fig. 4 D and E). Finally, PX-478 treatment completely inhibited “soft tissue” HO—extraskeletal bone, which forms within the proximal transected tendon and distal gastrocnemius but away from the calcaneus—after 9 wk, as shown by binary analysis (yes/no: χ2 = 9.5, P < 0.01) and quantitative comparison (0.90 mm3 vs. 0.00 mm3, P = 0.05). [2]

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Pharmacologic Inhibition of Hif1α Limits HO Caused by ACVR1 Constitutive Activity. [2]
Researchers next confirmed these findings in models of constitutive ACVR1 activity caused by expression of the caACVR1 (ACVR1 Q207D) mutation. caACVR1fl/fl mice injected with cardiotoxin and Ad.cre develop robust HO and this model has been used to study inhibitors of ACVR1 signaling. caACVR1fl/fl mice treated with PX-478 demonstrated near elimination of cartilage or bone based on pentachrome staining (Fig. 5A) after Ad.cre/cardiotoxin induction. Similarly, there was elimination of Hif1α and Sox9 based on immunostaining (Fig. 5B). Finally, microCT analysis confirmed the complete absence of HO in the PX-478 treated group based on binary analysis (yes/no; χ2 = 13.6, P < 0.001) and quantitative comparison (18.1 mm3 vs. 0.01 mm3, P = 0.01) (Fig. 5 C and D).
Finally, PX-478 was administered to mice with congenital HO (Nfatc1-cre/caACVR1fl/fl) every other day starting from birth for 2 wk. Treated mice had significantly less ectopic bone at the ankle joints compared with mutant mice treated with vehicle (6.8 mm3 vs. 2.2 mm3, P < 0.01).

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Loss of Hif1α Prevents Formation of Mesenchymal Condensations in HO Models. [2]
Similar to Hif1α knockout, PX-478 and rapamycin treatment substantially diminished the presence of mesenchymal progenitor cells and the formation of mesenchymal condensation as shown by H&E staining, as well as PDGFRα and Sox9 immunostaining (Fig. 8).

Clonogenic cell survival assay under normoxia [1]
To determine the effect of PX-478 in combination with radiation, cells were treated with the drug for 24 hr under normoxic condition, irradiated and plated after 1 hr. Colonies were stained with crystal violet after 12 days and the colonies of >50 cells were counted. For combination treatments, net survival was calculated by correcting the toxicity of PX-478 alone. Enhancement factor (EF) was calculated by dividing the dose of radiation required to reduce plating efficiency to 10% when cells were treated with radiation alone by the dose of radiation required to reduce plating efficiency to 10% when cells were treated with PX-478 and radiation.

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Hypoxia treatment [1]
Cells were plated in 70-cm2 glass flasks (for Western blotting) or small glass flasks (for clonogenic assay). Next day, media was removed and fresh media with or without PX-478 was added to the flasks. After incubating with the drug for 18–20 hr under normoxia, flasks were tightly sealed with rubber stopper. Two 19-gauge needles were inserted in the rubber stoppers to introduce hypoxic gas mixture and to interconnect flasks. Hypoxia was induced by gassing the flasks with a mixture of 95% N2 and 5% CO2 for 1 hr in a warm room.15,29 Needles were removed at the end of 1 hr gassing and the rubber-stopper sealed flasks were incubated for desired duration. Previous measurements with a Thermox probe indicated that the oxygen tension in flask was <10 ppm (0.02%) producing radiobiologic hypoxia.
After 1 hr of gassing, the cells were harvested by scraping for Western blot analysis or trypsinized and plated for clonogenic survival assay. For combination treatment, cells were pretreated with the drug for 20 hr under normoxia and subjected to 1 hr gassing as described. At the end of 1-hr hypoxic, gassing cells were irradiated under hypoxic condition and plated for clonogenic assay within 1 hr. To investigate the effect of prolonged treatment with PX-478 during hypoxia, in some experiments PX-478 was added to cells just before hypoxic gassing and cells were maintained under hypoxic condition with the drug for 18 hr. For clonogenic assays, cells were plated in drug-free media.

