4-hydroxyphenylpyruvate dioxygenase (4-HPPD) [1]

4-hydroxyphenylpyruvate dioxygenase (HPPD) with IC50 approximately 40 nM (rat liver) [2]

Nitisinone (compound 1) inhibited 4-HPPD activity in a concentration-dependent manner; at 100 nM, approximately 90% of enzyme activity was lost. Intracellular tyrosine accumulation in human fibroblasts (HfB) treated with Nitisinone increased by about 21% at 10⁻⁶ M and 32% at 10⁻⁵ M relative to control after 72 h, showing a dose-dependent effect. The LD50 after 24 h was >1 mM, and after 144 h was approximately 1 mM in HfB cells. [1]

Nitisinone (NTBC) at 50 nM reduced rat liver HPPD activity by >50% within 30 sec. The rate constant for inactivation (k) of rat liver HPPD by NTBC was 9.9 ± 2.5 × 10⁻⁵ sec⁻¹ nM⁻¹. The dissociation half-life (t½, 25°C) of the HPPD-NTBC complex was estimated at 63 h, with only 13.7 ± 1.0% of enzyme activity recovered over 7 h. [2]

In cultured human melanocytes from an OCA-1B patient, 1 mM tyrosine (not Nitisinone itself at 50 nM) increased pigmentation (P = 0.006). In CHO cells expressing H420R mutant tyrosinase, 1 mM tyrosine stabilized the mutant protein at 9 and 24 h (P < 0.05). In Melan-c cells expressing H420R tyrosinase, 1 mM tyrosine increased enzyme activity (P = 0.03). [3]

Over a daily distribution, nitisinone (5, 10 mg/kg; venous access; 5 weekly for 6 weeks) inhibits HPPD in a way that is both dose- and time-suppressive [2]. In OCA-1A and OCA-1B animals, nitisinone (4 mg/kg; venous access; once daily; 1 month apart) inhibits tyrosine [3]. In the OCA-1B model, nitisinone (4 mg/kg; once daily; one month apart) increases melanin concentration in ocular tissue melanosomes and pigmentation of the hair and iris [3]. Note: Type 1 Ocular-Clusive Albinism (OCA1). OCA-1A and OCA-1B are the two variants of OCA1. While some melanin is formed at night, the top lacks functional tyrosinase and so lacks melanin [3].

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Nitisinone (NTBC) administered orally to rats at 10 mg/kg caused elevation of plasma tyrosine (to about 2.5 mmol/mL, physiological level about 100 nmol/mL) and urinary excretion of 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA). [2]

In clinical trials (SONIA1), Nitisinone decreased urinary excretion of homogentisic acid (HGA) in a concentration-dependent manner; the observed increase in tyrosine was not clearly correlated with drug concentrations. [1]

In Tyrᶜ⁻ᵇ/ᶜ⁻ᵇ mice (OCA-1B model), oral Nitisinone at 4 mg/kg every other day for 1 month increased pigmentation in fur and iris, and increased the number of pigmented melanosomes in iris, choroid, and RPE (P < 0.001). Prenatal treatment (4 mg/kg daily from day 9-10 of pregnancy until birth, then every other day until weaning) also increased coat and iris pigmentation in pups. No effect was seen in Tyrᶜ⁻²/ᶜ⁻² mice (OCA-1A model). [3]

4-HPPD activity was measured using rat liver cytosol by monitoring oxygen consumption with an oxygen sensor. The reaction mixture contained 0.2 M sodium phosphate buffer (pH 7.4), 1.7 mM ascorbic acid, and 10.92 mg rat enzyme preparation, equilibrated at 37°C for 5 min. The reaction was initiated by adding 0.2 mM 4-hydroxyphenylpyruvate (HPP). Nitisinone or other test compounds were added at concentrations of 10⁻⁸, 10⁻⁷, 10⁻⁶, and 10⁻⁵ M. Percent inhibition was calculated as the ratio of oxygen consumption slopes (μM O₂/min) of samples versus untreated controls corrected with blanks. IC50 was calculated by sigmoidal curve fitting. [1]

