HIF-2α (IC50 = 9 nM)[1]

Belzutifan (PT2977) causes a rapid and quantitatively-dependent decrease in EPO expression and potently and quantitatively-dependently lowers the mRNA levels of human cyclin D1, a target gene regulated by HIF-2α [1].

Oral administration of PT2977 in mice, rats, dogs, and monkeys resulted in good plasma exposure.Comparison of mean plasma concentration–time profiles of PT2977 and PT2385 is shown in Figure 11, and summary of parent and glucuronide metabolite drug exposure (AUC) is provided in Table 5. Interestingly, the oral exposure of PT2977 was only slightly higher than PT2385 in rodents, while these two compounds behaved very differently in higher species. The dose-normalized AUC of PT2977 was 9- and 20-fold higher than that of PT2385 AUC in dogs and monkeys, respectively. This phenomenon turned out to be related to the relative rate of glucuronide metabolite formation for each analog in these species. As shown in Table 5, the AUC of the PT2977 glucuronide metabolite (PT3317) in dogs was about 30% of the parent, while the AUC of PT2639 (PT2385 glucuronide metabolite) was almost 2-fold higher than the parent. Similarly, a low amount of circulating metabolite was observed for PT2977 in monkeys, with a metabolite/parent ratio of 0.19. Both compounds formed significantly less amounts of glucuronide metabolites in rats, suggesting an alternative metabolic pathway in this species. For PT2385, dog pharmacokinetics were a better predictor of glucuronide metabolite PT2639 formation in humans. On the basis of these data, we predicted that PT2977 would have a reduced propensity for glucuronidation in humans and thus demonstrate a significantly improved pharmacokinetic profile over PT2385 in patients[1].

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A pharmacokinetic/pharmacodynamic (PK/PD) study was performed in mice bearing subcutaneous 786-O ccRCC tumors to correlate plasma drug levels of PT2977 with PD effects in the tumors. Six doses of PT2977 at 0.3, 1, and 3 mg/kg or PT2385 at 10 mg/kg b.i.d. were administered orally. Plasma and tumor tissue samples were collected 12 h after the last dose. Gene expression analyses in excised tumors by qPCR showed that PT2977 potently and dose-dependently reduced mRNA levels of human cyclin D1, a target gene regulated by HIF-2α (Figure 12A). Maximum PD response was achieved at 1 mg/kg for PT2977, while a much higher dose of 10 mg/kg was required for PT2385. And to achieve the same PD effect, the steady state trough plasma drug concentration of PT2977 was about 1/10 of PT2385 concentration (Figure 12B). Consistent with the improvement in potency and physical properties, compound PT2977 was about 10-fold more potent than PT2385 in vivo. PT2977 and PT2385 were subsequently evaluated for antitumor activity in the 786-O mouse xenograft model. Administration of PT2977 at 0.3, 1, and 3 mg/kg all led to rapid regression of established tumors (Figure 12C), confirming superior in vivo activity of compound PT2977 compared to PT2385[1].
aPT2385 and PT2977 were delivered as a suspension in 10% EtOH, 30% PEG400, 60% (0.5% methylcellulose, 0.5% Tween 80 (aq)).
bPT2385 and PT2977 were delivered as a suspension in 0.5% methylcellulose and 0.5% Tween 80 in water.
On the basis of its improved potency, free fraction, pharmacokinetics, and metabolite profile, compound PT2977 was predicted to have a low clinically active dose and less interpatient exposure variability. Therefore, PT2977 was chosen as our second-generation HIF-2α clinical candidate for further investigation as a potential new treatment for ccRCC and VHL disease[1].

UGT Phenotyping[1]
The test compound was incubated with a panel of individually expressed recombinant human UGT enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cell membranes. The incubation mixture contained the test compound at a final concentration of 50 μM, expressed UGT (0.2 mg protein/ml), Tris-HCl buffer (pH 7.5), magnesium chloride (10 mM), alamethicin (25 μg/mL), and UDPGA (2 mM). The mixture (without the UDPGA cofactor) was preincubated at 37 °C for 5 min, after which the reaction was started by the addition of UDPGA.
Incubations were performed at 37 °C. Aliquots (100 μL) were collected at 60 min and quenched with two volumes of ice-cold acetonitrile containing an internal standard. The samples were centrifuged at 10 000g at room temperature for 15 min to pellet precipitated proteins. The supernatants were then transferred to clean vials containing 200 μL of water and analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The glucuronide metabolite over internal standard peak area ratio was measured to quantify the glucuronide metabolite formation rate.

