Afatinib dimaleate(Gilotrif®)阿法替尼BIBW-2992Tablet, oral, EQ 20 mg/30 mg/40 mg afatinibAfatinib is a receptor tyrosine kinase inhibitor which was first approved in 2013 by FDA of US, indicated for the fist-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) whose tumors have epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21(L858R) substitution mutations as detected by an FDA-approved test.Developed and marketed by Boehringer-Ingelheim.The human recommended starting dose is 40 mg (EQ afatinib) orally once daily, taken at least 1 hr before a meal or 2 hrs after a meal.Worldwide Key Approvals Global Sales ($Million) Key Substance Patent Expiration 2013-Jul (US)2013-Sep (EU) 2014-Jan (JP) Not Available2018-Jul (US6251912B1)2021-Dec (EP1345910B1)2021-Dec (JP3827641B2)2021-Dec (CN1277822C)Mechanism of ActionAfatinib covalently bound to the kinase domains of EGFR (ErbB1), HER (ErbB2) and HER4 (ErbB4), and irreversibility inhibited tyrosine kinase autophosphorylation, resulting in down regulation of ErbB signaling.Target Binding Selectivity In Vitro Efficacy In Vivo EfficacyEGFR: IC50=0.5 nM.EGFR L858R/T790M: IC50=10 nM. HER2: IC50=14 nM, HER4: IC50=1 nM. H1666 cells with EGFR: IC50=60 nM.H3225 with EGFR L858R: IC50=0.7 nM.H1975 cells with L858R/T790M: IC50=99 nM.Significantly inhibited tumor growth in H1975(L858R/T790M) NSCLC xenograft model:Afatinib: 15 mg/kg/day, T/C% was 18%.PharmacokineticsParameters Rats Rabbits Minipigs Healthy HumansIn Vivo Dosage (mg/kg)4(i.v.)8(p.o.)1.95(p.o.)2(i.v.)2(p.o.)20(p.o.)30(p.o.)40(p.o.)50(p.o.)C max (nM) 1620 397 34 1190 29 8 14 24 37T max (hr) NA 4 1 0.083 4 5 5 5 5 AUC inf (nM·hr) 2920 2600 178 2000 214 189 327 549 724 T1/2 (hr) 5.2 4.5 2.6 13.8 10.8 30.7 32.9 29.6 28.5 CL(/F) (mL/min/kg) 55 108 467 35 NA1770mL/min1530mL/min1210mL/min1150mL/min Vd (L/kg) 16 44 110 12 NA 4700 L 4350 L 3110 L 2840 L F (%) - 44.5 NA - 11.2 NA NA NA NA Main component in plasma (%) Parent (59) Parent (36) NA Parent (NA)Major Metabolites in plasma (%) NAM2 (27,inactive)NA Trace amount (inactive) Excretion by Urine/Feces (%) 6/91 3/94 1/95 NA 2/93 1/85In Vitro Permeability Papp(A→B)=7.5-12×10-6cm/s in Caco-2 models.PPB (%Bound) 92.6 NA 92.9 95.0 Metabolic stability in LM NA Low metabolic rateDrug-Drug InteractionSubstrate Inhibitor InducerCYP EnzymesCYP3A4 (minor)Weakly inhibited CYP2C9NotOther EnzymesMichael addition (non-enzyme)Weakly inhibited UGT1A1, 2B7NATransportersP-gp, BCRPP-gp and BCRP,OATP-B, OCT3, OCT1NANon-clinical ToxicologySingle Dose Toxicity (MTD) Mice: 300 mg/kgRats: 300 mg/kgSafetyPharmacology•hERG potassium current inhibition (IC50=2.4 μM).•Increased arterial blood and heart rate pressure, prolonged gastric emptingbut no effects on respiration rate in telemetered rats.•Probable phototoxicity: PIF=3.Repeated Dose Toxicity (NOAEL)Rats: 1.5 mg/kg/day(0.19 & 0.06× MRHD)Minipigs: 0.5 mg/kg/day(0.02 & 0.01× MRHD)(Male & Female)Genotoxicity•Potential mutagenic effects in TA 98 Ames assay at 30 μg/plate but noclastogenic effect.ReproductiveToxicity•Fertility NOAEL: 6 mg/kg/day in rats.•Embryonic development: maternal NOAEL: 8 & 5 mg/kg/day (rats & rabbits)•Postnatal development: NOAEL: 8 mg/kg/day in rats.•Afatinib can cross placenta (very low concentration in fetal liver) in rats.