Patent Application: US-201213443073-A

Abstract:
the present invention relates to a pharmaceutical composition for promoting arteriogenesis , and preparation method and applications of the same , wherein said pharmaceutical composition comprises an effective amount of a drug , and a peptide hydrogel , and it forms a microenvironment for autologous cell recruitment and tissue regeneration .

Description:
the inventors reveal for the first time that the self - assembling peptide nanofibers ( nfs ) can function as a vehicle for controlled local release of vegf in the heart . also , they prove that the nf / vegf composition of the present invention can establish a microenvironment potent for vascular generation and endogenous myofibroblast recruitment to improve angiogenesis and arteriogenesis of myocardium after infarction . therefore , the nf / vegf composition of the present invention can improve cardiac performance after infarction and reduce the infarct area , and it has great potential for clinical use . the examples of the present invention are provided hereinafter , however , these examples are not used for limiting the scope of the present invention . seq id no : 35 ( synthesized by synbiosci , livermore , calif .) is used in the following examples . the powder of self - assembling peptide seq id no : 35 was formulated as a peptide solution of 1 % by weight by phosphate buffered saline ( pbs ), ph 7 . 4 , and sonicated ( 100 w , 10 minutes ) into a peptide hydrogel of 1 % by weight . during the synthesis process of vascular endothelial growth factor ( vegf ), a variety of vegf protein forms are produced due to different mrna splicing ways , such as vegf 121 , vegf 145 , vegf 165 , vegf 185 and vegf 206 , in which vegf 121 , vegf 145 and vegf 165 are secreted , soluble protein forms that directly act on vascular endothelial cells to promote the proliferation of vascular endothelial cells and increase vascular permeability . the vegf solution used in the following examples is vegf 165 solution having a concentration of 1000 ng / ml in pbs ( ph 7 . 4 ), but other vegf protein forms also can be used . the above - mentioned vegf solution and peptide hydrogel were mixed at a volume ratio of 1 : 9 to obtain the nf / vegf composition of the present invention . as shown in fig1 , the composition comprises a self - assembling peptide ( 1 ) and vegf ( 3 ). 6 - week - old male sd rats ( 250 g body weight ) were randomly divided into 4 groups ( n ≧ 4 in each group ) for the vascular permeability study of vegf . 80 μl of ( a ) pbs , ph 7 . 4 ( pbs group ), ( b ) vegf solution of example 1 ( vegf group ), ( c ) 1 % by weight peptide hydrogel of example 1 ( nfs group ) or ( d ) the nfs / vegf mixture of example 1 ( nfs / vegf group ) was given by intramyocardial injection at 6 different injection sites ( 211 ) in the region which could be damaged by infraction ( 21 ) in a mouse heart ( 2 ). the fig1 is a schematic illustration of the injection , in which the injection sites ( 211 ) are illustrative , not real injection sites . 45 minutes after the injection , 150 μl of red fluorescent fluospheres ( molecular probes , invitrogen ) was injected into the left internal jugular vein to assess the vascular permeability . after 30 minutes of fluosphere circulation , urine samples of the rats were collected . after that , systemic perfusion was performed to wash out the fluosphere in the blood of rats . following the perfusion , the organs such as brain , heart , lung , liver , spleen and kidney were collected for frozen embedding and further investigation . nanodrop 2000 ( thermo ) was used to analyze vascular permeability . frozen tissue sections were stained , and the fluorescence strength was quantified by fluorescence microscope and image analysis software . higher fluorescence strength indicates increased vascular permeability of the organ . the baseline ( 1x ) was set by the data from the control group ( pbs group ), and the relative fluorescence strength in folds of the other groups against the control group were calculated , as shown in table 1 . in brain , for example , the fluorescence strength of vegf group is 239 folds higher than the control group . the urine samples were analyzed based on the protein concentration of the samples . similarly , the baseline ( 1x ) was set by the data from the control group ( pbs group ), and the relative protein concentration in folds of the other groups against the control group were calculated . from table 1 , it should be clear that when vegf alone was given by intra - myocardial injection ( vegf group ), the other organs ( such as the brain , lung , liver , spleen and kidney ) in the rats had a greater vascular permeability than the control