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Cell cycle analysis [1]
Effect of PX-478 on cell cycle distribution was analyzed by flow cytometry by propidium iodide staining after treating cells with the drug for 24 hr. For BrdU staining, cells were incubated with 10 μmol/L BrdU for the last 1 hr of incubation and processed as described.31 Briefly, cells were trypsinized, washed with PBS and fixed in 70% ethanol overnight. Cells were pelleted and nuclei were isolated by pepsin/HCl digestion followed by treatment with 10 mmol/L borate (pH 8.6) to neutralize the acid. Cells were then incubated with anti-BrdU antibody as described in the manufacturer’s protocol followed by incubation with FITC-labeled antimouse IgG and PI staining. Cell cycle data were collected on BD FACSCalibur Flow Cytometer and analyzed using CellQuest/MOD-Fit software.

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Immunoflourescent staining for γH2AX [1]
PC3 cells were plated in 4-well chamber slides (20,000 cells/ml/well) and treated with PX-478. At desired time interval, PX-478 was removed by aspirating the drug media and cells were irradiated and further incubated in drug-free media. At 6- and 24-hr phosphorylated histone, H2AX (γH2AX) foci were analyzed by immunoflourescent staining as described.32 Briefly, cells were fixed in 4% paraformaldehyde, permeablized with 0.1% NP-40 and blocked with 5% Goat serum in 1% BSA. Cells were covered with antiphospho-histone H2AX primary antibody (1:2,000) and incubated overnight at 4°C. After washing with 1%BSA, cells were treated with FITC Goat anti-rabbit secondary antibody (1:100) for 1 hr followed by 30 min DAPI (1 μg/mL) staining in the dark. Coverslips were mounted with an antifade solution. Slides were examined on a Leica DMRXA fluorescent microscope. Images were captured by a photometrics Sensys CCD camera and imported into IP Labs image analysis software package running on a Macintosh G3 computer. For each condition, ~70–100 cells from 2 to 3 separate experiments were analyzed to determine the number of γH2AX foci per cell.

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Isolation and Culture of Mesenchymal Stem Cells. [2]
Mouse mesenchymal stem cells were harvested from the tendon transection site originating from the calcaneus to the confluence of the fibula and tibia in wild-type mice. All tissue was mechanically minced and digested with collagenase A and dispase, and subsequently plated. To test drug treatment on Hif1α expression, cells were cultured in a hypoxia chamber with 0.5% oxygen. Cell treatment with PX-478(10 μM) or rapamycin (5 μM) was initiated 24 h before hypoxia treatment and redosed in hypoxia for 24 h. Protein was harvested and analyzed using Western blot for Hif1α and α-tubulin. To test effect of PX-478 treatment on chondrogenesis, cells isolated from the tendon were cultured in chondrogenic differentiation medium (PT-3925 and PT-4121; Lonza). All in vitro experiments were performed in biologic and technical triplicate.

Mice bearing MCF-7 human breast cancer, HT-29 colon cancer, PC-3 prostate cancer, DU-145 prostate cancer, OvCar-3 ovarian cancer, A-549 non-small cell lung cancer, SHP-77 small cell lung cancer, and Caki-1 renal cancer, Panc-1, MiaPaCa, or BxPC-3 pancreatic cancer xenografts.

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Extraskeletal Bone Models. [2]
Burn/tenotomy mice received a 30% TBSA partial-thickness burn on the shaved dorsum followed by left hindlimb Achilles’ tendon transection. The dorsum was burned using a metal block heated to 60 °C in a water bath and applied to the dorsum for 18 s continuously. The tenotomy site was closed with a single 5-0 vicryl stitch placed through the skin only.
caAcvr1fl:fl mice received hindlimb cardiotoxin and Ad.cre injection at postnatal day 24. Mice were then killed after 22 d (PX-478) or 15 d (rapamycin). Separate controls were used for each drug treatment to account for differences in the day of killing.
Nfatc1-Cre/caAcvr1fl:wt mice were generated by crossing Nfatc1-Cre+ mice with caAcvr1fl:wt mice. Resulting mutants developed extraskeletal bone by postnatal day 4–5.

