Patent Abstract:
the present invention relates to methods and drugs for ameliorating insulin resistance in skeletal muscle , a major contributing abnormality to impaired glucose handling in such diseases as type 1 and type 2 diabetes , hypertension , obesity and critical care patients . this invention provides drugs , methods for screening those drugs and methods specifically targeted to ameliorating insulin resistance by increasing capillary blood flow in muscle .

Detailed Description:
in order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following non - limiting examples . methods for detecting insulin - mediated capillary recruitment and therefore the means by which the present invention acts . in laboratory rats three methods have been used by the applicants to demonstrate that insulin acts to recruit muscle capillary blood flow as part of its normal action in vivo . these are 1 - mx ( 14 ), ceu / microbubbles ( fig1 ) and ldf ( 19 ). only ldf has been used before by other researchers for this purpose but this has been to assess capillary blood flow changes in human skin . the other two methods 1 - mx and ceu / microbubbles are unique to the applicants &# 39 ; and the subject of co - pending applications . in humans , at this point in time , the ceu / microbubbles method can be used and approval has been granted for use of 1 - mx in humans by the danish authorities . the applicants and their collaborators are applying for wider approval to infuse 1 - mx in humans so that the 1 - mx method can be used generally . in principle , 1 - methylxanthine ( 1 - mx ) is an exogenous substrate for an enzyme located predominantly in the nutritive capillary endothelial cells ( much less so in the non - nutritive route and myocytes ). consequently , passage of blood borne 1 - mx through the nutritive vascular route leads to its conversion to the product 1 - methylurate ( 1 - mu ). chromatographic analysis of arterial and venous samples for 1 - mx and 1 - mu together with the total blood flow rate over the muscle bed allows the calculation of 1 - mx metabolism . a number of our studies using the perfused rat hindlimb have shown tight correlation between nutritive flow ( or the proportion of nutritive / non - nutritive flow ) and 1 - mx metabolism ( 12 , 13 ). from in vivo studies in rats using the hyperinsulinaemic euglycaemic clamp the applicants have shown that insulin acts to recruit capillary flow in muscle ( 14 ). deliberate impairment of capillary recruitment in an animal model gives rise to insulin resistance ( 15 ). at least one model of muscle insulin resistance in animals shows impaired insulin - mediated capillary recruitment ( zucker rat , unpublished ). exercise - training which is beneficial in treating and preventing muscle insulin resistance leads to enhanced insulin - mediated capillary recruitment ( unpublished ). the ultrasound method relies on the increased echogenicity of albumin microbubbles that are continuously infused intravenously during data acquisition . the acoustic signal that is generated from the microbubbles when exposed to ultrasound produces tissue opacification that is proportional to the number of microbubbles within the ultrasound beam . using harmonic pulsing methods essentially all microbubbles within the ultrasound beam are destroyed in response to a single pulse of high - energy ultrasound and an image is obtained . in the time interval between subsequent pulsing episodes , microbubbles flowing into the tissue are replenished within the beam and affect the intensity of the signal from the next high - energy pulse . repeating this process with pulse delays between 50 msec and 20 sec , the beam will be fully replenished and further increases in the time between each pulsing interval will not produce a change to tissue opacification . the rate of microbubble reappearance within the ultrasound beam provides an indication of capillary velocity and the degree of tissue opacification provides a measurement of capillary blood volume ( cbv or mw ). images are background - subtracted from images from a pulsing interval of 1000 ms which represents the replenishment of arteries and arterioles thus providing a measurement of capillary flow . the plateau tissue opacification ( measured as videointensity ) is the determination of capillary blood volume . using this approach , changes in capillary blood volume in response to insulin and exercise have recently been assessed in the skeletal muscle of the rat hindlimb in vivo and compared to data obtained using 1 - mx metabolism ( 16 ; fig1 ). compared to baseline values , saline - infusion resulted in little change in capillary blood volume whereas marked increases in capillary blood volume occurred during euglycemic insulin clamp ( 3 mu / min / kg ). recent studies have demonstrated that ceu data correlates well with 1 - mx metabolism data , and that capillary blood volume increases - 2 - 3 fold during these physiologic doses of insulin ( 16 ). a particular advantage of the ultrasound method is that it is relatively non - invasive and is suitable for human use ( 17 ). assay of new drugs acting to increase capillary recruitment in the presence of endogenous or exogenous insulin . this is done in an optional two tier manner , firstly in anaesthetized rats using the hyperinsulinaemic euglycaemic clamp ( 14 ) and secondly , in human forearm using a localized hyperinsulinaemic euglycaemic clamp ( 17 ). the initial testing in rats is optional , but allows rapid identification of those agents likely to be effective in humans . typically the means of assay in rats would involve infusion of a physiological dose of insulin that is sub - maximal ( e . g . 3 mu / min / kg body weight ) in animals that are instrumented to allow continuous monitoring of blood pressure , heart rate and femoral arterial blood flow . the drug to be tested would be infused commencing 1 hour before the infusion of insulin . arterial blood samples will be taken for glucose analyses in order to check that if the drug increases glucose disposal without insulin infusion . either way and within 10 minutes of commencing the infusion of the insulin , glucose infusion would be commenced . by assaying arterial blood samples every 15 minutes , the glucose infusion is adjusted to maintain euglycaemia ( i . e . 