- Plasma kallikrein (PKa). [2][3] - In HAE patients, a 150 mg once-daily dose of Berotralstat significantly reduces the HAE attack rate. [3]

- In a mouse whole blood (WB) thrombin generation (TG) assay triggered with silica to activate the contact pathway, 8 μM Berotralstat partially inhibited TG in both wild-type (F12+/+) and FXII-deficient (F12-/-) mouse WB. In F12-/- WB, Berotralstat significantly reduced the peak TG by approximately 35% compared to vehicle control. [1]
- Berotralstat (8 μM) had no observable effect on mouse WB TG initiated with a low dose of tissue factor (TF, 0.05 pM), indicating no significant impact on the extrinsic pathway. [1]
- In FXII-deficient mouse platelet-rich plasma (PRP), Berotralstat did not significantly reduce peak TG; however, when red blood cells (RBCs) were added back to PRP, Berotralstat significantly reduced peak TG, suggesting the RBC surface contributes to the FXII-independent function of PKa. [1]

- A phase 3 clinical trial in patients with hereditary angioedema (HAE) demonstrated that once-daily oral Berotralstat (110 mg or 150 mg) significantly reduced the rate of investigator-confirmed HAE attacks compared to placebo over 24 weeks. The model-based monthly attack rate for the 150 mg group was 1.31, compared to 2.35 for placebo (P < .001). [3]
- In HAE patients, the 150 mg Berotralstat group showed a significantly higher proportion of patients achieving a ≥50% reduction in attack rate from baseline (58% vs 25%, OR = 3.913, P = .005) and a ≥70% reduction (50% vs 15%, OR = 5.63, P = .0016) compared to placebo. [3]
- Both Berotralstat doses (110 mg and 150 mg) significantly reduced the rate of investigator-confirmed attacks treated with on-demand standard of care (SOC) medication and the rate of SOC medication use compared to placebo. For example, the model-based rate ratio for SOC doses per month for the 150 mg group was 0.46 (95% CI = 0.31-0.70, P < .001). [3]

- Mouse whole blood thrombin generation assay: ZGGR-AMC thrombin substrate was added to wells of a round-bottom 96-well plate containing whole blood and incubated at 37°C for 10 minutes. A trigger solution containing CaCl2 and an initiator (silica or tissue factor) was then added. The mixture was gently mixed, and 60 μL was dispensed into a polyvinyl chloride round-bottom 96-well plate. Fluorescence was measured continuously at 37°C using a fluorometer at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Data were acquired over 36 seconds with an integration time of 6 seconds. To assess Berotralstat, samples were supplemented with 8 μM Berotralstat or vehicle control before the assay. [1]
- Thrombin generation assay in platelet-rich plasma (PRP) and RBC-reconstituted PRP: Washed RBCs at 7×10^6/μL were added to an equal volume of PRP (two-fold dilution) and compared to PRP alone (three-fold dilution). TG was assessed using the same method as described for whole blood. [1]

- Mouse whole blood thrombin generation assay: Blood was collected from the inferior vena cava of anesthetized mice into sodium citrate (0.38% v/v) and corn trypsin inhibitor (CTI, 50 μg/mL). WB was used within 1 hour of collection. ZGGR-AMC substrate was added to WB and incubated at 37°C for 10 minutes, followed by the addition of a solution containing CaCl2 (final concentration 9 mM) and trigger (silica, final dilution 1:120; or TF, final concentration 0.05 pM or 0.5 pM). TG was monitored continuously using a fluorometer at 37°C. To evaluate Berotralstat, 8 μM of the inhibitor or vehicle control was added to the samples. [1]
- Phase 3 clinical trial (APeX-2) in HAE patients: A 24-week, randomized, double-blind, placebo-controlled, parallel-group, multicenter study. Eligible patients (C1-INH-deficient HAE, age ≥12 years, with ≥2 confirmed attacks during a 56-day run-in period) were randomized 1:1:1 to receive once-daily oral Berotralstat 110 mg, 150 mg, or placebo. Patients recorded HAE attacks daily in an electronic diary. The primary efficacy endpoint was the rate of investigator-confirmed HAE attacks during the 24-week treatment period. [3]

Absorption, Distribution and Excretion
Following the first dose, berottripstat reaches steady-state plasma concentrations within 6 to 12 days. With once-daily dosing, the peak steady-state plasma concentration (Cmax) and area under the curve (AUC) of berottripstat are approximately five times that of a single dose. After once-daily oral administration of berottripstat, the steady-state Cmax was 158 ng/mL (range: 110 to 234 ng/mL) at the 150 mg dose group and 97.8 ng/mL (range: 63 to 235 ng/mL) at the 110 mg dose group. The area under the curve (AUCτ) over the dosing interval was 2770 nghr/mL (range: 1880 to 3790 nghr/mL) and 1600 nghr/mL (range: 950 to 4170 nghr/mL) at the 110 mg dose. The median time to peak concentration (Tmax) in the fasting state is 2 hours, which is delayed to 5 hours after a high-fat meal. Tmax ranges from 1 to 8 hours. Following a single oral dose of 300 mg of radiolabeled berottripstat, approximately 9% of the drug is excreted in the urine, of which 1.8% to 4.7% of the radiolabeled compound is unmetabolized parent drug. Approximately 79% of the drug is excreted in the feces. Following a single dose of 300 mg of radiolabeled berottripstat, the plasma concentration-to-dose ratio is approximately 0.92. Clearance information is currently unavailable. Metabolites/Metabolites: Berottripstat is metabolized by CYP2D6 and CYP3A4. The metabolic pathways and metabolites of berottripstat are not well understood. Following a single oral dose of 300 mg of radiolabeled berottripstat, approximately 34% of the total plasma radioactivity is the parent drug, and approximately eight detectable metabolites account for 1.8% to 7.8% of the total radioactivity.

