A Mouse Model to Test the in vivo Efficacy of Chemical Chaperones

Line
Title: A Mouse Model to Test the in vivo Efficacy of Chemical Chaperones
Authors: Bai, Chunxue; Biwersi, Joachim; Verkman, Alan S.; Matthay, Michael A.
Publisher: Journal of Pharmacological and Toxicological Methods
Date Published: July 01, 1998
Reference Number: 486
Line
Abstract • Translation
In vitro studies in transfected cells have indicated that chemical chaperones including glycerol (0.5-1.2 M) and trimethylamine oxide (TMAO, 50-100 mM) can correct defective trafficking of some proteins, including deltaF508 CFTR in cystic fibrosis and AQP2 mutants in nephrogenic diabetes insipidus. To develop a mouse model to test the efficacy of chemical chaperones in vivo, glycerol and TMAO were administered by intraperitoneal (i.p.), subcutaneous (s.c.), and oral routes. Glycerol and TMAO assays that utilized 1-5 microL of tail vein blood were developed. Administration by the i.p. and s.c. routes gave maximum serum glycerol concentrations of approximately 100 mM, levels that were well below the effective in vitro concentrations. Single i.p. or s.c. doses of TMAO (7 g/kg, 8% solution in water) resulted in serum [TMAO] greater than 50 mM, with a long half-life (t1/2 approximately equal to 18-21 h). Sustained high serum and tissue [TMAO]. 52 mM for 3 days was achieved by s.c. administration of TMAO (7 g/kg) in water every 8 h. Although approximately 50% of the mice died with this multiple-dose regimen, the remaining mice had nearly normal liver, renal, and pancreatic function. A lower dose of TMAO (5 g/kg) given by the s.c. route every 8 h resulted in serum [TMAO] concentration of 22 mM, a level that was well tolerated by all mice for 72 h. These mice also had high [TMAO] in urine, 400 mM. These results demonstrate that potentially therapeutic concentrations of TMAO can be sustained in mice in vivo, permitting the testing of chemical chaperones in transgenic mouse models of diseases caused by defective protein trafficking.

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Chemical chaperones are low molecular compounds that can stabilize the shape of proteins in the face of thermal or chemical input that would otherwise break the proteins down. Some chemical chaperones have been shown to correct defects in the shapes of specific mutated proteins, thus allowing them to perform their function. (One such function is being able to move from one part of the cell to another. This is called protein trafficking.) However, these tests have been performed in laboratory cell cultures using high concentrations of the chemical chaperones. They have not been performed on living animals.

Trimethylamine oxide (TMAO) and glycerol are two of the most potent chemical chaperones, having shown in laboratory cell cultures that they can correct protein trafficking defects. In this paper, Bai, et al., report on their tests to determine if mice could tolerate TMAO and glycerol. If they could, mice could be used to see if these chemical chaperones could correct protein folding defects (i.e. proteins that end up misshaped as they are synthesized).

The chaperones were administered to the mice in several ways: orally, subcutaneously (s.c.), and intraperitonealy (i.p.). The researchers wanted to see if they could administer the necessary concentration of the chaperones (glycerol and TMAO) without causing a toxic reaction.

They found it was not possible to reach the concentration of glycerol in mice that researchers thought necessary for glycerol to correct protein folding defects. One-time low concentration s.c. and i.p. injections gave no indication of toxic side effects, but were too low to be effective. Larger doses caused lethargy and death. Oral administration of glycerol over a five day period resulted in glycerol concentration in the mice blood that was far too low.

The target concentrate of TMAO in the mice was 50 to 100 millimolars (mM) per mouse. Further, this dosage had to be sustained over a three-day period as cell culture models indicated that chemical chaperones had to be incubated with the protein whose defects they were intended to correct for one to three days.

To establish this concentration, 15 mice were given s.c. injections of TMAO equivalent to seven grams of TMAO for every kilogram of mouse weight. The mice received these injections every eight hours for a 72-hour period. Fifteen mice were given this regime. Eight died after the second or third injection. The seven surviving mice had lower TMAO concentrations than those that died. The researchers suggested that these lower concentrations (50 -100 TMAO) are well tolerated by the mice, as investigations showed these mice had nearly normal liver, kidney and pancreatic function.

The authors tested five other mice, giving them five grams of TMAO for every kilogram of their body weight every eight hours for 72 hours. This resulted in the mice having a blood serum TMAO level of 22mM. The mice tolerated this level over a 72 hour period.

These results suggest that mice can sustain potentially therapeutic concentrations of TMAO. This will allow researchers to test the effectiveness of TMAO's ability to help target proteins fold correctly and its ability to correct target proteins trafficking defects.