•Afatinib was excreted to milk (milk to plasma ratio >100 ) in lactating rats.Afatinib dimaleate 1 Afatinib dimaleate(Gilotrif ®)§1 General InformationAfatinib is a receptor tyrosine kinase inhibitor which wasfirst approved in 2013 by FDA of US.Developed and marketed by Boehringer-Ingelheim.Afatinib covalently bound to the kinase domains of EGFR(ErbB1), HER2 (ErbB2) and HER4 (ErbB4), and irre-versibly inhibited tyrosine kinase autophosphorylation,resulting in down regulation of ErbB signaling and inhibi-tion of tumor cell growth both in vitro and in vivo .Indicated for the first-line treatment of patients with met-astatic non-small cell lung cancer (NSCLC), whose tu-mors have epidermal growth factor receptor (EGFR) exon19 deletions or exon 21 (L858R) substitution mutations asdetected by an FDA-approved test.Available as orally tablet, containing 20 mg, 30 mg or 40mg afatinib and the human recommended starting dose is40 mg once daily.--Key Approvals around the World--#NSCLC: non-small cell lung cancer. Till May. 2015, it has not been approved byCFDA (China)--Worldwide Sales--*: The Sales of Afatinib dimaleate (Gilotrif ®) was not reported in the 2014Annual report of Boehringer-Ingelheim.--Active Ingredient-- Molecular formula: C 24H 25ClFN 5O 3• 2C 4H 6O 4 Molecular weight: 718.10 CAS No.:850140-72-6(Afatinib) 850140-73-7(Afatinib dimaleate) Parameters of Lipinski ’s “Rule of 5” 485.94 2 8 7 88.6Å2 1.569±1.363 a: molecular weight of anhydrous; b: Calculated by ACD/Labs software V11.02 --Drug Product-- Dosage route: Oral.Strengths: 20 mg/30 mg/40 mg (EQ to Afatinib). Dosage forms: Film-coated tablet. Inactive ingredients: Lactose monohydrate, Microcrystalline cellulose, Crospovidone, Colloidal silicon dioxide, Magnesium stearate. The tablet coating contains: Hypromellose, Polyethylene glycol, Titanium dioxide, Talc, Polysorbate 80, FD&C Blue No. 2 (40 mg and 30 mg tablets only). Recommended Dose: The human recommended starting dose is 40 mg (EQ to afatinib) orally, once daily.Take afatinib at least 1 hr before or 2 hrs after a meal.*Sourced from the FDA drug label information.NDA NO. 201292 EMEA/H/C/002280 22600AMX00018000/22600AMX00019000/22600AMX00020000Brand Name Gilotrif @ Giotrif @ Giotrif @Indication NSCLC NSCLC NSCLCAuthorization Holder Boehringer Ingelheim Boehringer Ingelheim BoehringerIngelheimThe Pharmaceutical Index – Worldwide 2013 NCEs 2 --Key Patents--Patent 1Substituted quinazoline derivativesProduct (SubstanceAmerican Cyanamid Co. NA NA 1997/8/1 NAUS6251912B1 1998/7/29 2001/6/29 2013/7/12 0 day 2018/7/29Patent 2Product (SubstanceBoehringer-Ingelheim WO0250043A1 PCT/EP01/14569 2000/12/20 2001/12/12USRE43431(7019012) 2001/12/17 2012/5/29 2013/7/12 36 days 2022/1/22EP1345910B1 2001/12/12 2009/4/29 2013/9/25 / 2021/12/12JP3827641B2 2001/12/12 2006/7/14 2014/1/17 5 years 2026/12/12CN1277822C 2001/12/12 2006/10/4 NA / 2021/12/12Afatinib dimaleate 3§2 ChemistryRoute 1: Original Discovery Route[1-4]1 Preparation of afatinib CN103755688A Key Ref.2 Quinoline and quinazoline derivative, preparation method, inter-mediate, composition and application CN103965120A3 Discovery of a novel Her-1/Her-2 dual tyrosine kinase inhibitor forthe treatment of Her-1 selective inhibitor-resistant non-small celllung cancerJ. Med. Chem. (2009),52,68804 Quinazoline derivatives, medicaments containing said compounds,their utilization and method for the production thereofWO0250043A1/CN1481370AThe Pharmaceutical Index – Worldwide 2013 NCEs 4 Route 2[5]:5 Process