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1、White Paper Diabetes Drug Discovery Utilizing in vitro panels and in vivo animal models to evaluate diabetes drug candidates1Diabetes mellitus is a serious chronic disease in which blood glucose levels rise due to the bodys inability to produce enough insulin or to use it effectively.The global prev
2、alence of diabetes continues to grow.Over 535 million adults(age 20-79)have diabetes,according to the International Diabetes Federation,that in 2021 caused 6.7 million deaths 1 death every 5 seconds.The number of people suffering from diabetes is projected to grow to 643 million by 2030,and reach 78
3、3 million by 2045.1 Among all diabetics,over 90%have Type 2 diabetes mellitus(T2DM or T2D);however,Type 1 diabetes mellitus(T1DM or T1D)is the most common form of diabetes in children(19 years of age),and over 1.2 million children have T1D in 2021.2T1D,insulin-dependent diabetes mellitus(IDDM),is pr
4、imarily a chronic autoimmune disease with potential autoantigens identified.It is characterized by progressive autoreactive T cell-mediated destruction and loss of insulin-secreting pancreatic islet cells,leading to deficient secretion of insulin and hyperglycaemia.3,4 A combination of genetic,immun
5、ologic,and environmental factors contributes to the onset and progression of T1D.Treatment of T1D focuses on managing blood sugar levels with insulin replenishment,diet,and lifestyle to prevent further complications.5 The research effort is underway to develop a treatment to prevent-cell loss and/or
6、 preserve-cell function.T2D,noninsulin-dependent diabetes mellitus(NIDDM),is due to a progressive loss of adequate-cell insulin secretion and the inability of insulin-sensitive tissues to respond appropriately to insulin,which is known as insulin resistance(Figure 1).6 The pathogenesis of T2D involv
7、es polygenic and environmental factors and is strongly linked with such risk factors as obesity and being overweight.Besides lifestyle changes to reduce blood sugar levels,the treatment for T2D is more diverse(see under“Diabetes Drug Discovery”)and more complicated than for T1D.7,8Diabetes-cell dysf
8、unctionInsulin resistanceIncreased glucagon release from-cells Increased FFA secretionIncreased lipolysisIncreased release inflammatory mediators(TNF)-cells degranulation Reduced insulin secretion from-cells HyperglycaemiaIncreased glucose productionDecreased glucose uptake and expression of GLUT4Fi
9、gure 1.Pathology of T2D.Insulin is a hormone synthesized by-cells of pancreatic islets,playing a key role in glucose homeostasis by reducing blood glucose levels.Insulin activates insulin receptors(INSR)in the liver,skeletal muscle,and adipose tissue to stimulate glucose transporter(GLUT4)translocat
10、ing from the intracellular site to the cell membrane,making the cells efficiently uptake glucose.-cell dysfunction follows damage from elevated free fatty acid(FFA)levels,obesity,insulin resistance,and inflammation.-cells can compensate by promoting insulin release initially;however,as time passes,t
11、he compensatory mechanism declines,and-cell mass decreases.The loss of-cell mass is due to cell degranulation,causing increased-cell glucagon and decreased-cell insulin.Meanwhile,skeletal muscle and adipocytes are incapable of modulating the increased glucose levels.In addition,overloaded adipocytes
12、 increase lipolysis and cytokine release of inflammatory mediators such as TNF-.Released FFAs also stimulate glycogenolysis in the liver.T2D is ultimately caused by a hyperglycaemia environment.Figure 1 is modified from Riddy DM,et al,2018.62Currently,there are nine major classes of antidiabetic age
13、nts for T1D and T2D(Table 1),including biguanide,DPP4 inhibitors(Dipeptidyl peptidase-4 inhibitor),SGLT2 inhibitors(sodium-glucose cotransporter 2 inhibitors),insulin/insulin analogues,GLP1(glucagon-like peptide 1),amylin analogues,sulfonylureas,thiazolidinediones,and-glucosidase inhibitors.7,8 Acco
