Methods of administering tetrahydrobiopterin, associated compositions, and methods of measuring

Abstract

The present invention is directed to treatment methods of administering tetrahydrobiopterin, including in oral dosage forms, in intravenous formulations, and with food. Also disclosed herein are biopterin assays for measuring the amount of biopterin and metabolites of biopterin in a sample.

1 . A method of orally administering tetrahydrobiopterin (BH4), comprising administering to a human in need thereof a therapeutically effective amount of BH4 or a pharmaceutically acceptable salt thereof, and informing said human that absorption of said BH4 or pharmaceutically acceptable salt thereof is increased when it is ingested with food compared to when ingested without food. 
     
     
         2 .- 12 . (canceled) 
     
     
         13 . A method of increasing gut retention time of tetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof in a subject comprising administering a formulation to said subject, said formulation comprising (1) BH4 or pharmaceutically acceptable salt thereof and (2) an agent that slows gut motility,
 wherein the BH4 or pharmaceutically acceptable salt thereof administered via the formulation has a longer gut retention time in comparison with a control formulation of BH4 or pharmaceutically acceptable salt thereof not having an agent that slows gut motility.   
     
     
         14 .- 19 . (canceled) 
     
     
         20 . An oral formulation of tetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof, comprising BH4 or pharmaceutically acceptable salt thereof and an agent that slows gut motility. 
     
     
         21 .- 25 . (canceled) 
     
     
         26 . A liquid formulation of tetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof, comprising an aqueous solution of BH4 or pharmaceutically acceptable salt thereof, an antioxidant, and a pH buffer. 
     
     
         27 . A dry powder formulation of tetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof for constitution into an aqueous solution, comprising a dry powder mixture of BH4 or pharmaceutically acceptable salt thereof, an antioxidant, and a pH buffer. 
     
     
         28 .- 37 . (canceled) 
     
     
         38 . A method of making a liquid formulation of tetrahydrobiopterin (BH4) or a pharmaceutically acceptable salt thereof, comprising
 providing an aqueous solution containing BH4 or pharmaceutically acceptable salt thereof;   adding an antioxidant and a pH buffer to the solution containing BH4 or pharmaceutically acceptable salt thereof;   sparging the aqueous solution containing BH4 or pharmaceutically acceptable salt thereof, before or after addition of antioxidant and pH buffer, with an inert gas or carbon dioxide; and   sealing the sparged solution containing BH4 or pharmaceutically acceptable salt thereof, antioxidant, and pH buffer in a container.   
     
     
         39 .- 44 . (canceled) 
     
     
         45 . A method of separating dihydrobiopterin and biopterin, or analogs thereof, from a mixture containing both base and dihydro forms, comprising:
 performing reverse phase HPLC using a mobile phase comprising an aqueous solution comprising methanol, sodium acetate, citric acid, EDTA, and 1,4-dithioerythritol, on a mixture containing dihydrobiopterin and biopterin, or an analog of dihydrobiopterin and an analog of biopterin.   
     
     
         46 .- 48 . (canceled) 
     
     
         49 . A method of measuring biopterins using reverse phase HPLC coupled with tandem mass spectrometry (LC/MS/MS), comprising:
 subjecting samples of blood, plasma, tissue homogenates, or urine comprising biopterins to oxidation;   subjecting the oxidized samples to iodometry;   passing the oxidized samples through an ion exchange column;   measuring total and oxidized biopterins in the samples using HPLC and tandem mass spectrometry; and   calculating the amount of reduced biopterin as the difference between the total biopterins less the oxidized form.   
     
     
         50 .- 51 . (canceled) 
     
     
         52 . A method of quantitating biopterins in a mixture of biopterin species, comprising:
 providing a mixture comprising biopterin and at least one of dihydrobiopterin and tetrahydrobiopterin, or analogs of biopterin and at least one of dihydrobiopterin and tetrahydrobiopterin;   separating the biopterin species in the mixture by reverse phase HPLC; and   in the case of tetrahydrobiopterin and analogs thereof, performing electrochemical detection by oxidizing the tetrahydrobiopterin and analogs thereof present by a first electrode to quinonoid dihydrobiopterin forms, followed by reducing the quinonoid forms back to tetrahydrobiopterin and analogs thereof present at a second electrode, and measuring current generated by the reduction reaction to determine the concentration of species; and/or   in the case of dihydrobiopterin, analogs thereof, biopterin, or analogs thereof, measuring such species by fluorescence detection following post-column oxidation of dihydrobiopterin species to biopterin.   
     
     
         53 . (canceled)