HPPD activity was also assayed by measuring oxygen consumption using a YSI Biological Oxygen Monitor. The chamber contained 0.2 M sodium phosphate buffer (pH 7.2), 7.1 μmol ascorbic acid, and 200 μL enzyme preparation (rat liver cytosol) in 4 mL total volume, equilibrated at 37°C for 5 min. Reaction was started by adding 200 μmol HPPA. For inhibition studies, Nitisinone (NTBC) was preincubated with enzyme for 3 min before substrate addition. Alternatively, progress curves were obtained by co-administering substrate and inhibitor. A kinetic model was used to fit the time course of oxygen consumption and derive the inactivation rate constant k. [2]

Release of ¹⁴CO₂ from [carboxyl-¹⁴C]-HPPA was measured to determine HPPD activity. Rat liver cytosol was incubated with [carboxyl-¹⁴C]-HPPA, and the decarboxylation rate was linear over 20 min at 37°C. This method was used to assess recovery of HPPD activity after dissociation of Nitisinone from the enzyme-inhibitor complex. [2]

Cell viability assay[1]
Cell Types: human primary fibroblasts (HFb)
Tested Concentrations: 0.01-10 μM
Incubation Duration: 72 h Nitisinone (0.01-10 μM; 72 h) promotes tyrosine accumulation in a dose-dependent manner[ 1].
Experimental Results: In human cell cultures, tyrosine accumulated in large amounts within the cells.

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Cytotoxicity of Nitisinone was evaluated in human fibroblasts (HfB, 12 × 10⁴ cells/well) exposed to concentrations of 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴, and 10⁻³ M for 24 h and 144 h. Cell viability was quantified by MTT assay. LD50 values were calculated by sigmoidal curve fitting. [1]

Intracellular tyrosine levels were measured in HfB cells (9 × 10⁴ cells/well) treated with Nitisinone at 10⁻⁸, 10⁻⁷, 10⁻⁶, and 10⁻⁵ M for 72 h. Cells were collected in methanol containing norvaline (10⁻³ M as internal standard), lysed by freeze-thaw cycles, and the supernatant analyzed by LC-MS/MS. Tyrosine amount per 9 × 10⁴ cells was plotted versus compound concentration. [1]

CHO cells expressing wild-type or mutant (R77L, H420R) mouse tyrosinase were treated with cycloheximide (2 μg/mL) with or without 1 mM tyrosine. Protein stability was assessed by Western blot at 0, 1, 3, 6, 9, and 24 h; GAPDH was used as loading control. [3]

Melan-c cells (mouse melanocytes) were transfected with wild-type or mutant tyrosinase constructs and cultured with or without 1 mM tyrosine for 24 h. Diphenol oxidase activity was measured spectrophotometrically using 7 mM L-DOPA in 0.1 M sodium phosphate buffer (pH 6.8) at 475 nm. [3]

Human melanocytes from OCA-1A and OCA-1B patients were cultured to confluency and treated with 1 mM tyrosine for 1 week. Melanin content was measured by solubilizing in 2 N NaOH at 80°C for 1 h and reading OD₄₇₅, normalized to protein content. [3]

Animal/Disease Models: Male Wistar rat (120-150 g) [2].
Doses: 5, 10 mg/kg.
Doses: po (oral gavage); 5 days a week for 6 weeks.
Experimental Results: HPPD was inhibited in rat liver in a dose- and time-dependent manner. (Rat liver animal model, incubated with 0-200 nM nitisinone for 3 minutes).