14、rding to international guidelines,patients should take metformin as the first-line T2D treatment.Some classes of antidiabetic agents offer benefits in addition to glucose-lowering;in particular,they may avoid low blood glucose(hypoglycaemia),protect the heart and kidneys,and prevent weight gain.Ther
15、efore,they may be combined with metformin for T2D patients with certain risk factors.9 Also,combination therapy with drugs of different mechanisms is often applied to better maintain blood glucose levels.10 However,even though myriads of therapeutic agents are clinically available,various adverse si
16、de effects are still associated with current therapies(Table 1).Diabetes Drug DiscoveryAntidiabetic Drug ClassRepresentative Clinical Agent Therapeutic TargetMechanisms of ActionAdverse EffectsBiguanideMetforminAMPK(Indirect)Improve insulin sensitivity,decrease glucose productionNausea,lactic acidos
17、isDPP4 InhibitorsSitagliptin,SaxagliptinDPP4Improve incretin secretion,improve insulin secretionGI discomfort,heart failureSGLT2 InhibitorsCanagliflozin,DapagliflozinSGLT2Inhibit renal glucose reabsorptionKetoacidosis,genital mycosis,bone fractureInsulin AnaloguesHumulin R,Novolin RINSRStimulate glu
18、cose uptake Hypoglycaemia,weight gain,injection site reactionGLP1 AnaloguesLiraglutide,SemaglutideGLP1RImprove insulin secretion Increase satiety,delay gastric emptyNausea,injection site reactionAmylin AnaloguesPramlintideAMY ReceptorDelay gastric empty,inhibit glucagon secretionNauseaSulphonylureas
19、GlyburideKATPImprove insulin secretionHypoglycaemia,CV risk,weight gainThiazolidinediones(TZDs)PioglitazonePPARImprove insulin sensitivity,stimulate glucose uptakeWeight gain,edema,heart failure-Glucosidase InhibitorsAcarbose-GlucosidaseInhibit carbohydrate degradationGI discomfort,diarrheaTable 1.C
20、urrent antidiabetic agents.3There are approximately 60 FDA-approved antidiabetic drugs,and many more are being evaluated in clinical trials.7,8 The enormous heterogeneity of diabetes drives more precise and appropriate treatment.Figure 2 shows antidiabetic clinical trials from 2010 to 2021,with a to
21、tal of 103 cases across 37 targets collectively.5,8,9,11 The majority of entries identified target GLP1R,GCK,GCGR,SGLT1,and SGLT2 receptors(47%in total),as well as the combination of these targets(e.g.,GLP1R/GCGP,GLP1R/GIPR,or GLP1R/GIPR/GCGR)(14%)(Figure.2A).Small molecules and peptides are the maj
22、or types of study drugs(55%and 31%,respectively)(Figure.2B).T2D is the dominant diabetes type for antidiabetic drug development(Figure.2C).Diabetes Drug DiscoveryGLP1R14%GLP1R/GCGRGLP1R/GIPRGLP1R/GIPR/GCGR14%GCK7%GCGR6%SGLT1/26%GPR405%GPR1195%Amylinreceptor4%11-HSD1 3%FGFR13%Fructose-1,6-Biphosphata
23、se3%INSR3%PTP-1B3%SIRT13%AMPK2%DGAT12%DPP42%IBAT2%Other targets16%ABCT2D88%T1D12%Small Molecules52%Peptides34%Biotherapeutics9%Others5%Figure 2.Diabetes drug discovery and development.Major antidiabetic drug classes are currently in various clinical phases of drug development.Percentages represent t
24、he percentage of unique compounds currently in development,per respective class based on A.Therapy targets,B.Compound types,and C.Diabetes types.Clinical trials started from 2010 to 2021,with a total of 103 cases across 37 targets collected.5,8,9,11 The discontinued clinical trials are excluded.4Eur
25、ofins Discovery offers two in vitro diabetes panels Insulin Release Panel and Insulin Sensitivity Panel,to be used to identify hits for drug discovery.Both panels cover approximately 70%of core therapeutic targets under clinical development for the treatment of diabetes(Figure 2).The Insulin Release
26、 Panel contains 10 therapeutic targets that are highly involved in insulin secretion(Table 2).The Insulin Sensitivity Panel comprises seven therapeutic targets that are highly involved in insulin resistance(Table 3).This panel contains 10 target-based assays and 1 phenotypic-based assay.The biologic