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Enzyme Kinetic Studies in Human Intestine Microsomes (HIM) and UGT2B17 Supersomes[1]
Similar experimental procedure to UGT phenotyping was used for enzyme kinetics studies of compounds in human intestinal microsomes and UGT2B17 supersomes except that a compound concentration range of 2–1000 μM was used in the incubations. The absolute glucuronide metabolite formation rate was measured by LC–MS/MS. The compound concentrations and its corresponding glucuronide metabolite formation rates were fitted to the standard Michaelis–Menten model to obtain the Vmax and Km by using Prism (version 6).

hERG Profiling of 2:[1]
The in vitro effects of compound 2 on the hERG (human ether-à-go-go-related gene) potassium channel current (a surrogate for IKr, the rapidly activating, delayed rectifier cardiac potassium current) expressed in mammalian cells were evaluated at near-physiological temperature. 2 inhibited hERG current by (Mean ± SEM) 6.7 ± 2.0% at 10 μM (n = 3) and 16.3 ± 0.4% at 50 μM (n = 3) versus 0.0 ± 0.6% (n = 3) in control. The IC50 for the inhibitory effect of 2 on hERG potassium current was not calculated but was estimated to be greater than 50 μM. The positive control (60 nM terfenadine) inhibited hERG potassium current by (Mean ± SD; n = 2) 85.2 ± 3.6%. This result confirms the sensitivity of the test system to hERG inhibition.

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Evaluation of 1 and 3 as Substrates of Human P-gp and BCRP Transporters: [1]
To determine if 1 (1 and 10 μM) and 3 (1 and 10 μM) are substrates of human efflux transporters (namely, P-gp [MDR1/ABCB1] and BCRP [ABCG2]), S5 the bidirectional permeability of PT2385 and PT2639 across MDCKII-MDR1 and MDCKIIBCRP cells was measured. Known substrates and inhibitors were included as positive controls in all experiments. The efflux ratios of 1 and 3 across MDCKII-MDR1 and MDCKII-BCRP cells was less than 2 at all conditions tested suggesting neither compound is a substrate of P-gp or BCRP.

Absorption, Distribution and Excretion
In patients with VHL disease-associated renal cell carcinoma, the mean Cmax and AUC0-24h at steady-state - which was achieved after approximately three days of therapy - were 1.3 µg/mL and 16.7 μg•hr/mL, respectively. The median Tmax is one to two hours following oral administration. The administration of belzutifan with food has a negligible effect on drug disposition - when given alongside a high-calorie, high-fat meal, the Tmax was delayed by approximately 2 hours with no other clinically meaningful effects observed.
The steady-state volume of distribution of belzutifan following oral administration is approximately 130 L.
The mean clearance of belzutifan following oral administration is 7.3 L/h.

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Metabolism / Metabolites
Belzutifan is primarily metabolized by UGT2B17 and CYP2C19, and to a lesser extent by CYP3A4.

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Biological Half-Life
The mean elimination half-life of belzutifan is 14 hours.

Hepatotoxicity
In the preregistration trials of belzutifan, serum aminotransferase elevations occurred in up to 20% of patients, but were invariably transient and mild (less than 3 times ULN) and were not accompanied by symptoms or jaundice. No patient required dose modification or discontinuation because of liver test abnormalities. Furthermore, since its more widespread use after its approval, there have been no published reports of clinically apparent liver injury attributed to belzutifan.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Plasma protein-binding is approximately 45%, although data regarding the specific proteins to which belzutifan binds are unavailable.

[1]. 3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile (PT2977), a Hypoxia-Inducible Factor 2α (HIF-2α) Inhibitor for the Treatment of Clear Cell Renal Cell Carcinoma. J Med Chem. 2019 Aug 8;62(15):6876-6893.