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Drug Treatment. [2]
Burn/tenotomy or hybrid HO mice were administered PX-478 (100 mg/kg) or rapamycin (5 mg/kg) in PBS solution via intraperitoneal injection. Mice received injections every other day for the duration of the study. Nfatc1-Cre/caACVR1fl:wt mice were administered PX-478 (100 mg/kg) every other day for a total of 2 wk.

[1]. PX-478, an inhibitor of hypoxia-inducible factor-1alpha, enhances radiosensitivity of prostate carcinoma cells. Int J Cancer. 2008 Nov 15;123(10):2430-2437.

[2]. Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification. Proc Natl Acad Sci U S A. 2016 Jan 19;113(3):E338-47.

PX-478 is a small molecule hypoxia-inducible factor (HIF)-1α inhibitor currently undergoing clinical trials in patients with advanced metastatic cancer and lymphoma. PX-478 has shown efficacy in both non-small cell lung cancer and small cell lung cancer models expressing HIF-1α. The HIF-1α inhibitor PX-478 is an orally effective small molecule drug with potential antitumor activity. Although its mechanism of action is not fully elucidated, the HIF1α inhibitor PX-478 appears to inhibit the expression of hypoxia-inducible factor 1α (HIF1A), which may lead to reduced expression of downstream target genes of HIF1A, which are crucial for tumor growth and survival, thereby reducing tumor cell proliferation and inducing tumor cell apoptosis. The inhibitory effect of this drug is independent of the tumor suppressor genes VHL and p53 and may be related to glucose uptake and metabolic disorders caused by inhibition of glucose transporter-1 (Glut-1).

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Drug Indications
Investigated for the treatment of cancer/tumor (not specified).

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Mechanism of Action
PX-478 is a novel small molecule compound that inhibits the activity of hypoxia-inducible factor (HIF)-1α. HIF-1α is a transcription factor that controls the expression of many genes crucial for cancer cell growth and survival. HIF-1α-regulated genes are involved in multiple functions, including angiogenesis, glucose supply, and resistance to apoptosis (programmed cell death). Preclinical data indicate that PX-478 induces apoptosis in experimental tumor models and downregulates angiogenesis regulators such as vascular endothelial growth factor (VEGF). Solid tumors often contain heterogeneous hypoxic regions. In addition to diffusion-restricted chronic hypoxia present in cells 100–150 mm from blood vessels, some tumor cells may also experience perfusion-restricted intermittent hypoxia due to intermittent blood supply caused by aberrant tumor vasculature. Our data show that the novel HIF-1α inhibitor PX-478 reduces the survival rate of prostate cancer cells under normoxic and hypoxic conditions and enhances their radiosensitivity. Furthermore, treatment of cells with PX-478 under normoxic conditions, followed by irradiation after 1 hour of hypoxia, resulted in increased radiosensitivity, indicating that cells experiencing intermittent hypoxia can also respond to radiation if pre-exposed to PX-478. In addition to inhibiting HIF-1α, PX-478 also altered cell cycle progression and prolonged γH2AX expression in irradiated cells. Our data show that the radiosensitivity of cells irradiated under both normoxic and hypoxic conditions was enhanced, suggesting that PX-478 is a promising radiation modulator. PX-478-induced histone H2AX phosphorylation suggests that the drug may cause DNA damage, indicating potential drug toxicity. A preclinical study described that in non-immunodeficient C57BL/6 mice, daily administration of PX-478 for 5 consecutive days resulted in neutropenia as the main acute toxicity. ¹³ The drug is currently in Phase I clinical trials (PX-478-001 NCT00522652, http://clinicaltrials.gov), making it a potential new drug for treating hypoxic cells in radiotherapy. [1]