27、al relevance of each target is described in Table 2.Representative results from the enzymatic glucokinase(GCK)activation assay and cell-based SGLT1 inhibition assay are demonstrated in Figure 3.In Vitro Panels For Diabetes Drug DiscoveryInsulin Release Panel(Item#PP277)ClassTargetItem#Biological Rel
28、evanceGPCRGLP1R231710GLP1R activation increases insulin secretion in a glucose-dependent manner and reduces glycaemia by inhibiting glucagon secretion.GIPRBy InquiryThe combination of GLP1 and GIP analogues improves glycaemic control and weight loss.Chimeric peptides that mimic GIP and GLP1 have bee
29、n developed for diabetes treatment.GPR40By InquiryGPR40 activation stimulates the intestinal secretion of GLP1 and GIP with the pancreatic secretion of insulin in a glucose-dependent manner.NPY1R257010NPY1R activation may particularly contribute to insulin secretion after weight-loss surgery for T2D
30、.The analogues of PYY(1-36),a selective NPY1R agonist,have been designed for diabetes treatment.GCGR231680GCGR antagonists that inhibit glucagon actions can counter high glycaemia in diabetes.Several GLP1R/GCGR dual agonists and GLP1R/GCGR/GIPR tri-agonists have been developed for diabetes treatment
31、.KinaseGCK199101-0GCK activation promotes hepatic glucose uptake,glycogen synthesis,and enhances glucose-stimulated insulin secretion from the pancreas.PeptidaseDPP4199007DPP4 plays a key role in the clearance of GLP1.DPP4 inhibition has been well-established for glycaemic control improvement in T2D
32、 patients.Ion ChannelKATP265600Inhibition of pancreatic KATP channels leads to depolarization and increases intracellular calcium levels,resulting in insulin release.TransporterIBAT314100-1IBAT inhibition prevents bile acid reabsorption,which leads to GLP1 secretion and GLP1R activation.SGLT1355710-
33、1SGLT1 inhibition delays glucose absorption in the small intestine and colon,resulting in glucose reduction,insulin secretion,and glycaemic control improvement.PhenotypicInsulin Release331500-0Pancreatic islet beta-cell line HIT-T15 is used to assess the ability to stimulate insulin release by test
34、compounds.Table 2.The biological relevance of each target included in the Insulin Release Panel.5GCK Activation-10-9-8-7-6-5-4-3-2020406080100120LogCompound(M)ControlSotagliflozinDapagliflozinCanagliflozinActivation(%)PF-04991532,EC50=0.34 MDorzagliatin,EC50=0.86 M-10-9-8-7-6-5-4-20020406080100Canag
35、liflozin,IC50=410 nMDapagliflozin,IC50=1200 nMLogCompound(M)Inhibition(%)Sotagliflozin,IC50=87 nMSGLT1 InhibitionACBFigure 3.Demonstrated results for the Insulin Release Panel.A.Enzymatic GCK activation assay(#199101-0).GCK is a promising therapeutic target in which several clinical candidates are u
36、nder clinical development for diabetes indication.7 The EC50 of the two reported GCK activators were validated via enzymatic GCK activation assay.PF-04991532 and Dorzagliatin are clinical candidates for T2D treatment in Phases 2 and 3,respectively.12,13 B.Cell-based SGLT1 inhibition assay(#355710-1)
37、.SGLT1 inhibition is an emerging antidiabetic strategy by reducing dietary glucose absorption in the intestine and increasing the release of gastrointestinal incretins like GLP-1.14 The IC50 of the approved antidiabetic agent was validated through the image-based SGLT1 uptake assay.Sotagliflozin(Zyn
38、quista),the first-in-class dual inhibitor of SGLT1/2,was approved by EMA for T1D indication in 2019.15 Canagliflozin and Dapagliflozin,the selective SGLT2 inhibitors,are antidiabetic agents approved by FDA in 2013 and 2014,respectively.16 C.Representative images for SGLT1 inhibition assay.Uptake of
39、the fluorescent substrate,1-NBDG(160 M),was performed by stably-expressed human SGLT1 cells(Control)and was significantly inhibited in the presence of Sotagliflozin(300 nM),Dapagliflozin(3 M),or Canagliflozin(1 M).The substrate uptake level was analyzed via high-content imaging.In Vitro Panels For D