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Animal/Disease Models: C57BL/6J mice (WT mice), Tyrc-2J/c-2J mice (OCA-1A model), and Tyrc-h/ch mice (OCA-1B model) (all 3 to 4 months old age) age) [3].
Doses: 4 mg/kg
Route of Administration: po (oral gavage); one time/day; once every other day for 1 month.
Experimental Results: After 1 month in the OCA-1A and OCA-1B models, plasma tyrosine levels increased 4- to 6-fold compared to placebo-treated controls. In the OCA-1B model, there is increased iris pigmentation and increased pigmentation in areas of physical new hair growth. The number of pigmented melanosomes was Dramatically increased in the OCA-1B model. The irises of Tyrc-h/ch pups born to nitisinone-treated mothers demonstrated extensive pigmentation.

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Nitisinone (NTBC) was dissolved in 0.1 M NaOH and adjusted to pH 7.0 with HCl. Male Wistar rats (120-150 g) were administered NTBC orally by gavage at 10 mg/kg (5 mL/kg), 5 days per week for 6 weeks. Urine was collected up to 24 h post-dose in containers cooled on dry ice. [2]

Nitisinone was dissolved in 2 M NaOH and brought to neutral pH before administration. C57BL/6J Tyrᶜ⁻²/ᶜ⁻² and Tyrᶜ⁻ᵇ/ᶜ⁻ᵇ mice (3-4 months old) received Nitisinone at 4 mg/kg every other day by oral gavage (volume 0.2-0.3 mL) for 1 month. A patch of fur was shaved to stimulate new hair growth. For prenatal treatment, pregnant Tyrᶜ⁻ᵇ/ᶜ⁻ᵇ females received Nitisinone 4 mg/kg daily by oral gavage starting at day 9-10 of pregnancy until birth, then every other day until weaning. [3]

Absorption, Distribution and Excretion
Capsules and liquid formulations are bioequivalent in terms of plasma concentration-time curves and maximum plasma concentration (Cmax). Biological Half-Life Approximately 54 hours.

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In clinical trials (SONIA1), pharmacokinetic parameters of Nitisinone such as area under the curve (AUC), maximum concentration (Cmax), and median oral clearance were obtained. [1]

In mice treated with Nitisinone at 4 mg/kg every other day for 1 month, plasma tyrosine levels increased from 109 ± 30 μM to 678 ± 73 μM in Tyrᶜ⁻²/ᶜ⁻² mice (P = 1 × 10⁻⁷), and from 74 ± 25 μM to 305 ± 35 μM in Tyrᶜ⁻ᵇ/ᶜ⁻ᵇ mice (P = 2 × 10⁻⁶). [3]

Hepatotoxicity
Type 1 tyrosinemia is a rare condition, and clinical experience with nitisidone is limited. Mild elevations in serum transaminase levels may occur during treatment, but are usually mild (below 3 times the upper limit of normal [ULN]) and often resolve spontaneously without dose adjustment. Elevated transaminase levels are usually asymptomatic and do not cause increases in serum alkaline phosphatase or bilirubin levels, thus rarely requiring dose adjustment. There are currently no reports of clinically significant liver injury in experimental studies of nitisidone treatment for tyrosinemia or for other tyrosine metabolism disorders (such as alkaptonuria). Probability Score: E (Unlikely to be the cause of clinically significant liver injury). Use during Pregnancy and Lactation ◉ Overview of Use During Lactation Blood concentrations of nitisidone in two exclusively breastfed infants of one mother were well below the therapeutic range and decreased from the first postpartum measurement to levels observed in the second week of breastfeeding. The infants experienced no adverse reactions and showed normal growth and development. If the mother needs to use nitisidone, breastfeeding does not need to be discontinued. Until more data are available, it is recommended to monitor the concentrations of nitisidone, tyrosine, and succinylacetone in the blood of breastfed infants.
◉ Effects on breastfed infants
A patient with type 1 tyrosinemia was taking nitisidone at a dose of 40 mg daily or 0.44 mg/kg daily, in addition to a low-tyrosine and phenylalanine diet. She continued taking the medication during her first pregnancy and postpartum. During her second pregnancy, the dose of nitisidone was increased to 60 mg daily at week 11 of gestation and to 80 mg daily at week 28, and continued at this dose postpartum. Both infants were exclusively breastfed for 2 weeks postpartum. They experienced no adverse reactions and showed normal growth and development.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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In human fibroblasts (HfB), short-term (24 h) LD50 of Nitisinone was >1 mM, and long-term (144 h) LD50 was approximately 1 mM. Intracellular tyrosine accumulation after 72 h treatment was dose-dependent: ~21% increase at 10⁻⁶ M and ~32% at 10⁻⁵ M relative to control. [1]