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Biological Half-Life
Following a single oral dose of 300 mg of radiolabeled berottripstat, the median elimination half-life of berottripstat is approximately 93 hours, ranging from 39 to 152 hours.
- In HAE patients taking once-daily oral Berotralstat 150 mg, the steady-state maximum plasma concentration (Cmax) is 158 ng/mL. [2]
- The half-life of Berotralstat is 3 to 4 days. [2]

Hepatotoxicity
In pre-registration trials, 2% to 5% of patients treated with berottripnical experienced mild, transient elevations in serum transaminases, compared to 10% in the placebo group. Transaminases exceeding the upper limit of normal (ULN) by 5 times were uncommon (less than 1%). Furthermore, bilirubin levels remained normal, and no patients developed symptomatic acute liver injury. In many cases, liver dysfunction is caused by other factors, including pre-existing non-alcoholic fatty liver disease, chronic viral hepatitis, or gallstones. Nevertheless, a small number of patients require discontinuation of the drug due to liver dysfunction. Since its approval and widespread clinical use, there have been no published reports of acute liver injury caused by berottripnical. Probability Score: E (Unproven, but suspected as a rare cause of clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of medication use during lactation
Bellotrastulstat is a plasma kallikrein inhibitor indicated for the prevention of hereditary angioedema. There is currently no information on whether berottractul is excreted into breast milk. Because berottractul binds to plasma proteins at a rate of approximately 99%, its concentration in breast milk is likely to be very low. If a mother needs to take berottractul, she should not discontinue breastfeeding. Until more data is available, especially during the nursing of newborns or premature infants, alternative medications may be preferred.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Plasma protein binding is approximately 99%.

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- In the 24-week clinical trial in HAE patients, the most frequent treatment-emergent adverse events (TEAEs) with Berotralstat were gastrointestinal symptoms (110 mg: 42% [17/41]; 150 mg: 50% [20/40]; placebo: 36% [14/39]), including abdominal pain, vomiting, and diarrhea. These were generally Grade 1 or 2, of short duration (median 2 days for the 150 mg group), and occurred primarily within the first month of treatment. [3]
- The percentage of patients experiencing any TEAE was similar in the Berotralstat 150 mg group (85%, 34/40) and the placebo group (77%, 30/39). Discontinuation due to TEAEs was also similar (150 mg: 3% [1/40]; placebo: 3% [1/39]). [3]
- No drug-related serious TEAEs occurred. No Grade 3 or 4 TEAEs were reported in the 150 mg group. [3]
- One asymptomatic Grade 4 alanine aminotransferase (ALT) elevation was reported during Berotralstat therapy (150 mg group) in a patient with prior androgen exposure, leading to study drug discontinuation. [3]
- Common side effects of Berotralstat (approximately 10%) include nausea, diarrhea, headache, and gastroesophageal reflux, typically low-grade. Minor asymptomatic hepatic transaminase elevations are occasionally seen. [2]

[1]. Plasma kallikrein supports FXII-independent thrombin generation in mouse whole blood. Blood Adv. 2024 Jun 25;8(12):3045-3057.
[2]. Reducing Brain Edema Using Berotralstat, an Inhibitor of Bradykinin, Repurposed as Treatment Adjunct in Glioblastoma. Neuroglia. 2024, 5(3), 223-233.
[3]. Oral once-daily berotralstat for the prevention of hereditary angioedema attacks: A randomized, double-blind, placebo-controlled phase 3 trial. J Allergy Clin Immunol. 2020;S0091-6749(20)31484-6.