40、iabetes Drug Discovery6Insulin Sensitivity Panel(Item#PP278)This panel contains seven target-based assays.The biological relevance of each target is summarized in Table 3.Representative results from the enzymatic AMPK activation assay and enzymatic 11-HSD1 inhibition assay are demonstrated in Figure
41、 4.ClassTargetItem#Biological RelevanceKinaseAMPK199100-0AMPK is the center of energy metabolism that regulates glucose uptake,glucose,and lipid metabolism.The first-line antidiabetic drug metformin,an indirect AMPK activator,is widely prescribed for T2D patients.INSR243000INSR activation causes the
42、 translocation of GLUT4 to the cellular membrane.This enhances glucose uptake and metabolism.Abnormally expressed INSR is highly associated with insulin resistance.GSK3176500GSK3 inhibition enhances glycogenesis and glucose metabolism.GSK3 signaling pathway is highly correlated with diabetes complic
43、ations such as diabetic neuropathy.NHRPPAR267500PPAR activation improves insulin sensitivity by increasing glucose uptake through GLUT4 in muscle and reducing free fatty acid by lipogenesis.This enhances the utilization of glucose.PhosphatasePTP1B192010PTP1B Inhibition suppresses activated INSR and
44、leptin receptors,leading to insulin sensitivity improvement.DeacetylaseSIRT1By InquirySIRT1 activation inhibits PTP1B activity and thus increases insulin sensitivity.SIRT1 activation also enhances insulin secretion.Dehydrogenase11-HSD1125710Abnormal high-cortisol levels are highly associated with in
45、sulin resistance and diabetes.11-HSD1 catalyzes the conversion of cortisone to cortisol.11-HSD1 inhibition reduces cortisol levels and results in insulin sensitivity improvement.Table 3.The biological relevance of each target included in the Insulin Sensitivity Panel.11-HSD1 Inhibition-9-8-7-6-5-4-3
46、-2-20020406080100LogCompound(M)Inhibition(%)PF-915275,IC50=0.041 MBVT-2733,IC50=39 M-10-9-8-7-6-5-4-3020406080100A769662,EC50=0.022 MAMP,EC50=4.0 MLogCompound(M)Activation(%)AMPK ActivationABFigure 4.Demonstrated results for the Insulin Sensitivity Panel.A.Enzymatic AMPK activation assay(#199100-0).
47、AMPK is the center of energy metabolism that regulates glucose uptake,glucose,and lipid metabolism.17 The first-line antidiabetic drug metformin,an AMPK activator,is widely prescribed for T2D patients.The EC50 of the two reported AMPK activators,A769662 and AMP,was validated via the enzymatic AMPK a
48、ctivation assay.18 B.Enzymatic 11-HSD1 inhibition assay(#125710).11-HSD1 is a critical metabolic enzyme that catalyzes pathophysiological processes like T2D and obesity.The 11-HSD1 inhibition represented an attractive therapeutic strategy for the treatment of T2D.19 The IC50 of the selective 11-HSD1
49、 inhibitors,BVT-2733 and PF-915275,was validated via the enzymatic 11-HSD1 inhibition assay.19,207In Vivo Models Of Diabetes From Pharmacology Discovery ServicesOral Glucose Tolerance Test(OGTT)OGTT Protocol In MouseThe ideal preclinical animal model of diabetes would reproducibly mimic the diverse
50、disease pathogenesis that is observed in diabetic patients,and the model should be readily available and should not be a cost burden.21 None of the currently available diabetes models fully replicates the disease.Animal models for T1D can broadly be divided into two types:genetic models and chemical
51、-induced models.Genetically T1D-predisposed models include nonobese diabetic(NOD)mice and diabetes-prone BioBreeding(BB)rats,which display many of the characteristics of human T1D.22 Streptozotocin(STZ),an antineoplastic agent,when given in a single injection at a large dose induces diabetes often r
52、eferred to as a model for T1D;it is used to study diabetic-cell glucotoxicity.STZ can cause partial destruction of pancreas-cell,leading to insulin deficiency and hyperglycaemia in mice and rats.Rodent animal models of T2D include genetic and diet-induced.These models have been shown to recapitulate