for preparing amino crotonyl compounds US20050085495A1/WO20050037824A2 Key Ref.Afatinib dimaleate 5 :Route 3[6]6WO2007085638A1 Key Ref.derivativesThe Pharmaceutical Index – Worldwide 2013 NCEs 6 Route 4[7-9]:7 Afatinib preparation method CN103242303A Key Ref.8 Process for preparation of afatinib and intermediates thereof WO2014183560A19 Process for preparation of afatinib CN103288808AAfatinib dimaleate 7 Route 5[10-13]:10 Preparation method of afatinib intermediate CN103254156A Key Ref.11 Method for preparing afatinib CN103254182A12 Method for preparing afatinib CN103254183A13 Process for preparation of afatinib and intermediates thereof WO2014180271A1The Pharmaceutical Index – Worldwide 2013 NCEs 8§3 Pharmacology✧ Mechanism of Action● Afatinib covalently bound to and irreversibility inhibited the auto-phosphorylation of, the kinase domains of EGFR (ErbB1, IC 50=0.5nM), HER2 (ErbB2, IC 50=14 nM) and HER4 (ErbB4, IC 50=1 nM),resulting in down regulation of ErbB signaling [14].● Afatinib was able to inhibit autophosphorylation and proliferation ofmultiple cell lines representing models of overexpression of wildtype EGFR, constitutively active HER2, and EGFR exon 19 deletionmutations or exon 21 L858R mutations, including the L858R/T790Mdouble mutation.● The potential of off-target inhibitory activity of afatinib was minimal.✧ In Vitro Efficacy● Afatinib was able to inhibit proliferation of multiple cell lines [14-16]:Cell lines with constitutively active HER2 or wild type EGFR, gIC 50= 12-60 nM.Cell lines with EGFR exon 19 deletion mutations, gIC 50= 0.7-4 nM.NIH-H1975 with L858R/T790M double mutant EGFR, gIC 50= 99 nM.✧ In Vivo Efficacy● Xenograft models in nude mice derived from multiple wild type EGFR- or HER2- overexpressing tumor celllines [14-16].In A431 (i.e. Epidermoid carcinoma) model: significant tumor growth inhibition and tumor regression at 20mg/kg/day.In SKOV-3 (ovarian carcinoma) model: significant efficacy at 20 mg/kg/day.In NCI-87 (gastric carcinoma) model: significant efficacy at 15 mg/kg/day.In NCI-H1975 (NSCLC with EGFR L858R/T790M double mutation) model: significant efficacy at 15 mg/kg/day. --Mechanism of Action--Target Binding Affinity, Selectivity and EfficacyTable 1: Effects of Afatinib and Other EGFR Inhibitors on Inhibition of EGFR Kinase Activity and EndogenousKinase Activity a WT EGFR KinaseHuman recombinant 0.5 1.0 0.9-1.7 0.3-17 0.3 EGFR L858RHuman recombinant 0.43 0.8 1.2 2 0.4-0.8 EGFR L858R/T790MHuman recombinant 10 1013 1520 >4000 18-36 HER2 kinaseHuman recombinant 14 1830 238-698 6-25 30 HER4 kinaseHuman recombinant 1 323 579 30 1 EndogenousPhosphorylationEGFRA431-EGFR cells 13 35 3-8 93-145 22 H1666 cells 7 72 87 No data 127 EGFR L858RH3255 cells 6 11 52 No data 5 EGFR L858R/T790M NCI-H1975 cells93 >4000 >4000 >4000 79 HER2 BT474 cells75 3600-3710 396-930 74-102 88-184 NIH-3T3-HER2 cells71 2330 No data No data 85 N87 cells 48 541 No data No data 288Note: afatinib covalently bound the EGFR at Cysteine 797 by x-ray structure analysis and mass spectrometry studies of EGFR-afatinib complex. In time course experiment using A431 cells, phosphorylation of EGFR recovered within 48 hrs post washout afatinib, as compared with reversible EGFR inhibitors (such as gefitinib) recovered within 8 hrs post washout. a:LYN 1.33-1.83 SAPK2α/p38α2000 H2 receptor 68 SRC 1.59-2.67 PHK 0.26-1.8M1 receptor78 LCK0.65-1.99a: kinases that >60% inhibition at 10 μM afatinib, screening >27 non-EGFR kinases. b: receptors that >50% inhibition at 5 μM afatinib, screening >50 receptors. c: at 5 μM afat-inib.