5 mm ). in the second hour of the 2 hour clamp markers for muscle glucose uptake ( radiolabelled 2 - deoxyglucose ) and capillary recruitment ( 1 - mx ) are infused . at the end of the clamp , arterial and femoral vein blood samples are taken from which capillary recruitment and leg glucose uptake can be calculated from glucose and 1 - mx values respectively . muscles of the lower leg are also removed and the radioactivity therein used to calculate muscle specific glucose uptake . a drug enhancing insulin &# 39 ; s action to increase muscle glucose uptake would be expected to increase each of the following : glucose infusion to maintain euglycaemia , leg glucose uptake , muscle specific glucose uptake , and capillary recruitment as indicated by increased 1 - mx metabolism ( or disappearance ). data for two founder drugs , zaprinast [ 1 , 4 - dihydro - 5 -( 2 - propoxyphenyl )- 7h - 1 , 2 , 3 - triazolo ( 4 , 5 - d ) pyrimidin - 7 - one ] and aicar [ 5 - aminoimidazole - 4 - carboxamide - 1 - β - d - ribofuranoside ] are shown in fig5 and 6 , respectively . zaprinast significantly ( p & lt ; 0 . 05 ) enhanced insulin - mediated capillary recruitment ( 1 - mx metabolism ), glucose appearance ( ra ) and glucose disposal ( rd ) ( fig5 ). fig5 . in this study zaprinast is infused into anaesthetised rats in conjunction with a sub maximal physiologic dose of insulin . fig5 a shows that zaprinast enhances insulin - mediated capillary recruitment as indicated by hindleg 1 - mx metabolism . zaprinast also enhanced insulin - mediated glucose appearance ( ra ) and most importantly , glucose disposal ( rd ). these changes were statistically significant with p & lt ; 0 . 05 . aicar recruited capillary flow on its own and when added with insulin , markedly enhanced hindleg glucose uptake ( fig6 ). fig6 . in this study aicar was infused into anaesthetised rats either alone ( fig6 a ) or with insulin ( fig6 b ). aicar increased capillary recruitment as indicated by ceu and enhanced insulin - mediated hindleg glucose uptake ( p & lt ; 0 . 05 ). drugs of greatest interest will be those that ameliorate insulin resistance in any one of a number of insulin resistant animal models . these might include the genetically obese zucker rat , and the intralipid ®- infused rat . second tier testing of those drugs found to act by enhancing insulin - mediated capillary recruitment in rats are to be tested in humans using the forearm clamp and contrast enhanced ultrasound / microbubbles ( ceu ) ( 17 ). the drug , in a form suitable for oral administration , would be taken one to two hours prior to testing . the patient &# 39 ; s response would be tested on two occasions , one with and one without drug administration and the two results compared . typically , in response to low doses of insulin 0 . 01 to 0 . 05 mu / min / kg infused locally into the brachial artery , plasma insulin rises by 70 - 350 pm in blood perfusing forearm muscle with little or no effect on the systemic insulin , glucose , ffa , catecholamines or amino acid concentrations . as a result , the isolated effect of local insulin on total blood flow into the arm and glucose balance across the arm can be measured . in addition , capillary recruitment in the forearm flexor muscle can be measured using ceu . total forearm blood flow is measured on the subject by two techniques : capacitance plethysmography and brachial artery ultrasound . for the doppler flow measurements , an ultrasound system ( sonos 5500 , hewlett - packard , andover , mass .) with a linear - array transducer is used with a transmit frequency of 7 . 5 mhz to allow 2 - d imaging of the brachial artery in the long axis . brachial artery diameter is measured 2 cm proximal to the tip of the arterial catheter at peak systole using online video calipers . a pulsed - wave doppler sample blood volume is placed at the same location in the center of the vessel and the mean brachial artery blood velocity measured using on - line angle correction and analysis software . brachial artery blood flow is calculated from 2 - d doppler ultrasound data using the equation : q = v □·( d / 2 ) 2 to measure capillary recruitment with ceu , a suspension of albumin microbubbles is infused intravenously in the contra - lateral arm while 2d imaging of the deep flexor muscles of the test forearm is performed . measurement is made in a trans - axial plane 5 cm distal to be antecubital fossa , using an ultrasound system ( sonos 5500 ) capable of harmonic imaging . intermittent imaging is performed with ultrasound transmitted at 1 . 8 mhz and received at 3 . 6 mhz . once the systemic microbubble concentration reaches steady - state ( 1 - 1 . 5 min ), intermittent imaging is begun , at pulse intervals ranging from 1 to 15 seconds , thus allowing progressively greater replenishment of the ultrasound beam elevation between destructive pulses . three images are acquired at each pulse interval . additional images are acquired with the same beam characteristics at a 30 hz sampling rate , at which there is replenishment of microbubbles only in vessels with very rapid flow , and these were used as background images . data are recorded digitally and analyzed using custom - designed software described elsewhere ( 25 ). averaged background frames ( acquired at a 30 hz frame rate ) are digitally subtracted from the averaged frames acquired at each pulsing interval . mean video intensity in the region of interest is measured from the background - subtracted images . pulsing interval vs . video intensity plots are generated and fitted to an exponential function : y = a ( 1 - e □□ t ). where y is the video intensity at a pulsing interval t , a is the plateau video intensity representing