53、 the hallmarks of the disease in humans:elevated fasting glucose and glucose intolerance.Drugs for T2D in humans are equally effective at reversing hyperglycaemia in these models.Thus,although differences in the progression of the disease exist between humans and model animals,T2D animal models are
54、extensively used in evaluating novel anti-diabetic drugs.The most commonly used genetic T2D models include ob/ob mice,db/db mice,Zucker diabetic fatty(ZDF)rats,and KK-Ay mice.23 These strains carry single gene spontaneous mutations in either the leptin(Lepob,ob/ob mice)or the leptin receptor(Leprdb,
55、db/db mice and Leprfa/fa,ZDF rats)genes in an inbred C57BL/6J,or a C57BLKS/J background,respectively,or A genes(KK-Ay mice).24 Diabetes and obesity in humans,however,is a polygenic event and very few diabetic patients carry mutations in their Lep,Lepr,or A genes.There are other polygenic diabetic mi
56、ce strains that develop diabetes due to a combination of multiple diabetes-susceptibility alleles,which may be more suitable for studies on drug candidates with different mechanisms of action.Patients with advanced diabetes if not properly treated often develop complications due to excessive blood g
57、lucose rise,including nephropathy,neuropathy,retinopathy,liver steatosis,impaired wound healing,and increased risk of cardiovascular diseases.Researchers have developed animal models to test for these complications,including Pharmacology Discovery Services(PDS).Pharmacology Discovery Services(PDS)cu
58、rrently offers one T1D model,i.e.,the STZ-induced diabetes model in both mice and rats,as well as three T2D rodent models,KK-Ay mice,db/db mice,and ZDF rat model.In addition to both T1D and T2D diabetes models,PDS also offers rodent models of diabetic complications,e.g.,diabetic nephropathy,wound he
59、aling in diabetic mice(item#595020),liver steatosis and NASH(items#546080 and#546082,learn more in our recent NASH White Paper).In this Diabetes White Paper,we will first discuss Oral Glucose Tolerance Test(OGTT),a test commonly used in diabetic research,and then describe animal models for T1D and T
60、2D.The glucose tolerance test is a medical test in which glucose is given and blood samples are taken afterward to determine how quickly it is cleared from the blood.The test is usually used to test for diabetes,insulin resistance,impaired beta cell function,and sometimes reactive hypoglycaemia and
61、acromegaly,or rarer disorders of carbohydrate metabolism.We use groups of six male or female ICR or C57BL/6 mice weighing 22 4 g.Animals are fasted overnight(16 hours),and test article is administered to test animals 30 minutes before oral glucose(1 g/kg)loading.Blood is collected from the tail vein
62、,and the blood glucose is measured by a Glucometer at-30(pre-treatment),0(before glucose loading),30,60,90,and 120 minutes after glucose loading.The area under the curve over 120 min(AUC0120 min)is determined.In addition,the peak blood glucose is compared at all time points.8Streptozotocin-Induced T
63、1D ModelsStreptozotocin-Induced T1D Models Protocol In MouseOGTT Protocol In RatStreptozotocin(STZ)can cause pancreatic-cell destruction after selectively being taken up by-cells via the GLUT2 glucose transporter,leading to insulin deficiency and hyperglycaemia in mice and rats.25 It is currently th
64、e most used diabetogenic agent in experimental animals for inducing insulin-dependent diabetes mellitus(IDDM),also known as T1D.However,STZ-induced diabetes does not seem to be autoimmune-mediated.STZ can also produce diabetes that is similar to T2D,when injected at a lower dose in rodents fed a hig
65、h-fat diet(HFD).26We use groups of six male or female ICR mice weighing 24 2 g.Test article is administered orally(PO)to a group of six male or female ICR mice weighing 24 2 g,48 hours after challenge with streptozotocin(160 mg/kg,IV).Serum glucose is determined by an automated biochemical analyzer