group ( pbs group ), which reached 6 ˜ 240 folds of the control group . and , the protein concentration of urine samples of vegf group was nearly twice higher than the control group . however , injection of the nf / vegf composition of the present invention ( nfs / vegf group ) significantly decreased the vascular permeability of these organs . it approximately reduced the vascular permeability caused by vegf to 1 ˜ 5 folds of the control group . on the contrary , injection of the nf / vegf composition of the present invention ( nfs / vegf group ) increased the vascular permeability of the hearts of rats to 7 folds of the control group . this result indicates that vegf comprised in the nf / vegf composition acts more effectively for the heart than for the other organs . in other words , the nf / vegf composition of the present invention has the ability to achieve a controlled local delivery of vegf in the target organ , the heart . this shows the nf / vegf composition of the present invention has high safety and effectiveness . 6 - week - old male sd rats ( 250 g body weight ) were randomly divided into 7 groups for the following study for a sustained release of vegf in the heart . group 1 was sham operation group , in which the chest cavity of rats was opened without coronary artery ligation ( n = 8 ). in groups 2 - 4 , the chest cavity of rats was opened with permanent ligation of the left anterior descending ( lad ) coronary artery ( experimental mi , for mimicking myocardial infarction ), and the rats were injected with phosphate buffer solution ( pbs ), or 100 ng / ml or 1000 ng / ml vegf solution in pbs ( mi + pbs , mi + v100 , mi + v1000 groups ; n = 8 ). in the groups 5 - 7 , the chest cavity of rats was opened with permanent ligation of the lad coronary artery ( to mimic myocardial infarction , mi ), and the rats were injected with 1 % by weight of peptide hydrogel , or 100 ng / ml or 1000 ng / ml nf / vegf composition of the present invention ( mi + nfs , mi + nfs / v100 , mi + nfs / v1000 groups ; n = 8 ). in all groups , the formulations including solution , hydrogel and composition were given by intramyocardial injection at 6 different sites in the infarct , and the volume of the formulations was 80 μl . after the operation for mimicking myocardial infarction and formulation injection of groups 1 ˜ 7 , left ventricular contraction function of rats was assessed by echocardiography on day 1 and 28 after the operation ( i . e ., post - mi ) under m - mode , and the left ventricular ejection fraction ( ef %) was assessed , as shown in table 2 . the rats were sacrificed on day 28 post - mi , the hearts were collected , embedded in paraffin wax and sectioned , and the circumference of left ventricle wall and the length of infarcted left ventricular wall were measured . the size of infarcted area was calculated by the following formula , as shown in table 2 . the greater infarcted area indicates a greater damage of myocardium . although there was a marginal dose - dependent amelioration of the cardiac contraction functions in mi + v100 and mi + v1000 groups on day 28 post - mi against the control group , there was no significant difference in the size of infarct area between these vegf - treated groups and the control group ( mi + pbs ). and , as the data shown in table 2 , the delivery of vegf alone was not sufficient to improve the cardiac functions or to decrease the infarct size . similarly , when the peptide hydrogel of the present invention alone was injected ( mi + nfs group ), the cardiac contraction functions on day 28 post - mi were not improved . however , when the nf / vegf composition of the present invention was injected ( mi + nfs / v100 and mi + nfs / v1000 groups ), the cardiac functions were improved and the infarct size was reduced . furthermore , immuno - staining was performed using antibody against isolectin ( invitrogen ) to specifically label the endothelial cells in the sections , and antibody against sm22α ( abeam ) to specifically label the smooth muscle cells in the sections . after that , the capillary and artery densities around the infarcted area of the myocardial sections were calculated , in which the artery was defined as arteriole if the diameter of vessel was less than 75 μm , and as artery if the diameter was greater than 75 μm . the results are shown in table 3 . in comparison with the control group ( mi + pbs ), the capillary densities of groups of vegf , nfs and nfs / vegf of the present invention were obviously improved on day 28 post - mi ; however , the arteriole densities were improved only when the nf / vegf compositions of the present invention were injected ( mi + nfs / v100 and mi + nfs / v1000 groups ), and the artery