Patent Application: US-84698007-A

Abstract:
the invention is intended for a treatment of severe infections using an injectable drug - delivery system comprising nanoparticles of a biodegradable polymer with incorporated antibacterial drug .

Description:
nanoparticles with incorporated antibiotics were prepared by double emulsion technique , or by nanoprecipitation at different drug - to - polymer ratios and water soluble coadjuvants were added to water phase in various concentrations . after elimination of organic solvents , a suspension of formed nanoparticles was concentrated and filtered through a microporous filter membrane . the particle size was measured by photon correlation spectroscopy ( malvern zetasizer nano - s ). for evaluation of drug loading in np , a free drug was separated by ultrafiltration ( separation membrane with molecular cutoff 30 , 000 or 300 , 000 nmwl ) and its concentration was measured by hplc . streptomycin in biodegradable polymeric nanoparticles ( examples 1 - 34 ). 50 - 500 mg of antibiotic ( streptomycin sulfate usp ) was dissolved in 0 . 5 - 1 . 0 ml of purified water and emulsified in 5 - 10 ml of organic solvent ( water saturated ethyl acetate or methylene chloride ), containing dissolved d , l - lactide - glycolide copolymer ( resomer ® 503h , boehringer ingelheim , germany ) with help of short sonication ( 30 sec ) at 20 khz using titanium indenter or high shear rotor - stator mixer ( ultra - turrax t10 , ika , germany ). a formed emulsion was added to continuous water phase , containing surfactants and may contain other water soluble adjutants and further homogenized ( 30 sec . sonication , 3 - 5 cycles of high pressure homogenization ( avestin emulsiflex c5 or similar machine ). the obtained fine emulsion was evaporated under decreased pressure ( 2 - 100 mm ) to eliminate organic solvent and concentrate product . the final suspension of nanoparticles was centrifugated ( 10 minutes , 1000 g ) to remove big particles and aggregates , and filtrated through microporous membrane . the particle size was measured by photon correlation spectroscopy ( malvern zetasizer nano - s ) in water . for the purposes of evaluating a drug as an np , a free unbond drug was separated by transmembrane ultracentrifugation ( separation membrane with molecular cutoff 30 , 000 or 300 , 000 nmwl ) and its concentration was measured by hplc . nanoparticles with vancomycin were prepared using the same methods , as for streptomycin loaded nanoparticles ( see examples 1 - 34 ). vancomycin dissolved in 0 . 5 - 1 ml of water phase or butter ( ph & lt ; 10 ), containing surfactant . some of prepared composition are presented in the table 4 . nanopailicles with levofloxacin were prepared using the same methods , as for streptomycin loaded nanoparticles ( see examples 1 - 34 ). levofloxacin was dissolved in water phase with ph adjusted to 2 . 5 using 1n hcl . composition of example 73 was prepared by precipitation of dissolved combination of polymer , lipid , surfactants , counter - ion and drug from solution in acetone , followed by evaporation of solvent and water . formulations of examples 84 and 85 were prepared by precipitation of dissolved combination of polymer , lipid , surfactants , counter - ion and drug from solution in acetone , followed by evaporation of solvent and water . polymeric nanoparticles with rifampicin and plga were prepared using the same methods , as for streptomycin loaded nanoparticles ( see examples 1 - 34 ), with methylene chloride as a solvent . lipid nanoparticles ( example 96 ) were obtained using hot high pressure homogenization . nanoparticles with doxorubicin were prepared using the same methods , as for streptomycin loaded nanoparticles ( see examples 1 - 34 ). composifions of examples 100 and 101 were prepared by precipitation of from solution in acetone , followed by evaporation of solvent and water . kinetics of release of associated drug from colloidal formulations into phosphate buffered saline ( pbs ) was investigated using dialysis tube ( spectra pore ®) with cellulose membrane ( mw cutoff 50 , 000 dalton ) in usp dissolution apparatus ii ( paddles , 50 rpm ) at 37 ° c . results are presented at graphs 2 - 6 . tuberculosis model : extremely lethal mycobacterium tuberculosis strain h 37 rv ( atcc 27294 ) in dose 10 7 cfu / mice , causing 100 % lethality in spf balb / c mice in 72 hours after inoculation , was used . sepsis ( septicemia ) model : escherichia coli o157 was chosen as a model infection being one of the most common nosocomial pathogens . female balb / c mice were infected by the intraperitoneal injection of 2 . 5 × 10 8 cells ( ld 90 ). treatment started 2 hours post bacterial inoculation pneumonia model : streptococcus pneumonia serotype 3 strain ( atcc 6303 ), administrated intratracheally into swiss webster mice ( 10 5 - 10 6 cfu / mice ) was used as a model of community acquired pneumonia ( cap ), with treatment beginning 24 hours after disease initiation . drug - loaded np formulations and control antibiotics in solution were administrated according to predetermined route and schedule . spf balb / c female mice ( 18 - 20 g , n = 65 ) were infected with m . tuberculosis ( h 37 rv , atcc27294 , 10 7 cfu / mouse , iv ). poly ( lactide - glycolide ) nanoparticulate formulations , stabilized with bsa ( bovine serum albumin ) ( example ) and cremophor ( polyethoxylated castor oil ) ( example ), were tested . infected mice ( 12 per group ) were treated ip as follows : 1 . untreated ( saline ), 5 times per week 2 . sm sulfate usp , 200 mg / kg ( calc . as streptomycin base ), 5 times per week ( positive control ) 3 . sm sulfate usp , 100 mg / kg , twice weekly ( comparative control ). 