66、TBA-120FR,obtained from each non-fasted animal,5 minutes before and 90 minutes after test article administration.We use groups of six male or female Wistar or Sprague Dawley(SD)rats weighing 200 40 g.Animals are fasted overnight(16 hours)and test article is administered to test animals 30 minutes be
67、fore oral glucose(1 g/kg)loading.Blood is collected from the tail vein,and blood glucose is measured by a Glucometer at-30(pre-treatment),0(before glucose loading),and at post-glucose loading at 30,60,90,and 120 minutes.The area under the curve over 120 min(AUC0120 min)is also determined.In addition
68、,the peak blood glucose is compared at all time points.AB0500Vehicle(1%Tween 80),10 mL/kg,POGlibenclamide,1 mg/kg,POGlucoseloadedTreatment-300*Time(minutes)Blood glucose levels(mg/dL)*p0.05,treated vs.vehicle control;two-way ANOVA followed by Bonferronis test.050100150200Vehicl
69、e(1%Tween 80),10 mL/kg,POGlibenclamide,1 mg/kg,POGlucoseloadedTreatment-300306090120*Time(minutes)Blood glucose levels(mg/dL)*p0.05,treated vs.vehicle control;two-way ANOVA followed by Bonferronis test.Figure 5.Demonstrated data for OGTT test.A.OGTT test in mice(#540100)or B.OGTT test in rats(#54011
70、0).9Streptozotocin-Induced T1D Models Protocol In RatT2D ModelsWe use groups of six male or female Wistar rats weighing 250 50 g.Test article is administered orally(PO)to a group of six male or female Wistar rats weighing 250 50 g,48 hours after challenge with streptozotocin(65 mg/kg i.v.).Serum glu
71、cose is determined by an automated biochemical analyzer TBA-120FR,obtained from each non-fasted animal,5 minutes before and 90 minutes after test article administration.Some of the most commonly used genetic T2D models include ob/ob mice,db/db mice,Zucker diabetic fatty(ZDF)rats,and KK-Ay mice.23 Mi
72、ce homozygous for the Lepob(ob/ob mice)or Leprdb mutation (db/db mice)are hyperphagic,rapidly gain weight and become obese and hypoglycaemia.Hyperglycaemia in ob/ob mice is transient and mice become normoglycaemia,yet hyperinsulinemic by 14 to 16 weeks of age,and therefore can only be used for diabe
73、tic research before 14 weeks of age and not for long-term studies.The db/db mice have genetically determined obesity and such diabetic syndromes as hyperglycaemia,hyperinsulinemia,glucosuria,and severe insulin resistance.Diabetes in db/db mice is more severe than ob/ob mice and shows advanced stages
74、 of the disease.Mice become severely diabetic by 6 weeks of age,suffering pancreatic islet degeneration and atrophy,resulting in lethality sometimes seen as early as 16 to 20 weeks of age.Further,the db/db mice can also be used for studying diabetic nephropathy.23The Zucker fatty(ZF)rat harboring a
75、missense mutation(fatty,fa)in the leptin receptor gene(Lepr)develops obesity without diabetes;Zucker diabetic fatty(ZDF)rats derived from the ZF strain develop obesity with diabetes at 10 to 12 weeks of age and are most widely used for research on T2D among rat models of diabetes.27 Only male ZDF ra
76、ts develop diabetes.KK-Ay mice are a cross between diabetic KK mice and lethal yellow(obese)(Ay)mice,and carry a heterozygous mutation of the agouti gene from Ay mice for obesity with polygenic origin in KK mice for diabetes.28 KK-Ay mice show altered adipokine expression,obesity,insulin resistance,
77、hyperglycaemia,hyperinsulinemia,and dyslipidemia by 8 weeks of age,closely resembling obesity-linked T2D in humans.AB00500*p0.05,treated vs.vehicle control;one-way ANOVAfollowed by Dunnetts test.Pre-treatmentPost-treatmentSerum glucose levels(mg/dL)Vehicle10 mL/kg,SCInsulin1 U/kg,SC020040
78、0600*p0.05,treated vs.vehicle control;one-way ANOVAfollowed by Dunnetts test.Pre-treatmentPost-treatmentSerum glucose levels(mg/dL)Vehicle10 mL/kg,SCInsulin0.3 U/kg,SCFigure 6.Demonstrated data for the Streptozotocin-induced T1D Models.Streptozotocin(STZ)-induced diabetes in A.mice(#541000)or B.rats