--In Vitro Efficacy--BT474 Cells Human ovarian carcinoma HER2 anchor-age-dependent 12 1070 599-899 12-128 66 N87 Cells Human gastric carcinoma L858R EGFR 4 690 No data No data 23 H1666 Cells NSCLC EGFR anchor-age-dependent 60 157 110 534 198 H3225 Cells NSCLC EGFR L858R anchor-age-dependent 0.7 5 40 63 1 NCI-H1975 CellsNSCLCEGFR L858R/T790M anchorage-dependent99>4000>4000>4000101In a 72 hrs cell growth assay. NSCLC: non-small cell lung cancer.Table 4: Inhibition of Afatinib in Survival of EGFR-dependent Ba/F3 Cells Ectopically Expressing Various EGFRL858R 4 16 L747_A750del4insP+T790M 60 >10000 L858R+T790M 119 >10000 L747_P750del7insS 2 0.3 E746_A750del5 0.9 5 L747_P750del7insS+T790M 49 >10000 E746_A750del5+T790M 64 >10000 E746_S752del7insV 0.2 25 S752_I759del8 0.2 33 E746_S752del7insV+T790M 102 >10000 S752_I759del8+T790M 103 >10000 VIII (variant III deletion)0.9 144 L747_A750del4insp15Note: Cell line: B-lymphoid mouse Ba/F3 cell, ectopic expression of active EGFR mutants can overcome IL-3 dependent survival. Afatinib inhibited IL-3 independent survival of EGFR exon 19 deletion mutant expressing Ba/F3 cells with low nM concentrations regardless of their T790M status. Erlotinib had no effect on cells expressing EGFR T790M mutants.--In Vivo Efficacy--[14-16, 18]Human Epidermoid CarcinomaA431EGFR overexpressingNude 3 p.o., 15 20100Tumor growth wassignificantly inhibited at 20 mg/kg/day or more, induced tumor regressions.Nude 10 p.o., 15 80 NMRI-nude 20 p.o., 25 2 Nude 30 p.o., 15 2 Human Gastric CarcinomaNCI-N87HER2 overexpressingNude10 p.o., 38 2064Tumor growth wascompletely inhibited and the regression of tumors was induced at 20 mg/kg/day afat-inib.Nude 20 p.o., NA b 4 Nude 20cp.o., 21 NA b Nude30 p.o., 24 NA b Human Ovarian CarcinomaSKOV-3EGFR/HER2Nude 15 p.o., 271513Afatinib suppressedtumor growth, simi-larly to canertenib.Nude20 p.o., 33 3 NSCLC cNCI-H1975EGFR L859R/T790MNMRI-nude15p.o., 201518Afatinib was shownto efficacy in inhibi-tion of tumor growth in NSCLC xenograft models.20p.o., 2012 Note: Afatinib (orally) also strongly inhibited breast tumor growth in MDA-MB-451(HER2), SUM149 (triple-negative) and SUM190 (EGFR/HER2 overexpressing) xenograft mice models, but moderate in MCF-7 (HER2 negative) xenograft mice model. a: ED effect dose at which showed significant difference compared with vehicle. b: NA=not availa-ble. c: NSCLC: non-small cell lung cancer, afatinib also had efficacy in NCI-H1781 (HER2 G776insV G/C) xenograft mice model (T/C not available).Figure A: Tumor Volume in NCI-H1975 Xenograft Model Administrated with Afatinib [16]Study: NCI-H1975 xenograft mice model Animal: NMRI-nu/nu mice (female)Xenograft: NCI-H1975 human non-small cell lung cancer cells containing L850R/T790M double mutant EGFR were inoculated into female NMRI nude mice.Administration: Treated daily, p.o., afatinib 15 or 20 mg/kg/day, vehicle control, and reference gefitinib (75 mg/kg/day), lapatinib (100*2 mg/kg/day, twice daily).Starting treatment: Mice bearing established tumors (40-130mm 3) Test: Tumor volumes = π/6×length× (width)2, 3 times a week.Result : Xenograft tumor was effectively controlled by afatinib, with a T/C value of 12% for doses of 20 mg/kg/day, while it was resistant to lapatinib and gefitinib.