Patent Application: US-80633610-A

Abstract:
a method for inducing neovascularization in which a pharmaceutically effective amount of polyethylene glycol is injected into the tissue of the subject for purposes of accelerating wound healing and mimicking age related macular degeneration , cancer , tumors , and atherosclerosis for further research purposes .

Description:
with reference to fig1 - 8b , the preferred embodiment of the present invention may be described . male c57bl / 6 mice ( 7 - 9 weeks old ) were anesthetized with ketamine ( 80 mg / kg ) and xylazine ( 20 mg / kg ) prior to subretinal injection . one eye was decompressed with 27g needle by inserting the needle through the limbus and sclera . microinjector ump3 equipped with nanofil syringe 100 μl and 33g blunt needle ( word precision instruments ) was used for injections . the needle was inserted into the incision created by decompression . movement of the needle was stopped when light resistance was felt by operator . solutions ( single injection of 2 μl ) were injected slowly for 5 seconds in the subretinal space . all manipulations were performed under visual control using microscope . detachment of the retina was visible through the lens . the second eyes of mice were not injected . to investigate dose dependent effect of peg - 8 ( polyethylene glycol 400 ) the inventors divided the animals into 7 groups : one control group ( n = 3 mice ) which was not treated at all , group 2 ( n = 3 mice ) received a single subretinal injection of vehicle ( 2 μl of 0 . 9 % sterile sodium chloride ), and five experimental groups which were injected with different doses of peg - 8 : 2 . 0 mg , 1 . 0 mg , 0 . 5 mg , 0 . 25 mg and 0 . 125 mg . peg - 8 was dissolved in vehicle before injection . doses of peg - 8 were delivered to the one experimental eye in 2 μl respectively . to determine optimal dose of peg - 8 , the inventors performed histological examination and investigation of flat mounted rpe - choroid - sclera complexes after perfusion of animals with fitc - dextran solution at day 14 after injection . the mice were sacrificed at day 14 after subretinal injection of peg - 8 . as shown in table 1 , the inventors found cnv in all investigated groups . the 1 . 0 mg dose induced cnv of the largest size and frequency ( 4 . 4 sites of cnv per eye ) ( fig1 ). to investigate the time course of cnv after peg - injection , the animals were injected with 1 . 0 mg of peg - 8 subretinally and 5 mice from each group were sacrificed at day 1 , 3 , 5 , 7 , 14 , 21 , 28 , and 42 post - injection . as shown in fig2 , first signs of cnv were detected as early as day 3 after treatment . cnv reached maximal size at day 5 and was present until at least day 42 . to evaluate the cnv size , the animals were perfused through their heart with 0 . 75 ml of phosphate - buffered saline ( pbs ) containing 50 mg / ml of fluorescein - labeled dextran ( fitc - dextran , 2 million average mw ) before they were sacrificed . the eyes were harvested and fixed for 4 hours in 10 % phosphate buffered formalin and rpe - choroid - sclera flat mounts were prepared . the rpe - choroid - sclera complexes were mounted in prolong antifade reagent with the sclera facing down and were examined under a zeiss lsm 510 laser confocal microscope . confocal microscopy of flat mounted rpe - choroids perfused with fitc - dextran was performed and investigated with semi thin ( 1 μm ) embedded sections at day 5 after injection when cnv was fully developed . analysis of flat mounts and semi thin sections showed that in naive mice rpe formed single layer of cells ( fig3 a , c , e ) and vessels were located exclusively within choroid ( fig3 a , c , e ). after peg - 8 injection , cnv was located between two layers of rpe cells in subretinal space ( fig3 b , d , f ). no vessels originated from retina was found ( fig3 d ). 5 μm paraffin section of control ( saline ) and peg - 8 injected mice at day 1 were investigated , 3 and 5 after treatment . rpe cells in control groups formed single layer of cells and endothelial cells were found within choroid ( fig3 g ). starting from day 1 after peg - 8 treatment , zones of affected rpe cell structure were discovered with increased size , irregular shape and presence of space between neighboring rpe cells ( fig3 h ). isolectin - ib4 positively labeled cells located between affected rpe cells were found at day 1 , 3 and 5 after injection ( fig3 h - j ). isolectin - ib4 positive had contact with choroidal isolectin - ib4 positive capillaries and formed vessels in subretinal tissue between existing and newly formed rpe cell layers ( fig3 j ). to investigate dependence of peg - 8 induced cnv on complement system activation , the complement system in the mice was depleted by intraperitoneal administration of cobra venom factor . four groups of animals were used ( n = 5 mice in each group ). group 1 and 2 were treated intraperitoneal with 4 u of cobra venom factor as described in the inventors &# 39 ; previous publication [ 11 ] for 2 days before and every day after subretinal injections . group 3 and 4 were injected intraperitoneal with vehicle ( 0 . 