Patent Application: US-2904805-A

Abstract:
a method of lowering homocysteine level of an individual and of reducing the vascular pathology associated therewith , by administering a ligand of the peroxisome proliferator activate receptor gamma . the peroxisome proliferator activated receptor preferably may be rosiglitazone .

Description:
ten - week - old female sprague dawley rats were obtained from harlan laboratories ( indianapolis , ind .). after an acclimatization period of one week , they were fed a powdered control diet with or without methionine for one week . after one week on the normal or methionine diet , an aqueous preparation rosiglitazone ( 3 . 0 mg / kg / d ) or vehicle was administered by gavage . the methionine diet was prepared with 10 g of l - methionine obtained from sigma chemical company in st . louis , miss ., added to 10 kg of powdered feed and thoroughly mixed . the rats were divided into four groups ( n = 7 / group ) and fed a ) control diet ; b ) control diet with administration of rosiglitazone ; c ) methionine diet ; and d ) methionine diet with rosiglitazone . balloon catheter injury of to the carotid artery was induced as previously published ( 27 ). briefly , a 2 . 0 mm ptca obtained from boston scientific , inc . in quincy , mass . was introduced through the femoral artery to the left carotid . the balloon was inflated to 4 atmospheres for 20 seconds and then deflated to 2 atmospheres and dragged down to the aorta . the animals were continued on their respective diets and rosiglitazone treatment was continued as indicated for three weeks after the surgery . the surgical procedures were conducted under anesthesia in a totally aseptic atmosphere . all rats had free access to the feed and water , and were maintained in a 12 hour day / light cycle . before the beginning of the experiment , and at the time of sacrificing the animal a blood sample was obtained from each rat . at the end of the experiment , rats were sacrificed using a carbon dioxide chamber for harvesting tissues . samples of blood and liver were collected and immediately processed . the serum and liver samples were stored frozen . the hcy levels were measured by high pressure liquid chromatography according to an established method ( 28 , 29 ). cystathionine - β - synthase ( cβs ) was assayed in the liver samples , and the procedure followed was as previously published ( 26 ). the carotid arteries were separated and flushed with zinc buffered formalin . each artery was cut from top into 4 equal segments and placed sequentially for embedding in paraffin . sections no thicker then 6 um were cut and stained with hemotoxalin and eosin for microscopy . all four segments were read and the intimal / medial ( i / m ) ratio was calculated using image - pro software ( nih ). the mean of the i / m ratios of the 4 sections reflected the i / m for one animal . representative micrographs of sections of the carotid artery of sprague dawley rats sacrificed 21 days following the balloon catheter - induced injury are presented in fig1 . micrograph a is from an animal that was fed the control diet . as can be observed , development of intimal hyperplasia occurred over the three - week interval following catheter injury ; the im ratio was 0 . 13 . there was not a significant change in the medial area following catheter injury in any of the animals . therefore , a change in i / m represents a change in the intimal area . micrograph b is from a carotid artery of an animal on the methionine diet ; the i / m was 1 . 37 . micrograph c is from an animal that was on the control diet and also administered rosiglitazone ; the i / m ratio was 0 . 19 . micrograph d is from an animal that received the methionine diet as well as rosiglitazone ; the i / m ratio was 0 . 17 . thus the animal on the methionine diet exhibited augmented development of intimal hyperplasia and this augmentation was attenuated by treatment with rosiglitazone . summary data for the effect of rosiglitzone on the rate of development of intimal hyperplasia are presented in fig2 . the group of animals received the control diet had an i / m of 0 . 18 +/− 0 . 05 ; the group on the control diet that received rosiglitazone exhibited an i / m of 0 . 21 +/− 0 . 03 ; this was not statistically significant , as compared to the control diet group without rosiglitazone , ( p = 0 . 997 ). however , in the group that received the methionine diet , the development of intimal hyperplasia was markedly augmented ; this group exhibited an i / m of 0 . 82 +/− 0 . 21 which was significant , as compared to the control diet group ( p = 0 . 005 ). administration of rosiglitazone to the group receiving the methionine diet inhibited the development of intimal hyperplasia seen in the presence of the methionine diet alone , i / m of 0 . 28 +/− 0 . 12 , ( p = 0 . 018 ). moreover , the development of intimal hyperplasia in the group with methionine diet plus rosiglitazone treatment was not statistically different from that seen in the group on the control diet without rosiglitazone ( 0 . 28 +/− 0 . 12 vs . 0 . 18 +/− 0 . 05 , respectively ( p = 0 . 918 ). the i / m in animals that did not undergo balloon catheter - induced injury was zero . summary data for serum homocysteine levels are present in fig3 . homocysteine levels in animals on the control diet were 6 . 3 +/− 0 . 04 μm . administration of rosiglitazone to animals on the control diet did not affect the homocysteine levels , 5 . 1 +/− 0 . 6 μm . the group receiving the methionine diet exhibited markedly enhanced levels of homocysteine , 28 . 9 +/− 3 . 2 μm . this increase was statistically significant as compared with the control diet ( p = 0 . 002 ). administration of rosiglitazone to the animals on the methionine diet significantly reduced the serum homocysteine concentration , 16 . 4 +/− 1 . 7 μm . this effect was statistically significant as compared to the methionine diet alone ( p = 0 . 001 ); 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