§4 ADME & Drug-Drug Interaction✧Absorption•Oral dosing produced non-linear increased in AUC exposure in humans. The increase in C max and AUC inf in the dose range of 20 to 50 mg afatinib appeared to be more than dose-proportional.•Afatinib had a moderate oral bioavailability in rats (44.5%) and a low oral bioavailability in minipigs (11.2%).•Afatinib was absorbed slowly with the T max occurring 4 to 5 hrs in non-clinical species and humans but rapidly ab-sorbed in rabbits (T max=1 hr).•The half-life of afatinib in humans (29.6 hrs) was longer than them in rats (4.54 hrs), rabbits (2.6 hrs) and minipigs(10.8 hrs).•The clearance of afatinib in rats (55.3 mL/min/kg) and minipigs (35.3 mL/min/kg) were higher comparable to liver blood flow, but lower in humans (1210 mL/min).•The apparent volume of distribution was high in humans (3110 L), rats (43.6 L/kg), rabbits (110 L/kg) and minipigs(12.4 L/kg), so afatinib had an extensive distribution in tissues in all species.•The permeability of afatinib was high with the Papp (A-B) (7.5-12) ×10-6cm/s in Caco-2 cells model.✧Distribution•Afatinib exhibited high plasma protein binding in humans (95.0%), rats (92.6%), mice (94.3%) and minipigs (92.9%), and the Cc:Cp were more than 1 in all species, suggesting penetration into red blood cells. In human plasma, afatinib mainly bound to HGB.•Albino and Pigmented male rats following a single intravenous Administration:Afatinib was rapidly and well distributed from blood into most of the tissues except for the central nervous system (CNS) since the blood-brain barrier was crossed by a very small extent.The high concentration tissues were kidney, adrenal, brown fat, spleen, pituitary, and accessory sex organs of the male. It was similar between pigmented and albino rats. However, in pigmented rats, the concentration of afatinib inthe retina of the eye was very high and kept constant over the investigation period, indicative of melanin binding.Some accumulation was observed in the liver and kidney, adrenal, pituitary and spleen.Minipigs following a single oral administration: the majority of the dose was recovered from the liver and spleen, and the autoradiography of the eyes demonstrated afatinib could bind to melanin.✧Metabolism•The metabolism of afatinib was low in human liver microsomes.•The major component was parent and the major metabolites were the covalent adducts to endogenous proteins in human plasma. A few metabolites of afatinib were detected in trace amount in human plasma.•All the metabolites found in humans could be found in other species. However, the major metabolite was M30 with ra-dioactivity 31.4% of dose in feces of mice.•Afatinib metabolism catalyzed by CYP450 enzymes was to a minor extent in vitro and in vivo. The metabolite M10 catalyzed by CYP3A4 was in trace amount. The major metabolites were produced by Michael addition(non-enzymatic).✧Excretion•The predominant elimination route of afatinib was via feces and the major component in feces and urine was parent in all species.•Approximately 88% of the excreted [14C] radioactivity was afatinib, followed by 6.7% as M4, 3.7% as M13 and 0.4% as M15 in human urine and feces after oral administration.✧Drug-Drug Interaction•Afatinib was a weak inhibitor of CYP2C9 (IC50 =79.3 μM), UGT1A1 (IC50 =24.2 μM) and UGT2B7 (IC50 =73.7 μM).