R261Q PAH Mouse

Description

PAH-R261Q Mouse data for sepiapterin

Steps to reproduce

20 (10/sex) mice were used to determine the effect of sepiapterin (SP) when administered one time by oral gavage. Animals were randomly assigned to each group, with five mice in each group. The test article was administered orally via oral gavage. Oral administration is the intended route of administration to humans. Phenylalanine was administered by i.p for the Phe-challenge. SP in vehicle (Oral Mix by Medisca) was administered one time by oral gavage. Phenylalanine was administered once by i.p. The dosage volumes were calculated based on the body weight measured the day before. The formulations were vortexed before each administration. Mice (Mus musculus) / Pah-R261Q from a C57Bl6/J background were used. Up until the day before treatment the mice were supplied with rodent feed from a contract vendor, ad libitum. The day before treatment started, the mice were switched to a specially produced Phe-free diet (from Research Diet, made by request). This switch was made to avoid fasting the animals before blood samples. Animals were provided purified water (filtered through a 1.2 µm filter) ad libitum by water bottles. SP and vehicle doses were administered by oral gavage. The dose level (5 mL/kg) was well below the maximum volume for oral gavage in mice. Phenylalanine was administered by i.p. with a 30G insulin syringe. The dose level (8.33 mL/kg) was below the maximum volume for i.p. injections in mice. Blood samples were collected from all study animals five times during the experiment; on -1 Day and 0.5h, 1.5, 3h, and 5h after Phe injection and at euthanasia (6h). Microsampling sticks (Mitra sticks from Neoteryx) was used to collect 10 µL from the saphenous vein at each blood collection. Phe levels in the blood samples were measured by extracting amino acids from dried blood and using fluorometric detection to analyze the samples on a high performance liquid chromatography (HPLC) system. After the fifth blood sample, the mice were euthanized by cervical dislocation. Livers were harvested within 2 min and flash frozen in liquid nitrogen and stored at -80°C. The livers were ground into a fine powder, homogenized, and then divided into tubes of ~200 mg to allow for several fresh lysates to be made. Liver powder was stored at -80°C. The same lysate was used for measuring PAH enzyme activity. In vitro PAH activity was measured by extracting PAH from powdered liver. The liver lysates were made fresh from powdered liver aliquots. The liver extract/lysate was mixed with Fe(II) and Phe to activate PAH, before BH4 was added to start the catalyzed reaction. After the reaction had stopped, the amount of L-tyrosine produced in the sample was quantified by fluorometric detection on a high performance liquid chromatography (HPLC) system. The reaction is repeated three times for each sample.

Institutions

• PTC Therapeutics (United States)

  New Jersey, South Plainfield

Categories

Funders

• PTC Therapeutics (United States)

  New Jersey, South Plainfield

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