Patent Application: US-46962299-A

Abstract:
this invention relates to a method of treating an inherited disease due to a point mutation producing a stop codon by administering an effective dose of an aminoglycoside antibiotic or a derivative thereof . mdx mouse , which is an animal model for duchenne muscular dystrophy , has been successfully treated with intramuscularly administered 1 and 5 mg gentamicin , which had for effect to suppress the premature stop mutation by inserting an amino acid at the stop codon . dystrophin positive muscle fibers not different in number from those of normal mouse were detected at the dose of 5 mg gentamicin .

Description:
in the present series of experiments , 4 mdx mice were treated with i . m . injections of gentamicin , two of them received 1 mg / day and the other two 5 mg / day during 7 days . control normal mice and mdx mice did not receive any gentamicin injections . all mice were then sacrificed , their skeletal muscle and their heart were frozen and cryostat sectioned . the presence of dystrophin in these sections was investigated by immunohistochemistry . strong dystrophin immunostaining was observed in the normal skeletal and heart muscles . in the untreated mdx muscles , dystrophin immunostaining was observed only in a few revertant fibers ( hoffman et al . 1990 ). dystrophin was detected by immunohistochemistry on all muscle fibers and all heart muscle cells of the mdx mice treated with gentamicin . in some muscles of mice treated with 5 mg / day of gentamicin the staining appeared as intense as that observed in the normal mouse muscles . the dystrophin staining was less intense in the muscles of mice treated with 1 mg / kg . this is the first demonstration that an aminoglycoside suppressed in vivo a premature stop codon . therefore a dose of 8 to 40 mg per kg of body weight per day successfully suppressed the premature stop codon by inserting an amino acid , and continuing translation of gentamicin - encoding nucleic acids subcutaneous and intravenous administration would provide the same results . the duration of treatment may vary . for the purpose of the present demonstration , the i . m . treatment lasted for 7 days . sub - cutaneous or intra - muscular daily treatments of 7 to 14 days or longer have been equivalently successful . in humans , the usual antibiotic dosage rate of gentamicin is of about 3 - 5 mg / kg / day , non - divided dose , for intramuscular route of administration . to avoid toxicity , it is recommended that gentamicin dosage rates be adjusted to avoid blood concentrations higher than 12 μg / ml for prolonged periods of time . for the purpose of using an optimally effective gentamicin dose , it may be necessary to administer maximal gentamicin doses achieving 12 μg / ml or higher blood concentration levels in mdx mice , a blood concentration which is about 65 μg / ml was achieved with a dose of about 35 - 40 gentamicin mg / kg / day ). no toxicity signs appeared in mdx mice . the blood circulation levels of gentamicin that would be effective in humans have not been calculated , but allometric calculation could bring a dose effective in mice close to an equivalent of about 1 . 5 to about 6 mg per kg per day in humans . it is possible to co - administer agents to counteract the oto - or nephrotoxicity of high doses of gentamicin , such as 2 , 3 dihydroxybenzoate ( dhb ) as disclosed by song and schacht ( 1996 ) and by pearce et al . ( 1985 ). alternatively , aminoglycoside derivatives having an activity towards inserting an amino acid instead of stopping translation may be designed , taking gentamicin as a reference compound . such gentamicin - like or derived molecules may be more potent than gentamicin and provide fewer toxic side effects . further , any mean by which gentamicin or gentamicin - like or derived molecules can be driven more directly to the site of action is within the scope of this invention . this would lead to more selective treatment with lower side effects liposomes or immunoliposomes may be envisageatle , for example , or else , conjugating a gentamicin - related molecule to a ligand specific to muscle tissue ( targeting muscle tissue as opposed to systemic dispersion ). any other disease caused by a stop mutation in nucleic acids of interest , such as certain types of hemophilia , are under the scope of this invention although the present invention has been described hereinabove by way of a preferred embodiment thereof , this embodiment can be modified at will , within the scope of the appended claims , without departing from the spirit and nature of the subject invention . anderson , m . d . s . and l . m . kunkel . 1992 . the molecular and biochemical basis of duchenne muscular dystrophy . elsevier science publishers 17 ; 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