In rats, oral administration of Nitisinone (NTBC) at 10 mg/kg caused marked elevation of plasma tyrosine (to ~2.5 mmol/mL) and excretion of HPPA and HPLA in urine, indicating chemically induced tyrosinemia. [2]

In mice, Nitisinone at 4 mg/kg every other day for 1 month was well tolerated with no discernible side effects. No congenital malformations, systemic illnesses, or behavioral abnormalities were observed in pups from prenatally treated mothers. [3]

[1]. Inhibition of para-Hydroxyphenylpyruvate Dioxygenase by Analogues of the Herbicide Nitisinone As a Strategy to Decrease Homogentisic Acid Levels, the Causative Agent of Alkaptonuria. ChemMedChem. 2016 Apr 5;11(7):674-8.

[2]. Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione. Toxicol Appl Pharmacol. 1995 Jul;133(1):12-9.

[3]. Nitisinone improves eye and skin pigmentation defects in a mouse model of oculocutaneous albinism. J Clin Invest. 2011 Oct;121(10):3914-23.

[4]. Nitisinone: a review. Orphan Drugs: Research and Reviews, 2017, 7.

Nitisinone is a cyclohexanone compound with a structure of cyclohexane-1,3-dione substituted at the 2-position with a 2-nitro-4-(trifluoromethyl)benzoyl group. It is used to treat type 1 hereditary tyrosinemia. Nitisinone is an EC 1.13.11.27 (4-hydroxyphenylpyruvate dioxygenase) inhibitor. It belongs to the cyclohexanone, C-nitro, (trifluoromethyl)benzene, and mesotrione classes. Nitisinone is a synthetic, reversible 4-hydroxyphenylpyruvate dioxygenase inhibitor. It is used to treat type 1 hereditary tyrosinemia. Its trade name is Orfadin. Nitisinone is a 4-hydroxyphenylpyruvate dioxygenase inhibitor. The mechanism of action of nitisidone is as an inhibitor of phenylpyruvate dioxygenase, cytochrome P450 2C9, cytochrome P450 2E1, organic anion transporter 1, and organic anion transporter 3. Nitisidone is a tyrosine catabolism inhibitor used to treat hereditary tyrosinemia type 1, in which the accumulation of tyrosine metabolic intermediates leads to severe, progressive liver and kidney damage. Nitisidone is associated with mild, transient elevations in serum transaminases, but has not been found to be associated with clinically significant acute liver injury or jaundice. Drug Indications: For adjunctive treatment of hereditary tyrosinemia type 1, in conjunction with dietary restrictions on tyrosine and phenylalanine. FDA Label: For the treatment of adults and children (any age) diagnosed with hereditary tyrosinemia type 1 (HT1), in conjunction with dietary restrictions on tyrosine and phenylalanine.
For the treatment of adult and pediatric patients diagnosed with hereditary tyrosinemia type 1 (HT1), dietary restriction of tyrosine and phenylalanine is required.
Orfadin is indicated for the treatment of adult and pediatric patients (any age) with hereditary tyrosinemia type 1 (HT1). Diagnosis of hereditary tyrosinemia type 1 (HT1) combined with dietary restriction of tyrosine and phenylalanine. Alkaloid Urinary Ulcers (AKU) Orfadin is indicated for the treatment of adult patients with AKU.
Treatment of Type 1 Tyrosinemia

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Mechanism of Action
Nitrazone is a competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase, an upstream enzyme of fumaroacetoacetate hydrolase (FAH) in the tyrosine catabolism pathway. Nitrazone prevents the accumulation of catabolism intermediates maleate and fumaroacetoacetate by inhibiting normal tyrosine catabolism in patients with hereditary tyrosinemia type 1 (HT-1).

