the a3 adenosine receptor ( a3ar ) belongs to the family of the gi - protein coupled receptors ( gpcr ) . much evidence including preclinical and clinical studies has accumulated showing anti - inflammatory effects that are mediated via the a3ar [ 18 ] . a3ar agonists induce anti - inflammatory effects in both murine and rat models of autoimmune arthritis [ 2 , 9 ] , via downregulation of nuclear factor kappa b ( nf-b ) and related proteins as well as tumor necrosis factor- ( tnf ) , resulting in the inhibition of inflammatory cytokines [ 1 , 10 , 11 ] . a3ar is overexpressed in cells from inflammatory tissues whereas normal cells have a low expression of the receptor [ 12 , 13 ] . furthermore , a3ar was found to be upregulated in the peripheral blood mononuclear cells ( pbmcs ) of patients with autoimmune inflammatory diseases [ 1316 ] . pbmcs drawn from rheumatoid arthritis ( ra ) , psoriasis , and crohn 's disease ( cd ) patients showed a3ar upregulation compared to that of healthy subjects , suggesting that the high a3ar expression levels in the inflammatory tissues are reflected in the pbmcs . a3ar agonists such as cf101 ( ib - meca ) and cf102 ( cl - ib - meca ) have been investigated in several phase ii clinical studies including ra , psoriasis , and hepatocellular carcinoma , showing clear evidence of efficacy and an excellent safety profile [ 1719 ] , proof of the validity of the a3ar as a therapeutic target . while a3ar agonists bind at an orthosteric receptor binding site to induce activation , a positive allosteric modulator ( pam ) would bind to an additional binding site on the receptor that is distinct from the agonist binding site , thereby enhancing the affinity and/or efficacy of the natural ligand adenosine at the a3ar . the advantage of allosteric modulators is their capability to target specifically areas where adenosine levels are increased such as at inflammatory and tumor sites , whereas normal body cells and tissues are in theory refractory to the allosteric modulators due to lower adenosine levels . n-(3,4-dichloro - phenyl)-2-cyclohexyl - lh - imidazo [ 4,5-c]quinolin-4-amine ( luf6000 ) is an imidazoquinolinamine allosteric enhancer of the human a3ar that upon binding changes the native ligand interaction with the receptor and raises its maximal effect by 45%   ( figure 1 ) . in a [ s]gtps binding assay , luf6000 was able to convert the nucleoside antagonist mrs542 into an a3ar agonist and was found to be highly effective in raising the maximal effect of low - efficacy agonists . luf6000 alone did not induce receptor activation and therefore is potentially safer than orthosteric agonists . in this study we present data showing that luf6000 has a specific and potent anti - inflammatory effect in vivo . a molecular mechanism associated with these effects the allosteric modulator luf6000 ( n-(3,4-dichloro - phenyl)-2-cyclohexyl - lh - imidazo [ 4,5-c]quinolin-4-amine ) was synthesized for can - fite biopharma at leiden academic centre for drug research ( leiden , the netherlands)/haoyuan chemexpress co. , ltd ( shanghai , china ) . a stock solution of 10  mm was prepared in dmso and further dilutions were prepared in pbs . cona ( canavalia ensiformis , jack bean hemagglutinin ) was purchased from calbiochem - emd millipore ( san diego , ca ) . rabbit polyclonal antibodies against rat a3ar , phosphoinositide 3-kinase ( pi3k ) , ib kinase ( ikk ) , ib , nuclear factor kappa b ( nf-b ) , janus kinase 2 ( jak-2 ) , and signal transducer and activator transcription 1 ( stat-1 ) , glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , and -actin were purchased from santa cruz biotechnology , inc . female lewis rats , 9 weeks of age , were obtained from harlan laboratories ( jerusalem , israel ) . experiments were performed in accordance with the guidelines established by the institutional animal care and use committee at can - fite biopharma , petach tikva , israel . the rats were injected subcutaneously ( sc ) at the tail base with 100  l of suspension composed of incomplete freund 's adjuvant ( ifa ) with 10  mg / ml heat killed mycobacterium tuberculosis , ( mt ) h37ra , ( difco , detroit , usa ) . luf6000 ( 100  g / kg ) treatment was orally administered by gavage , thrice daily , starting with the disease onset . the control group received vehicle only ( dmso at a dilution corresponding to that of the dmso stock solution of luf6000 ) . a clinical disease activity score was assessed , blinded , as follows : the animals were inspected every day for clinical signs of arthritis . the scoring system ranged from 0 to 4 of each limb : 0 , no arthritis ; 1 , redness or swelling of one toe / finger joint ; 2 , redness and swelling of more than one toe / finger joints , 3 , the ankle and tarsal - metatarsal joints involvement ; 4 , redness or swelling of the entire paw . the clinical score was calculated by adding the four individual legs ' score to a maximum . at the end of the study in addition , blood samples were collected and subjected to a ficoll - hypaque gradient . the pbmcs were then washed with pbs and protein extracts were prepared as is detailed below . the bony tissue was broken into pieces , snap frozen in liquid nitrogen , and stored at 80c until use . the paw tissues were added to ( 4  ml / g tissue ) radioimmunoprecipitation assay ( ripa ) extraction buffer containing 150  mm nacl , 50  mm tris , 1% np40 , 0.5% deoxycholate , and 0.1% sds . tissues were homogenized on ice with a polytron , centrifuged