•Afatinib was not an inducer of CYP450 (CYP3A4, CYP2D6, CYP1A2, CYP2C19, CYP2B6, CYP2C8, CYP2E1, CYP4A11).•Afatinib was a substrate of P-gp and BCRP, and had inhibition for P-gp and BCRP.•Afatinib was not a substrate of OATP2, OATP8, OATP-B, OAT1, OAT3, OCT1, OCT2, OCT3, and had inhibition for OATP-B (IC50=6.05 μM), OCT3 (IC50=11.8 μM) and OCT1 (IC50=20 μM).--Absorption--Table 6: Pharmacokinetic Parameters of Afatinib BS after Single Intravenous or Oral Dose Administration in all Spe-[15, 19]Wistar Rats/Male i.v. 4 NA 1620 2920 5.22 55.3 16.2 - p.o. 8 4 397 2600 4.54 108 43.6 44.5 Himalay Rabbits/Female p.o. 1.95 1 34 178 2.6 467 110 NA Gottingen Minipigs/ Male& Femalei.v.. 2 0.083 1190 2000 13.8 35.4 12.4 - p.o. 2 4 29.1 214 10.8 NA NA 11.2 Healthy Humans/ Male& Femalep.o.20 mg 5.0 7.78 189 30.7 1770 mL/min 4700 LNA p.o. 30 mg 5.0 13.7 327 32.9 1530 mL/min 4350 L NA p.o 40 mg 5.0 24.3 549 29.6 1210 mL/min 3110 L NA p.o.50 mg5.037.172428.51150 mL/min2840 LNAa: Formulation: tablet for humans. NA=not applicable. The unit of C max for humans was ng/mL. The unit of AUC inf for humans was ng/mL/mg.Figure B: Mean Concentration-Time Profile in Plasma and Blood of [14C] Afatinib after a Single Intravenous Dose of 4 mg/kg and Oral Dose of 8 mg/kg Administration to Male Wistar RatsFigure C: Plasma Concentration-Time Profile of Afatinib after Oral or IV Dose of 2 mg/kg Afatinib Administration to Minipigs and Oral Dose of 20-50 mg Afatinib to Healthy Male Subjects[14]Afatinib MA27.5-12High--Distribution--14[14, 15]Rats 0.05-0.5 92.6 Rats p.o. 8 24 129.9 Minipigs 0.05-0.5 92.9 Minipigs p.o. 2.46 168 27.8 Humans 0.05-0.5 95.0 Rabbits p.o. 4 96 7.4 Mice0.05-0.594.3Humansin vitro150 μM2 mins2.21Cc:Cp= RBCs to plasma concentration ratio.Table 9: In Vivo Distribution Study of [14C] Afatinib in Albino Rats, Pigmented Rats and Minipigs after a Single Intra-[16]Blood 3105 740 1 2402 884 1 NA NA Brain 81 15 0.02 88 36 0.03 NA NA Kidney45697 2903 5.1 22509 2918 4.42 366 297 Liver 24427 1210 2.4 11662 1200 2.16 1180 953 Lungs 25590 620 2.06 8513 838 1.39 392 343 Spleen 10668 1792 4.09 33007 2897 4.93 619 1020 Pancreas 24607 479 1.4 9028 488 1.16 NA NAAdrenal 54190 1650 3.39 24284 ND 2.82 125 107 Access.genital gl. 12583 ND 2.71 14922 3214 2.86 NA NA Pituitary 29978 132 4.78 40490 1211 8.4 NA NA Brown fat 41881 537 1.97 9229 377 1.3 NA NA Retina NA NA NA 50224 51940 37 NA NA Skin25192780.5136469661.4354.359.8Sampling time: 0.083, 4, 24, 96 hr for albino rats; 4, 24, 96 hr for pigmented rats. NA=not applicable.--Metabolism--Table 10: Metabolites in Humans vs. Non-clinical Test Species Plasma, Urine, Bile and Feces after Oral Administra-[15, 16]PlasmaRats c p.o. 8 0-24 58.8 NA NA NA NA NA NA NA NA - - - Rabbits c p.o. 1.95 0-24 36 - 26.6 1.8 - 5 5.1 NA NA NA - - Humans p.o. 15 NA majority NA NA NA - NA NA NA NA NA - -UrineMice p.o. 8.5 0-24 1.04 - NA NA - 0.03 0.05 - 0.02 0.04 - - Rats a i.v. 4 0-24 13 1.5 4.9 5 2.4 NA NA 1.7 1.3 - - NA Minipigs p.o. 2.46 0-96 0.45 - - 0.26 - - 0.33 - 0.05 1.1 - - Humans b p.o. 15 0-72 88 NA 0.2 0.2 - NA NA 3.7 NA 0.4 - -BileMice i.d. 8.5 0-6 7.48 NA 4.9 4.9 NA 0.25 0.2 - 0.03 NA - 0.55 Rats a i.v. 4 0-6 13 1.5 4.9 5 2.4 NA NA 1.7 1.3 - - NA Minipigs p.o. 2.46 NA 0.82 NA NA NA NA NA 0.27 NA 0.11 0.19 - NAFecesMice p.o. 8.5 0-48 59.97 - NA NA - 1.6 NA - NA 0.25 - 31.4 Minipigs p.o. 2.46 0-96 68.85 - - 3.59 - - 