79、(#541010),and the effects of insulin.10T2D KK-Ay Mouse Model ProtocolT2D db/db Mouse Model ProtocolT2D-ZDF Rat Model ProtocolWe use groups of 6 non-insulin dependent diabetic mellitus(NIDDM)male or female mice(KK-Ay/Ta Jcl weighing 50 5 g.Test article is administered by oral gavage(PO)once daily for
80、 three consecutive days to groups of six non-insulin dependent diabetic mellitus(NIDDM)male or female mice(KK-Ay/Ta Jcl)weighing 50 5 g(12 to 14 weeks old;serum glucose=400 50 mg/dL,serum insulin=13.0 2.0 ng/mL).All animals are allowed free access to normal laboratory chow and water.Serum glucose an
81、d insulin levels are determined by automated biochemical analyzer TBA-120FR and ELISA(mouse insulin assay kit)before(pre-treatment)and 90 minutes after the last vehicle and/or test article administration(post-treatment)and percent change is determined.The mean SEM of pre-and post-treated values are
82、then calculated.Groups of 6 non-insulin dependent diabetic mellitus(NIDDM)male or female mice(C57BLKS/J Iar-+Leprdb/+Leprdb)weighing 50 10 g are used.Test article is administered by oral gavage(PO)once daily for three consecutive days to groups of 6 non-insulin dependent diabetic mellitus(NIDDM)male
83、 or female mice(C57BLKS/J Iar-+Leprdb/+Leprdb)weighing 50 10 g(10 to 15 weeks old;serum glucose=500 50 mg/dL,serum insulin=13.0 2.0 ng/mL).All animals are allowed free access to normal laboratory chow and water.Serum glucose and insulin levels are determined by automated biochemical analyzer TBA-120
84、FR and ELISA(mouse insulin assay kit)before(pre-treatment)and 90 minutes after the last vehicle and/or test article administration(post-treatment),and percent change is determined.Serum glucose and insulin percentage of post-treatment relative to pre-treatment group values obtained on the third day
85、are calculated.We use groups of six obese Zucker diabetic fatty rats(ZDF/Gmi-fa/fa)12 1 weeks of age.After pre-treatment blood collection,test animals receive treatment of vehicle and/or test article by oral gavage(PO)once daily for a total of 7 consecutive days.All animals are allowed free access t
86、o normal laboratory chow and water.Blood samples are collected again 90 minutes after the last dosing for serum glucose and insulin determination.Serum glucose and insulin levels are measured by automated biochemical analyzer TBA-120FR and ELISA(rat insulin assay kit),and percent change is determine
87、d.Serum glucose and insulin percentage of post-treatment relative to pre-treatment group values obtained on the seventh day are calculated.AB0 hr72 hrs0200400600Vehicle control,10 mL/kg x3,POMetformin,300 mg/kg x3,PO*p0.05,treated vs.vehicle control;two-way ANOVAfollowed by Bonferroni test.Serum glu
88、cose levels(mg/dL)*Day 0Day 70100200300*Vehicle control,10 mL/kg QDx7,POMetformin,300 mg/kg QDx7,PO*p0.05,treated vs.vehicle control;two-way ANOVAfollowed by Bonferroni test.Serum glucose levels(mg/dL)Figure 7.Demonstrated data for the T2D Models.A.db/db mouse model(#541630)and B.ZDF rat model(#5417
89、00)of T2D and the effects of metformin.11Summary Of In Vivo ModelsDiabetes and NASHThe complex and not yet fully understood pathogenesis of diabetes makes it a challenging disease for effective therapeutic intervention.With dozens of candidates in the clinic that target different stages of pathology
90、,efforts to further elucidate disease progression are necessary to achieve success in the clinic.It is also essential to develop novel in vitro assays and in vivo models to accurately evaluate diabetes drug candidates.Eurofins Discovery and PDS have both in vitro and in vivo services to aid clients
91、with their diabetes drug discovery programs.The insulin release,insulin sensitivity,and glucose uptake in vitro panels provide the opportunity to quickly test compounds across multiple targets.To enhance research flexibility,all the assays in both panels can be ordered individually.With several targ