9 % sodium chloride ). the mice were then injected with subretinal peg - 8 and investigated at day 5 after treatment . total complement activity in serum of cvf treated mice was significantly reduced compared to saline injected animals ( p & lt ; 0 . 05 , fig4 a ). rpe - choroids of these mice were stained for mac by ihc and investigated flat mounts using confocal microscopy . areas of high mac deposition in animals treated with saline ( fig4 b ) were found but not in cvf treated group ( fig4 c ). mac positive fluorescence in saline treated animals was significantly higher compared to cvf injected mice ( fig4 h , p & lt ; 0 . 05 ). analysis of cnv size showed that treatment with cvf significantly ( 3 fold , p & lt ; 0 . 05 , fig4 i ) reduced area of fitc - dextran perfused vessels . activation of the complement system by peg - 8 was also studied in the rpe - choroid and in the blood . peg - 8 or saline was injected subretinally and animals were sacrificed at day 1 , 3 and 5 after treatment . total complement activation was studied in the serum . levels of c3 split products and mac were investigated in rpe - choroid tissue . peg - 8 did not affect total and alternative complement activity in mouse serum at all investigated time points ( fig5 a , b ). a three - fold increase of c3 split products levels in peg - 8 injected animals compared to saline injected mice was observed only at day 1 post - injection ( fig5 c , d ). peg - 8 and saline injected animals at day 3 and 5 had the same levels of total c3 ( 43 kda and 23 kda ), but levels of c3 ( 43 kda ) were higher in peg - 8 treated animals compared to saline injected mice ( fig5 c , d ). western blotting showed that in peg - 8 treated mice mac levels were approximately three fold higher compared to saline treated animals at all investigated time points ( fig5 c , e ). an ihc colocalization experiment was performed to find the relationship between c3 / mac deposition , vegf secretion and initiation of cnv growth . mac was found mostly in sites of c3 deposition ( fig6 ). it was discovered that cnv growth took place in sites of extremely high deposition of c3 and mac on the surface of rpe cells and in choroid ( fig6 ). at the same locations , release of vegf from rpe cells and distribution of this growth factor within bruch &# 39 ; s membrane and choroid was observed ( fig7 ). choroidal capillaries were colocalized with vegf ( fig7 ). elisa analysis of vegf levels showed increase of vegf in intra - and extracellular fraction of proteins obtained from posterior part of the eyes ( fig7 g , h ). comparison of new vessel growth is shown in fig8 a - b ; laser induced model ( a ) and peg - induced model ( b ) cnv . green color shows the size of new vessel growth in both the figures . damage of rpe - choroid after subretinal injection is much smaller compared to laser treatment or surgical procedures . local ( intraocular ) stimulation of vegf expression and secretion by peg - 8 mimic changes in human amd . for purposes of mimicking cancer , tumor formation , atherosclerosis , and aiding in wound healing , peg can be locally injected into the tissue or , if applicable , applied directly to the tissue in the form of a gel or ointment . 1 . coleman h r , chan c c , ferris 3rd f l , chew e y : age - related macular degeneration . lancet 2008 , 372 : 1835 - 1845 2 . visual impairment and blindness , word health organization , fact sheet no 282 , may 2009 . 3 . campochiaro p a : retinal and choroidal neovascularization . j cell physiol 2000 , 184 : 301 - 310 . 4 . gehrs k m , anderson d h , johnson l v , hageman g s : age - related macular degeneration - emerging pathogenetic and therapeutic concepts . ann med 2006 , 38 : 450 - 471 . 5 . zarbin m a . current concepts in the pathogenesis of age - related macular degeneration . arch ophthalmol . 2004 april ; 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