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Pathological extraosseous formation, or heterotopic ossification (HO), occurs after mechanical trauma, burns, orthopedic surgery, and in patients with hyperactivation mutations of type I bone morphogenetic protein receptor ACVR1 (activin type 1 receptor). Extraosseous formation is achieved through endochondral ossification, with cartilage as an intermediate, leading to the hypothesis that hypoxia signals present during cartilage formation drive heterotopic ossification (HO), and that HO precursor cells originate from the mesenchymal lineage defined by the pairwise related homeobox 1 (Prx). This study demonstrates that hypoxia-inducible factor-1α (Hif1α)—a key mediator of cellular adaptation to hypoxia—is highly expressed and active in three distinct HO mouse models: trauma-induced, genetic, and a hybrid genetic and trauma-induced model. In all these models, Hif1α expression was consistent with the expression of the cartilage master transcription factor Sox9 (Y-box 9, the sex-determining region). Pharmacological inhibition of Hif1α using PX-478 or rapamycin significantly reduced or inhibited extraosseous bone formation. Importantly, in treated mice, new soft tissue HO was eliminated or significantly reduced. Lineage-tracing mouse experiments showed that the cells forming ectopic ossification belong to the Prx lineage. Burn/tendon resection in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in a significant reduction in ectopic ossification, and again, no new soft tissue ectopic ossification was observed. Deletion of the Hif1α gene in Prx-cre labeled mesenchymal cells prevents the formation of mesenchymal aggregates, as confirmed by routine histology and immunostaining with Sox9 and PDGFRα. Pharmacological inhibition of Hif1α has a similar effect on the formation of mesenchymal aggregates. Our results suggest that Hif1α is a promising target for the prevention and treatment of pathological extraosseous lesions. [2]

C13H20CL4N2O3

392.0230

392.022

C, 39.62; H, 5.12; Cl, 35.98; N, 7.11; O, 12.18

685898-44-6

685898-44-6 (HCl); 685847-78-3;

11234794

Off-white to yellow solid powder

4.262

4

4

8

22

304

1

C1=CC(=CC=C1C[C@@H](C(=O)O)N)[N+](CCCl)(CCCl)[O-].Cl.Cl

GIGCDIVNDFQKRA-LTCKWSDVSA-N

InChI=1S/C13H18Cl2N2O3.2ClH/c14-5-7-17(20,8-6-15)11-3-1-10(2-4-11)9-12(16)13(18)19;;/h1-4,12H,5-9,16H2,(H,18,19);2*1H/t12-;;/m0../s1

4-[(2S)-2-amino-2-carboxyethyl]-N,N-bis(2-chloroethyl)benzeneamine oxide;dihydrochloride

PX478; PX-478; PX 478 dihydrochloride; 685898-44-6; PX-478; PX-478 2HCl; PX478; PX 478; PX-478 HCl; UNII-T23U22X160; (S)-4-(2-amino-2-carboxyethyl)-N,N-bis(2-chloroethyl)aniline oxide dihydrochloride; PX478 HCl;PX478 Hydrochloride;

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 (e.g. under nitrogen), 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: 78 mg/mL (197.9 mM) Water:78 mg/mL (197.9 mM) Ethanol: N/A

Solubility in Formulation 1: ≥ 5 mg/mL (12.69 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 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (12.69 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 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (12.69 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (6.34 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.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (6.34 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.

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Solubility in Formulation 6: ≥ 0.5 mg/mL (1.27 mM) (saturation unknown) in 1% DMSO 99% 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.

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Solubility in Formulation 7: 10% DMSO: 40% PEG300: 5% Tween-80: 45% Saline: ≥ 5 mg/mL (12.7 mM)

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Solubility in Formulation 8: 16.67 mg/mL (42.30 mM) in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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