13.75 - 1.44 - 3.33 - Humans b p.o. 15 0-72 88 NA NA 6.7 - NA NA 3.7 NA 0.4 - -a: rat: the sum of urine and bile. b: human: the sum of feces and urine. c: rats and rabbits: % of total radioactivity. NA=not applicable.Figure C. Proposed Pathways for the in Vivo Biotransformation of Afatinib in Rats, Minipigs, Rabbits and Humans[15, 16]Table 11: Identification of Enzymes Involved in the Biotransformation of Afatinib with Human Liver Microsomes[14]Conjugate Formation via Michael Addition - M2, M3, M4, M1342 Enzymatic Phase I MetabolismFMO3 M15 48 CYP 3A4 M10 trace CYP450M6, M149--Excretion--Table 12: Species Comparison of Excretion Data of [14C] Afatinib after a Single Oral or Intravenous[14-16]CD-1 Mice Male & Female8.5 p.o. 96a 10.1 1.2 95.4 96.8 Albino Wistar Rats Male 8 p.o. 96b 14.6 2.7 93.6 96.5 4 i.v. 96b 28.3 5.5 90.8 96.7 Himalayan Rabbits Femle 1.95 p.o. 96d 22.8c 0.8 95.4 96.8 Gottingen Minipigs Male 2.46 p.o. 168 ND e 2.2 92.9 95.7 HumansNA15p.o.312NA0.785.4NAa: 6 hrs for biliary excretion. b: 4 hrs for biliary excretion and 192 hrs for urine. c: following intraduodenal administration. d: 168 hrs for faeces excretion. e: in minipigs only spot samples of bile were taken that provide no meaningful data for excretion balance. NA=not applicable.--Drug-Drug Interaction--[15, 16]CYP1A2 >100 0-5NO CYP2A6 >100 0-5 NO CYP2B6 >100 0-5 NO CYP2C8 >100 0-5 NO CYP2C9 79.3 0-5 NO CYP2C19 >100 0-5 NO CYP3A4 >100 0-5 NO CYP2D6 >100 0-5 NO UGT1A1 24.2 NA NA UGT2B773.7NANANA=not applicable.Table 14: In Vitro Evaluation of Afatinib as an Inhibitor and a Substrate of Uptake Transporters and Efflux Trans-[15, 16]as a Substrate Yes Yes Yes NO NO NO NO NO NO NO NO as an Inhibitor IC 50 (μM)241.60.7582.871.26.05>100>10020>10011.8a: Caco-2 cells. b: LLC-PK1 cells.§5 Non-Clinical Toxicology✧ Single Dose Toxicology• Single dose oral administration of afatinib in different species: MTD in mice and rats: 300 mg/kg.Mainly toxicity found was GI track toxicity.✧ Repeat Dose Toxicology• Repeated dose oral administration of afatinib in different species from 2 to 52 weeks: For rats: The NOAEL was 1.5 mg/kg/day (0.19× and 0.06× MRHD for male and female respectively), determinedby 26-weeks repeated dose toxicity study, main toxicity was skin lesion, GI track and kidney toxicity. For Göettingen Minipigs: The NOAEL was: 0.5 mg/kg/day (0.02× and 0.01× MRHD for male and female respec-tively), determined by 52-weeks repeated dose toxicity study, main toxicity was skin lesion, corneal eye, GI trackand kidney toxicity. ✧ Safety Pharmacology• Modified IRWIN test in mice: no effects on general behavior or motility.• Afatinib prolonged gastric empting and increased serum and liver enzymes in rats following single dose administration. • The IC 50 of afatinib in the hERG potassium current was 2.4 μM , suggesting low potential for QTc prolongation at clini-cally relevant concentrations.• In telemetered rats, afatinib administration resulted in increased arterial blood pressure and heart rate at 100 mg/kg, however, no effects were noted on respiration rate and tidal volume.• In domestic pigs, afatinib intravenous infusion administration resulted in decreased LVdP/dt-max contractility at 10 and 30 mg/kg.• PIF (photo irritation factor) =3, classified as probable phototoxicity. ✧ Genetic Toxicology• The genotoxic potential of afatinib was tested in both in vitro and in vivo assays.