Belzutifan is an inhibitor of hypoxia-inducible factor 2α (HIF-2α) used in the treatment of von Hippel-Lindau (VHL) disease-associated cancers. The HIF-2α protein was first identified in the 1990s by researchers at UT Southwestern Medical Center as a key player in the growth of certain cancers. Initially considered to be undruggable, a binding pocket was eventually discovered in the HIF-2α molecule which allowed for compounds to bind and inhibit these proteins. This discovery led to the initial development of belzutifan (at the time called PT2977), which was further developed by a spin-off company named Peloton Pharmaceuticals (which itself was eventually acquired by Merck in 2019). Belzutifan inhibits the complexation of HIF-2α with another transcription factor, HIF-1β, a necessary step in its activation - by preventing the formation of this complex, belzutifan can slow or stop the growth of VHL-associated tumors. Belzutifan received FDA approval for the treatment of select VHL-associated cancers on August 13, 2021.
Belzutifan is a Hypoxia-inducible Factor Inhibitor. The mechanism of action of belzutifan is as a Hypoxia-inducible Factor 2 alpha Inhibitor, and Cytochrome P450 3A4 Inducer.
Belzutifan is a small molecule inhibitor of hypoxia-inducible factor 2 alpha used to treat solid tumors in patients with von Hippel-Lindau disease. Belzutifan is associated with a low rate of minor serum enzyme elevations during therapy, but has not been linked to cases of clinically apparent liver injury.
Belzutifan is an orally active, small molecule inhibitor of hypoxia inducible factor (HIF)-2alpha (HIF-2a), with potential antineoplastic activity. Upon oral administration, belzutifan binds to and blocks the function of HIF-2alpha, thereby preventing HIF-2alpha heterodimerization and its subsequent binding to DNA. This results in decreased transcription and expression of HIF-2alpha downstream target genes, many of which regulate hypoxic signaling. This inhibits cell growth and survival of HIF-2alpha-expressing tumor cells. HIF-2alpha, the alpha subunit for the heterodimeric transcription factor HIF-2, is overexpressed in many cancers and promotes tumorigenesis.

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Drug Indication
Belzutifan is indicated for the treatment of adult patients with von Hippel-Lindau (VHL) disease who require therapy for associated renal cell carcinoma (RCC), central nervous system (CNS) hemangioblastomas, or pancreatic neuroendocrine tumors (pNET), who do not require immediate surgery.

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Mechanism of Action
Hypoxia-inducible factor 2α (HIF-2α) is a transcription factor which aids in oxygen sensing by regulating genes that promote adaptation to hypoxia. In healthy patients, when oxygen levels are normal, HIF-2α is broken down via ubiquitin-proteasomal degradation by von-Hippel Lindau (VHL) proteins. In the presence of hypoxia, HIF-2α translocates into cell nuclei and forms a transcriptional complex with hypoxia-inducible factor 1β (HIF-1β) - this complex then induces the expression of downstream genes associated with cellular proliferation and angiogenesis. Patients with von-Hippel Lindau (VHL) disease lack functional VHL proteins, leading to an accumulation of HIF-2α, and this accumulation is what drives the growth of VHL-associated tumors. Belzutifan is an inhibitor of HIF-2α that prevents its complexation with HIF-1β in conditions of hypoxia or impaired VHL protein function, thereby reducing the expression of HIF-2α target genes and slowing/stopping the growth of VHL-associated tumors.

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Pharmacodynamics
Belzutifan exerts its therapeutic effects by inhibiting a transcription factor necessary for the growth of solid tumors associated with VHL disease. It is taken once daily at approximately the same time each day, with or without food. Both severe anemia and hypoxia have been observed following therapy with belzutifan, and patients should be monitored closely before and during therapy to ensure patients can be managed as clinically indicated. There are no data regarding the use of erythropoiesis-stimulating agents for the treatment of belzutifan-induced anemia, and as such these therapies should be avoided. Belzutifan may cause embryo-fetal toxicity when administered to pregnant women. Female patients and male patients with female partners of reproductive potential should ensure that an effective form of contraception is used throughout therapy and for one week after the last dose - as belzutifan appears to decrease the efficacy of systemic hormonal contraceptives, patients should be advised to use an additional method of contraception (e.g. condoms) to eliminate the possibility of pregnancy during therapy.

C17H12F3NO4S

383.341693878174

383.04

C, 53.27; H, 3.16; F, 14.87; N, 3.65; O, 16.69; S, 8.36

1672668-24-4

117947097

White to light yellow solid powder

2

1

8

3

26

675

3

CS(=O)(=O)C1=C2[C@@H]([C@@H]([C@@H](C2=C(C=C1)OC3=CC(=CC(=C3)C#N)F)F)F)O

LOMMPXLFBTZENJ-ZACQAIPSSA-N

InChI=1S/C17H12F3NO4S/c1-26(23,24)12-3-2-11(13-14(12)17(22)16(20)15(13)19)25-10-5-8(7-21)4-9(18)6-10/h2-6,15-17,22H,1H3/t15-,16-,17+/m1/s1

3-[(1S,2S,3R)-2,3-Difluoro-1-hydroxy-7-methylsulfonylindan-4-yl]oxy-5-fluorobenzonitrile

PT2977; MK 6482; PT-2977; MK-6482; PT 2977; MK6482; Belzutifan; 1672668-24-4; PT2977; Welireg; MK-6482; MK6482; PT-2977; Belzutifan [INN]; Welireg

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)

Acetone : 50 mg/mL (~130.43 mM)
DMSO : ~50 mg/mL (~130.43 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 0.5 mg/mL (1.30 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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 (Please use freshly prepared in vivo formulations for optimal results.)