92、ets,multiple assays(e.g.,binding,enzymatic,functional,and phenotypic)may be used to fully interrogate the in vitro pharmacological profile of a compound.For testing of novel anti-diabetics,we have both T1D rodent models(streptozotocin-induced diabetes in mouse,#541000/in rat,#541010)and T2D rodent m
93、odels(db/db mouse,#541630/KK-Ay mouse,#541620/ZDF rat,#541700)available.Parameters measured include serum glucose,insulin,total cholesterol(T-CHO),low-density lipoprotein(LDL),triglyceride(TG)levels,oral glucose tolerance test(OGTT),as well as fat and liver weight,etc.Nonalcoholic fatty liver diseas
94、e(NAFLD)is the most common chronic liver disease worldwide.It includes a variety of progressive liver diseases ranging from nonalcoholic fatty liver(NAFL)to nonalcoholic steatohepatitis(NASH).29 Diabetes promotes the progression of NAFL to NASH and increases the risk of cirrhosis and hepatocellular
95、carcinoma(HCC).Notably,NAFLD is associated with an increased risk of T2D,affecting approximately 45-75%of NAFLD patients with T2D.30 The dynamic association between NAFLD and T2D is bidirectional and has led to the clinical trials of several diabetes candidates(e.g.,GLP1R agonists,AMPK activators,IB
96、AT inhibitors,PPAR agonists)for the treatment of NAFLD/NASH.31,32,33 Figure 8 shows the overlap of in vitro assay targets between Diabetes Panels and NASH Panels.Due to the extensive regulatory requirements for T2D drug development,pharmaceutical companies tend to drift their T2D clinical developmen
97、t to NAFLD/NASH with less strict regulatory requirements.8 Eurofins Discovery offers both in vitro assay panels and in vivo models for Diabetes and NASH,which can be applied for new drug discovery identification or cross-verification(Tables 5 and 6).Disease ModelSpeciesModel NumberGlucose,Blood,Oral
98、 Glucose Tolerance Test(OGTT)Mouse540100Rat540110Type 1 DiabetesStreptozotocin(STZ)-Induced Mouse541000Streptozotocin(STZ)-Induced Rat541010Type 2 DiabetesKK-Ay Mouse541620db/db Mouse541630Obese Zucker Diabetic Fatty Rat541700Table 4.Summary of in vivo diabetes models.12In vitro Diabetes PanelsItem#
99、Insulin Release PanelPP277Insulin Sensitivity PanelPP278In vivo Diabetes ModelsItem#Oral Glucose Tolerance Test(OGTT)Mouse Model540100Oral Glucose Tolerance Test(OGTT)Rat Model540110Streptozotocin(STZ)-Induced T1D Mouse Model541000Streptozotocin(STZ)-Induced T1D Rat Model541010T2D db/db Mouse Model5
100、41630T2D KK-Ay Mouse Model541620T2D ZDF Rat Model541700Table 5.Summary of in vitro diabetes panels and in vivo diabetes models.In vitro NASH PanelsItem#Liver Steatosis PanelPP270Hepatitis PanelPP271Liver Fibrosis PanelPP272In vivo NASH ModelsItem#MCD-induced NASH Model546080CDAHFD-induced NASH Model
101、546082Table 6.Summary of in vitro NASH panels and in vivo NASH models.KATPSIRT1SGLT1PPARFGFR1FGFR4ACC1THRFXRTGR5CCR2/5PDE4A/4BCaspase 3/7/8CysLT1ASK1NRF2LOXL2VAP1LipasePPARGLP1RIBATAMPKPPARGPR40NPY1INSRGSK3GCKDPP4GCGRPTP-1BGIPR11-HSD1Diabetes Panels(17 Targets)NASH Panels(25 Targets)Figure 8.Therape
102、utic targets are included in the in vitro diabetes panels and in vitro NASH panels.Eurofins Discovery and our partner lab,Pharmacology Discovery Services,offer standalone and integrated Diabetes Drug Discovery Services.Eurofins Discovery focuses on the in vitro characterization of compounds,and drug
103、 efficacy could be further evaluated via in vivo diabetes models.Our Technical Directors and Study Directors are available to consult with clients for their in vitro and in vivo pharmacology studies and design studies to assess the efficacy and mechanism of action of drug candidates.We can also cust
104、omize in vitro assays or in vivo studies or develop new assays/models to meet the specific needs of your diabetes programs.For more information on in vitro services,please visit:For more information on in vivo models,please visit: 2023 Pharmacology Discovery Services.All rights reserved.All trademar
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