4 . sm np example 20 , 100 mg / kg , twice weekly . 5 . sm np example 32 , 100 mg / kg , twice weekly . sm formulations were administered ip for 28 days . four mice from each group were assessed for cfu count and organ weights on days 14 , 28 and 56 . all animals survived in np - sm ( example 32 ) group , received 800 mg cumulative dose of sm , while survival rate for positive control ( sm usp , cumulative 4000 mg ) was 92 %, and for comparative control ( sm usp solution , total 800 mg ) was 58 % only . bacterial count in lung and spleen was also significantly lower fornp groups . same model was used for evaluation of anti - tuberculosis activity of rifampicin in biodegradable nanoparticles . rifampicin . in plga nanoparticles , given orally ( twice a week , 20 mg / kg , 4 weeks treatment ) was significantly more efficient in elimination of mycobacter tuberculosis in lungs and spleen than same doses of rifampicin solution in saline ( see graph 10 ) e . coli atcc 25922 was stored at − 80 ° c . until use in this study . the bacterium was transferred onto trypticase soy agar ( tsa ) plates and incubated for 18 h at 37 ° c . a suspension of the bacterium was prepared in pbs and added to sterile 5 % hog mucin . an aliquot of the suspension was added to 5 % hog gastric mucin to obtain the required concentration of inoculum ( 3 . 5 × 10 6 cfu / ml ). each mouse was inoculated with 0 . 5 ml of the appropriate inoculum preparation by ip injection . 2 hours later mice were treated with a single injection of the appropriate concentration of gentamicin sulfate in dose 10 mg / kg ( calculated by base ). animals were observed for six days and mortality was recorded . one of the tested formulations ( example 42 , see graph 11 ) showed better protection against e . coli induced septicemia in mice than gentamicin solution ( survival rates 90 % and 50 %, respectively ; for untreated group survival rate is 11 %) levofloxacin and azithromycin in np formulations ( examples 67 and 80 ) showed increase levels in lungs , liver and spleen in healthy animals compared with drug solution , administrated in same doses ; auc ( 0 - 24 hr ) increased 73 % and 161 %, respectively . administration of doxorubicin in plga nanoparticles ( example 97 ) in glioblastoma model improved survival rate to 40 % at day 100 after tumor inoculation , while doxorubicin in solution , administered in the same dose and schedule , did not provide any protection ( 0 % survival ). 1 . eng r h k , et al . antibiotic killing of bacteria : comparison of bacteria on surfaces and in liquid , growing and non - growing . chemotherapy 1995 ; 41 : 113 - 20 2 . huguette pinto - alphandary , antoine andremont , patrick couvreur international journal of antimicrobial agents 13 ( 2000 ) 155 - 168 targeted delivery of antibiotics using liposomes and nanoparticles : research and applications 3 . u . s . pat . no . 4 , 897 , 384 janoff , et al . drug preparations of reduced toxicity ; 4 . u . s . pat . no . 5 , 759 , 571 hersch , et al . antibiotic formulation and use for drug resistant infections ; 5 . schiffelers r , et al ., liposome - encapsulated aminoglycosides in pre - 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nasr , m . rad - malekshahi , n . samadi , f . atyabi , r . dinarvand , “ preparation and antibacterial activity evaluation of rifampicin - loaded poly lactide - co - glycolide nanoparticles ”. nanomedicine : nanotechnology , biology , and medicine 3 ( 2007 ) pp . 161 - 167 28 . j . vandervoort , a . ludwig “ biocompatible stabilizers in the preparation of plga nanoparticles : a factorial design study ” international journal of pharmaceutics 238 ( 2002 ) 29 . h . l . wong , r . bendayan , a . m . rauth , h . y . xue , k . babakhanian , x . y . wu “ a mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer - lipid hybrid nanoparticle system ” the journal of pharmacology and experimental therapeutics 2006 vol . 317 , no . 3 , pp . 1372 - 1381 graph 1 describes increase of streptomycin binding to nanoparticles along with increase of sucrose concentration graph 2 and graph 3 demonstrate different dependence of streptomycin release patterns from formulation type : solution , micellar solution and nanoparticulate formulations graph 4 presents release of gentamicin from solution and nanoparticulate formulations graph 5 shows release of rifampicin from solution and nanoparticulate formulations graph 6 illustrates release of levofloxacin from solution and several nanoparticulate formulations graph 7 displays survival rate of mice , infected with mycobacter tuberculosis ( h 37 rv , strain atcc27294 ), treated with different streptomycin formulations graph 8 presents results of counting number of mycobacter tuberculosis in lungs of animals , treated with streptomycin in nanoparticulate formulations and in solution graph 9 shows count of mycobacter tuberculosis in spleen of animals , treated with streptomycin in nanoparticulate formulations and in solution graph 10 reveals number count of mycobacter tuberculosis in lungs and spleen of animals , treated with rifampicin in nanoparticulate formulations and in solution graph 11 describes the survival rate in sepsis model in mice , caused with e . coli ( atcc 25922 ) and treated with gentamicin in solution and nanoparticulate formulations