Berotralstat is a selective kallikrein inhibitor used to prevent attacks of hereditary angioedema (HAE). Its mechanism of action involves blocking the enzymatic activity of kallikrein, inhibiting the release of bradykinin. Bradykinin is the main bioactive peptide that promotes swelling and pain associated with HAE attacks. Berotralstat is for the prevention of HAE attacks only and is not intended for treatment. Developed by BioCryst Pharmaceuticals and marketed under the brand name Orladeyo, berotralstat is available in oral capsule form. On December 3, 2020, berotralstat received its first approval from the U.S. Food and Drug Administration (FDA), becoming the first once-daily oral HAE prevention medication for adults and children aged 12 years and older. On April 30, 2021, berotralstat received approval from the European Commission; and on June 6, 2022, it received approval from Health Canada. Berotralstat is a kallikrein inhibitor. Bellotrostat's mechanism of action is as a kallikrein inhibitor, cytochrome P450 2D6 inhibitor, cytochrome P450 3A4 inhibitor, and P-glycoprotein inhibitor. Bellotrostat is a small-molecule plasma kallikrein inhibitor used to prevent acute exacerbations of hereditary angioedema (HAE) in adults and children aged 12 years and older. Mild to moderate elevations in serum transaminase levels occasionally occur during treatment with bellotrostat, but have not been found to be associated with clinically significant liver damage (with symptoms or jaundice). See also: Bellotrostat hydrochloride (active ingredient). Drug Indications Bellotrostat is indicated for the prevention of acute exacerbations of hereditary angioedema (HAE) in adults and children aged 12 years and older. It is not used to treat acute exacerbations of hereditary angioedema (HAE). Orladeyo is indicated for the routine prevention of relapses of hereditary angioedema (HAE) in adults and adolescents aged 12 years and older.
Treatment of Hereditary Angioedema

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Mechanism of Action
Hereditary angioedema (HAE) is a rare genetic disorder characterized by severe swelling of the skin and upper respiratory tract. It is caused by mutations in the regulatory or coding regions of the gene encoding a C1 inhibitor (SERPING1), resulting in a deficiency (Type I) or dysfunction (Type II) of the C1 inhibitor (C1 esterase inhibitor, C1-INH). The C1 inhibitor is a serine protease inhibitor that normally regulates bradykinin production by covalently binding to and inactivating plasma kallikrein. Plasma kallikrein is a protease that cleaves high molecular weight kininogen (HMWK) to generate cleaved high molecular weight kininogen (cHMWK). During an episode of hereditary angioedema (HAE), plasma kallikrein levels decrease, leading to the cleavage of high molecular weight kininogen and the release of bradykinin. Bradykinin is a potent vasodilator that increases vascular permeability. Bradykinin plays a crucial role in promoting edema and pain associated with hereditary angioedema (HAE). Due to a deficiency or dysfunction of serum C1 inhibitors, HAE patients are unable to properly regulate plasma kallikrein activity, leading to an uncontrolled increase in plasma kallikrein activity and triggering recurrent episodes of angioedema. Berottroxate is a potent plasma kallikrein inhibitor whose mechanism of action is to control excessive bradykinin production by binding to plasma kallikrein and blocking its proteolytic activity.

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Pharmacodynamics
Berotroxate prevents angioedema attacks by inhibiting plasma kallikrein, thereby regulating excessive bradykinin production in patients with hereditary angioedema (HAE). Clinical trials have shown that berottroxate has a rapid onset, long duration of action, and good tolerability. Berottroxate inhibits plasma kallikrein in a concentration-dependent manner. Clinical trials have shown that berottroxate reduces the incidence of HAE attacks at 24 weeks, and the effect can last up to 48 weeks. Clinical trials have also found that once-daily doses of berottrastatin exceeding 150 mg can lead to QT interval prolongation in a concentration-dependent manner.

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- Berotralstat (brand name Orladeyo™) is approved by the FDA and EMA for prophylaxis of hereditary angioedema (HAE) attacks in adults and pediatric patients 12 years and older. It is the first approved oral plasma kallikrein inhibitor. [2][3]
- In the context of glioblastoma (GB), Berotralstat could be beneficial by inhibiting bradykinin generation, leading to potential effects such as inhibiting tumor cell migration and proliferation, reducing peritumoral brain edema, and potentially reducing the need for corticosteroids like dexamethasone, whose use is associated with shorter survival in GB patients. [2]

C30H26F4N6O

562.560659885406

562.21

C, 64.05; H, 4.66; F, 13.51; N, 14.94; O, 2.84

1809010-50-1

Berotralstat dihydrochloride;1809010-52-3

137528262

White to off-white solid powder

4.5

3

9

9

41

938

1

FC1C=CC(=CC=1NC(C1=CC(C(F)(F)F)=NN1C1C=CC=C(CN)C=1)=O)[C@@H](C1C=CC=C(C#N)C=1)NCC1CC1

UXNXMBYCBRBRFD-MUUNZHRXSA-N

InChI=1S/C30H26F4N6O/c31-24-10-9-22(28(37-17-18-7-8-18)21-5-1-3-19(11-21)15-35)13-25(24)38-29(41)26-14-27(30(32,33)34)39-40(26)23-6-2-4-20(12-23)16-36/h1-6,9-14,18,28,37H,7-8,16-17,36H2,(H,38,41)/t28-/m1/s1

1-[3-(aminomethyl)phenyl]-N-(5-{(R)-(3- cyanophenyl)[(cyclopropylmethyl)amino]methyl}-2- fluorophenyl)-3-(trifluoromethyl)-1H-pyrazole-5- carboxamide

ORLADEYO; BCX-7353; BCX 7353; Berotralstat; 1809010-50-1; BCX7,353; BCX-7,353; XZA0KB1BDQ; BCX7353

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 : ~100 mg/mL (~177.76 mM)

Solubility in Formulation 1: ≥ 8.5 mg/mL (15.11 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 85.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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