microvascular blood volume , and □ is the rate constant reflecting the rate of rise of video intensity ( and mean microbubble velocity , or microvascular flow velocity ) ( fig7 ) ( 25 , 26 ). fig7 . this figure illustrates in more detail how the microvascular blood volume or capillary volume and microvascular flow velocity are determined using ceu . fig7 a illustrates the successive filling of a capillary over time after all microbubbles in the capillary have been lysed by a high energy harmonic ultrasound pulse . as the delay time prior to signal detection increases ( t0 through t5 ) the number of microbubbles and hence the videointensity increases . fig7 b plots this data for typical signals collected over forearm muscle . the tangent to the upward sloping hyperbolic function is a measure of the rate of microvessel filling ( mvfv ) while the asymptote that intercepts the y - axis is a measure of the maximal signal seen when the vessels are filled and is determined by the microvascular volume ( mw ) i . e . capillary volume . in order to derive values for the mvfv and mw , the time versus video intensity plots are fitted to the function : y = a ( 1 - e − βt ), where y is the video intensity at time t , a is the plateau intensity which represents mw , and p is the time constant of rise and reflects velocity . fig7 c shows a typical experiment done before ( open circles ) and after ( filled circles ) infusing insulin ( 3 mu / min / kg ) to an anesthetized rat . the plateau videointensity ( a ) is clearly higher , with no change in the rate of microvascular filling ( β ). a positive effect of the drug would be seen as enhancing glucose uptake across the arm and enhanced capillary recruitment typified by an increase in the microvascular volume from ceu over insulin alone . as above , those drugs most useful in treating insulin resistance will be effective in insulin resistant subjects . a positive result in normal healthy individuals is not essential and probably not desirable . examples of drugs acting to increase capillary recruitment based on mechanism . these may act by inhibiting cyclic gmp degradation in those smooth muscle cells of the terminal arterioles controlling blood flow entry to the nutritive capillaries . as an example , the drug would be targeted to the specific isoenzyme form of cyclic gmp phosphodiesterase expressed in those same smooth muscle cells . the concept for this mechanism is analogous to that accounting for the action of viagra ®. alternatively , these drugs may act by altering gene expression over a period of time so that insulin &# 39 ; s action to recruit capillary blood flow in muscle is enhanced . a mechanism envisaged here would encompass the induction of enzyme ( s ) responsible for the production of no in endothelial cells of the terminal arterioles controlling blood flow entry to the nutritive capillary networks of muscle . equally , repression of enzyme ( s ) responsible for no destruction at these sites , is envisaged . combined , or separate , such chronic effects of an administered drug would resemble the effects of exercise training as recently reported by us where both insulin - mediated capillary recruitment and muscle glucose uptake was increased ( 27 ). alternatively , these drugs may act by enhancing focal production of no in the vicinity of the smooth muscle cells of the terminal arterioles controlling blood flow entry to the nutritive capillaries . the process of enhanced no production is identical to that normally used by insulin . general or global production of no in skeletal muscle is counter - productive and would very likely dilate arterioles controlling blood flow to the non - nutritive route . as a further alternative , these drugs may act by enhancing the focal production of endogenous vasodilators from muscle glucose metabolism . as an example , adenosine is thought to be one of the vasodilators produced by exercising muscle and responsible for the reactive hyperaemia . a logical drug targeted at enhancing the effect of adenosine would act to block adenosine degradation ; i . e . an inhibitor of adenosine deaminase . as a further alternative , these drugs may act using site - specific delivery of a micro - encapsulated nitrovasodilator with the intention of releasing no in the vicinity of the smooth muscle cells of the terminal arterioles controlling blood flow entry to the nutritive capillaries . there are several enzymes located in the aforementioned specific regions including angiotensin converting enzyme , alkaline phosphatase , and uridine diphosphatase that could be used to hydrolyse polymers constituting the micro - encapsulated nitrovasodilator . as a further alternative , these drugs may act by blocking substance ( s ) in the blood that are preventing the normal effect of insulin to recruit capillary flow . for example , we have shown the inflammatory cytokine , tnfα to completely block insulin - mediated capillary recruitment and 50 % of the insulin - mediated muscle glucose uptake . it follows that an agent that blocks tnfα would under these circumstances restore normal insulin responses . finally , these drugs may act through a central acting mechanism to modify vasomotor neural output thus increasing capillary recruitment by site - specific vasodilatation . any discussion of documents , acts , materials , devices , articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention . it is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in australia before the priority date of each claim of this application . it will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described . the present embodiments are , therefore , to be considered in all respects as illustrative and not restrictive . 1 . baron a d , laakso m , brechtel g , edelman s v : mechanism of insulin resistance in insulin dependent diabetes mellitus : a major role for reduced skeletal muscle blood flow . j . clin . endocrinol . metab . 73 : 637 - 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