densities of these two groups were also higher than the other groups . next , a study was conducted to explore the mechanism of arteriogenesis following nf / vegf injection . mi was induced in rats , followed with intramyocardial injection of pbs , v100 , nfs or nf / v100 . the rats were sacrificed on day 3 , 7 or 14 post - mi . then , immunostaining of smooth muscle cells using anti - α smooth muscle actin ( α - sma + ) antibody ( sigma - aldrich ) was conducted , as shown in fig2 a . α - sma + myofibroblasts were recruited in the infarcted myocardium of all groups on day 3 post - mi . myofibroblasts were recruited into the myocardium on day 7 post - injection of nfs , with or without vegf ( mi + nfs and mi + nfs / v100 groups ), which implies that myofibroblasts were mostly acquired through the microenvironmental benefits gained by the nfs of the present invention rather than the influence of prolonged vegf delivery . interestingly , on day 14 post - mi , the nf / vegf group also recruited more α - sma + cells that were capable of vessel integration , and this suggests that the recruited myofibroblasts had differentiated into mural cells , which enveloped nascent capillaries . this process is required for arteriogenesis . together these results support that for functional arteriogenesis it requires not only vegf - dependent angiogenesis but also mural cells recruitment by nf injection . therefore , the nf / vegf composition of the present invention not only promotes arteriogenesis , but also establishes an intramyocardial microenvironment suitable for myofibroblast engraftment . a further study was then designed to verify the capacity of the self - assembling peptide of the present invention ( i . e ., nfs ) to capture circulating bone marrow cells ( bmcs ). bmcs are the main source of myofibroblasts . rats received intravenous injections of 1 × 10 7 allogeneic dii - labeled bmcs on day 7 post - mi along with intramyocardial injection of pbs , v100 , nfs or nf / v100 . the rats were sacrificed 1 day after bmc injection , and then the hearts were embedded and sectioned for fluorescence immuno - staining . the results are shown in fig2 b . more dii + bmcs were retained within the myocardium that received injections of nfs . notably , the level of myocardial dii + bmc infiltration was significantly enhanced by nf / vegf injection . this result suggests that nf / vegf injection of the present invention creates an intramyocardial microenvironment capable of recruiting circulating bmcs to promote arteriogenesis . cardiac troponin - i - positive ( ctni + ) cells or putative renewed cardiomyocyte - like cells were detected within the injected myocardium on day 28 post - mi following the nf / v100 injection . to test whether these cardiomyocyte - like cells were derived from either non - myogenic stein / progenitor cells or just debris of remnant cardiomyocytes , a genetic fate - mapping approach was conducted using mer - cre - mer / zeg mice which were hybridized from the cardiac - specific inducible cre mice ( the mer - cre - mer mice ) and a dual reporter zeg transgenic mice . through induction of tamoxifen , cardiomyocytes were marked with green fluorescence protein ( gfp ). on the contrary , stem cell - derived new cardiomyocytes were not marked by gfp . the mer - cre - mer / zeg mice were injected with 15 μl of pbs , v100 , nfs or nf / v100 after experimental mi . on day 28 post - mi , mice were sacrificed and the hearts were sectioned and stained by ctni antibody . the gfp ( green ) and ctni ( red ) fluorescence signals were observed using fluorescence microscopy ( gfp : abeam ; ctni : dshb ), in which gfp - negative and etni - positive cells were stem / progenitor cell origin . the results are shown in fig3 . as shown in fig3 , hearts from the nf / vegf injection group recruited more gfp − / ctni + cardiomyocyte - like cells than the other groups . there were also gfp - negative mature cardiomyocytes observed in the mi + nf / v100 group with no significant difference compared with that in the control . again , these results suggest that nf / vegf injection provides an intramyocardial microenvironment favorable for the induction of endogenous cardiomyocyte regeneration . in summary , the nf / vegf composition of the present invention can provide a controlled local delivery of vegf in the heart without vascular leakage in other organs . thus , it has high safety and effectiveness . in addition , the nf / vegf composition of the present invention effectively improves cardiac functions and decreases infarct size after mi . these benefits may be through recruitment of myofibroblasts for arteriogenesis and cardiomyocyte - 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