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Pharmacodynamics
Hereditary tyrosinemia type 1 is caused by a deficiency of fumaroacetylacetase (FAH), the last enzyme in the tyrosine catabolism pathway. Nitisidone inhibits tyrosine catabolism by preventing the formation of catabolism intermediates. In HT-1 patients, these catabolism intermediates are converted into the toxic metabolites succinylacetone and succinylacetate, leading to the observed hepatotoxicity and nephrotoxicity. Succinylacetone can also inhibit the porphyrin synthesis pathway, leading to the accumulation of 5-aminolevulinic acid, a neurotoxin and the culprit behind the porphyria crisis specific to HT-1.

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Nitisinone (also known as NTBC) is a potent time-dependent (tight-binding) inhibitor of 4-HPPD. It has been approved by the FDA for use “under exceptional circumstances” as a lifesaving agent in infants with hereditary tyrosinemia type I (HT-1), but may cause side effects such as visual disorders, liver failure, convulsions, and cognitive difficulties related to chronic high plasma tyrosine levels. Four clinical trials (NCT00107783, NCT01390077, NCT01828463 SONIA1, NCT01916382 SONIA2) have investigated Nitisinone for alkaptonuria (AKU); it consistently decreases homogentisic acid (HGA) but also causes serum tyrosine accumulation. [1]

Nitisinone (NTBC) was discovered to inhibit HPPD, leading to its clinical use as the first effective pharmacological therapy for hereditary tyrosinemia type I. The inhibition is reversible, with slow dissociation from the enzyme. [2]

Nitisinone elevates plasma tyrosine by inhibiting tyrosine catabolism. It is being investigated for oculocutaneous albinism type 1B (OCA-1B) to increase pigmentation and potentially improve visual function. Standard doses in humans are 1-2 mg/kg/day; in mice up to 160 mg/kg/day have been tolerated. [3]

C14H10F3NO5

329.23

329.051

104206-65-7

115355

Light yellow to yellow solid powder

1.5±0.1 g/cm3

486.2±45.0 °C at 760 mmHg

129-131°C

247.9±28.7 °C

0.0±1.2 mmHg at 25°C

1.535

1.37

0

8

2

23

524

0

MVOGOEKATQJYHW-CIAYNJNFSA-M

InChI=1S/C38H50N6O9S.K/c1-6-22-19-38(22,35(47)43-54(49,50)25-13-14-25)42-32(45)29-18-24-20-44(29)34(46)31(37(2,3)4)41-36(48)53-30-16-21(30)10-8-7-9-11-27-33(52-24)40-28-17-23(51-5)12-15-26(28)39-27;/h6,12,15,17,21-22,24-25,29-31H,1,7-11,13-14,16,18-20H2,2-5H3,(H3,41,42,43,45,47,48);/q;+1/p-1/t21-,22-,24-,29+,30-,31-,38-;/m1./s1

potassium ((1R,2S)-1-((33R,35S,91R,92R,5S)-5-(tert-butyl)-17-methoxy-4,7-dioxo-2,8-dioxa-6-aza-1(2,3)-quinoxalina-3(3,1)-pyrrolidina-9(1,2)-cyclopropanacyclotetradecaphane-35-carboxamido)-2-vinylcyclopropane-1-carbonyl)(cyclopropylsulfonyl)amide

MK5172 K; MK 5172 potassium; MK-5172 potassium, Trade name: Zepatier‎.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~41.67 mg/mL (~126.57 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.59 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 (7.59 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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 (Please use freshly prepared in vivo formulations for optimal results.)