and the supernatants were subjected to western blot analysis . each group included 10 animals , and the study was repeated at least 3 times . male wistar rats ( 150175  g ) were obtained from harlan laboratories ( jerusalem , israel ) . experiments were performed in accordance with the guidelines established by the institutional animal care and use committee at can - fite biopharma , petach tikva , israel . oa experimental model was induced with mia , 2  mg at a total volume of 50  l . the mia was injected intra - articularly through the patellar ligament of the right knee using a 26  g needle under anesthesia . the left knee joint ( control ) was injected with saline . oral treatment with luf6000 , 100  g / kg , bid started on day 7 ( after mia injection ) and lasted until the termination of the study . each group included 10 animals , and the study was repeated at least 3 times . the diameter of the knees was measured every other day using a digital caliper ( mitotoyo , tokyo , japan ) . male c57bl/6j mice 8 weeks of age were obtained from harlan laboratories ( jerusalem , israel ) . experiments were performed in accordance with the guidelines established by the institutional animal care and use committee at can - fite biopharma , petach - tikva , israel . male c57bl/6j mice were injected intravenously ( tail vein ) with concanavalin a ( con a ) ( 20  mg / kg ) . luf6000 was orally administered by gavage , in a dose of 10 and 100  g / kg , twice daily starting 8  h after con a administration . the control group received only dmso in a dilution corresponding to a 100  g / kg dose of luf6000 . blood samples were collected 21  h after con a administration from the retroorbital vein and serum levels of liver enzymes ( sgot and sgpt ) were determined . icr male mice ( 2325  g ) were treated thrice daily with luf6000 , 100  g / kg for 48 hours . blood samples were withdrawn 24 and 48 hours after the last treatment . a differential blood cell count was performed . samples were rinsed with ice - cold pbs and transferred to ice - cold lysis buffer ( tnn buffer , 50  mm tris buffer ph = 7.5 , 150  mm nacl , np 40 ) . cell debris was removed by centrifugation for 10  min , at 7500  g . equal amounts of the sample ( 50  g ) were separated by sds - page , using 12% polyacrylamide gels . the resolved proteins were then electroblotted onto nitrocellulose membranes ( schleicher & schuell , keene , nh , usa ) . membranes were blocked with 1% bsa and incubated with the desired primary antibody ( dilution 1  :  1000 ) for 24  h at 4c . blots were then washed and incubated with a secondary antibody for 1  h at room temperature . bands were recorded using bcip / nbt color development kit ( promega , madison , w1 , usa ) . units were determined by calculation of the ratio between the housekeeping gene and the subjected protein . -actin was used in the mia and aia models , while gapdh was used in the liver inflammation model due to previous experiments indicating a much accurate results with the gapdh in the liver . the results were evaluated using student 's t - test , with statistical significance set at p < 0.05 . luf6000 reduced the ra clinical score in an adjuvant induced arthritis rat model , demonstrating a significant anti - inflammatory effect ( figure 2 ) . a3ar expression levels in paw extracts and in the pbmcs were downregulated in the luf6000-treated rats compared to the vehicle - treated ones ( figure 3(a ) ) , demonstrating that a3ar levels in the pbmcs are a reflection of the receptor level in the remote inflammatory organ . pi3k , ikk and ib expression levels in the pbmcs were downregulated upon treatment with luf6000 resulting in a decrease in nf-b expression levels ( figure 3(b ) ) . luf6000 inhibited osteoarthritis development in an experimental rat model manifested by a decrease in knee swelling and edema in the luf6000-treated group compared to the vehicle ( figure 4 ) . a3ar expression levels in the pbmcs were down regulated in the luf6000-treated group ( figure 5(a ) ) followed by down regulation of the expression levels of the inflammatory proteins jak-2 and stat-1 compared to the vehicle - treated group ( figure 5(b ) ) . the a3ar agonist , cf102 , had been found to have a protective effect in a liver inflammation model of acute hepatitis in mice . in the current study we explored the effect of luf6000 on liver inflammation induced by con a induction . a decrease was observed in serum glutamic pyruvate transaminase ( sgpt ) and serum glutamic oxaloacetic transaminase ( sgot ) levels , compared to the vehicle - treated group ( figure 6 ) . luf6000 showed a protective effect in both 10 and 100  g / kg groups , resulting in dose - dependency with the 100  g / kg dose demonstrating a better protective effect than at 10  g / kg . luf6000 administration to normal icr mice for 2 sequential days resulted in an increase in white blood cell ( wbc ) count at 48  h after the last treatment . neutrophil counts were increased at both 24  h and 48  h after luf6000 administration demonstrating a slight stimulatory effect on bone marrow myeloid cells ( figure 7 ) . small molecules that bind to topographically distinct sites on gpcrs are known to generate a conformational change in the orthosteric site of the receptor , thereby modulating the affinity or efficacy of natural ligands . such allosteric modulators , which may alternately enhance or reduce the effect of the native agonist , are considered as safe and efficacious drug candidates . luf6000 is an imidazoquinolinamine allosteric modulator at the human a3ar , known to enhance the efficacy of receptor agonists acting at the receptor 's orthosteric site . this was shown in both [ s]gtps binding assays   and a number of other functional assays . accumulation of the endogenous agonist adenosine in the microenvironment of inflammatory sites has been extensively documented and serves as a good basis to utilize luf6000 as an allosteric ligand of a3ar to evoke a specific anti - inflammatory effect . luf6000 was also shown to enhance the agonist effect of inosine , which serves as a second , albeit weak , endogenous agonist at the human a3ar [ 2123 ] . in this study we have looked at the effect of the allosteric modulator luf6000 on three different experimental animal models , sharing common mechanistic pathways . in the aia and mia models , an antiarthritic effect , manifested by a reduced clinical score of the disease , interestingly a3ar expression levels in the animals ' pbmcs were found to be downregulated , demonstrating that receptor modulation , most probably internalization and degradation took place . the downstream molecular mechanism entailed inhibition in the expression of pi3k , ikk , and ib resulting in inhibition of nf-b . this mechanistic pathway corroborates with the one described for a3ar agonists , supporting the notion that the natural ligand adenosine mediated the response and that luf6000 is a positive allosteric modulator , other mechanisms can not be excluded though , and it should be kept in mind that a3ar ligand pharmacology may differ between rodent ( in the present study ) and man [ 22 , 23 ] . this study is the first to present data showing the anti - inflammatory effect of luf6000 in vivo . the role of a3ar in mediating inflammatory responses has been extensively described in preclinical and clinical studies . a3ar upregulation has been described in patients with ra   and in animals in which arthritis was induced . receptor upregulation has been attributed to transcription factors such as nf-b , known to be overexpressed in arthritis . treatment with a3ar agonists such as cf101 and cf502 led to a marked improvement in disease parameters in vivo [ 2 , 4 , 25 ] . in experimental animal models , downregulation of the nf-b and the wnt signaling pathways has been shown to mediate the anti - inflammatory effect of the a3ar agonists [ 2 , 25 ] . the mechanistic pathway described in this study shows that luf6000 induces its anti - inflammatory effect via a similar mechanistic pathway as described earlier for a3ar agonists . an additional mechanism explored in the oa model entailed a decrease in the inflammatory proteins stat-1 and jak-2 . inhibition of jak-2 is known to block stat-1 activation as well as matrix metalloproteinase 13 in chondrocytes , resulting in the protection of chondrocytes and cartilage in oa . we next checked the effect of luf6000 in a liver inflammation model of con a - induced hepatitis in mice . luf6000 administration resulted in a decrease of liver enzymes , thus counteracting the effect of con a in a dose - dependent manner . earlier studies showed that a3ar agonists have a hepatoprotective effect on the liver mediated via downregulation of tnf levels and inhibition of the apoptotic proteins bax and bad [ 26 , 27 ] . the effect of luf6000 on normal cells was explored upon its administration to icr mice and the followup on peripheral wbc and neutrophil counts . interestingly , wbc counts were normal upon treatment with luf6000 , and even a slight increase in neutrophil numbers was noted . this effect demonstrated that luf6000 has differential effects on normal and pathological cells , again , similar to the effect of the a3ar agonists ib - meca and cl - ib - meca , which were mediated via the secretion of granulocyte colony - stimulating factor ( g - csf ) [ 2732 ] . luf6000 has been shown to be effective when given orally , inducing specific anti - inflammatory effects , rendering this molecule to be considered as a potential drug candidate .
the a3 adenosine receptor ( a3ar ) is overexpressed in inflammatory cells and in the peripheral blood mononuclear cells of individuals with inflammatory conditions . 
 agonists to the a3ar are known to induce specific anti - inflammatory effects upon chronic treatment . 
 luf6000 is an allosteric compound known to modulate the a3ar and render the endogenous ligand adenosine to bind to the receptor with higher affinity . 
 the advantage of allosteric modulators is their capability to target specifically areas where adenosine levels are increased such as inflammatory and tumor sites , whereas normal body cells and tissues are refractory to the allosteric modulators due to low adenosine levels . 
 luf6000 administration induced anti - inflammatory effect in 3 experimental animal models of rat adjuvant induced arthritis , monoiodoacetate induced osteoarthritis , and concanavalin a induced liver inflammation in mice . the molecular mechanism of action points to deregulation of signaling proteins including pi3k , ikk , ib , jak-2 , and stat-1 , resulting in decreased levels of nf-b , known to mediate inflammatory effects . 
 moreover , luf6000 induced a slight stimulatory effect on the number of normal white blood cells and neutrophils . 
 the anti - inflammatory effect of luf6000 , mechanism of action , and the differential effects on inflammatory and normal cells position this allosteric modulator as an attractive and unique drug candidate .