• Afatinib demonstrated mutagenic potential at 30 μg/plate in TA 98 in Ames assay by the plate incorporation method only both with and without mammalian microsomal activation; it was negative in all other tests. ✧ Reproductive and Developmental Toxicity• Fertility toxicity: The NOAEL was 6 mg/kg/day for both male and female rats.• Fetal embryonic developmental toxicity: The NOAEL was 8 and 5 mg/kg/day for rats and rabbits maternal, respective-ly.• Postnatal developmental toxicity: The NOAEL was 8 mg/kg/day.• Milk excretion of afatinib was also found in lactating rats and excretion ration was: milk/plasma=137.0 (AUC 0-24 hr ), 124.7 (C max ).• No relevant levels of [14C] afatinib were found in embryos and foetuses except very low concentrations in foetal liver.--Single Dose Toxicity--[14, 15]Crl:NMRI Mice 300, 600, 1200 300 Adverse clinical and lethal effects were seen at the dose of 1200 mg/kg. The gastrointestinal tract was the target organ system. CrlGlxBrlHan:WlRats300, 600, 1200300Macroscopic changes were mostly seen in gastrointestinal tract.Vehicle: demineralized water.--Repeated Dose Toxicity--[14, 15, 20]Wistar Rats20, 8, 16, 32NANANAMortality was observed at 32 mg/kg/day. Target organs were he-molymphopoietic system, GI, kidney and liver, starting with 16mg/kg. Most of changes were resolved at the end of the recovery period except for renal papillary necrosis.40, 4, 8.5, 184772:558NAMortality occurred at 18 mg/kg/day. The toxicological main targetorgans were the gastrointestinal tract and the kidneys.Dose-dependent neutrophil, diminished erythropoiesis in the bone marrow were observed at 8.5 and 18 mg/kg/day.13 0, 2, 5, 10 2 264:126 NASkin was a target toxicity organ. Gross pathology changes of thekidney such as enlargement or discoloration.26 0, 1.5, 3, 6 1.5 303:97.7 0.19:0.06 Skin lesions (folliculitis), and kidneys toxicity including papillarynecrosis; repeat dosing resulted in more pronounced accumulationin males than females.Göettin-genMinipigs2 0, 2, 4.5, 10 NA NA NA Toxicity target organ including skin, kidney, and GI track. 40, 1, 2.45, 61109:92.6NAGI related toxicities, a shortening in QT-interval which led to aslight increase in heart rate.13 0, 0.5, 2, 7/5.5a0.5 25.3:15.4 NA Atrophic epithelia of GI, upper respiratory tract, mucinous glands,male genital tract and slight atrophy of the corneal epithelium of the eyes. Target organ was GI track.520, 0.5, 1.5, 50.526.2:19.50.02:0.01Upper GI tract and corneal eye considered targets.Vehicle: demineralized water. a: In the high-dose group, due to observed clinical the dose was reduced from 7.0 to 5.5 mg/kg /day on Day 32, but erroneously increased again to 7.0 mg/kg /day on Day 43, 44 and 46-77.b: safety margin was calculated with MRHD 40 mg QD administration in NSCLC patients and drug exposure AUCss=920 ng ·hr/mL. NA=not applicable.--Safety Pharmacology--[14, 15]Studynocturnal motility in rats 0, 30, 100, 300 mg/kg No serious adverse effects on behavior Cardiovascular Safety Pharmacology StudyVital physiological functions inconscious rats. Single p.o. 0, 10, 30, 100 mg/kg Dose dependent increased blood pressure with significant increase at 100 mg/kg.hERG-mediated potassiumcurrent assay 0.1, 0.3, 1, 3, 10 μM The IC 50 of afatinib in the hERG assay was 2.4 μM.No changes on the action potential configuration.Modified Irwin study in maleand female rats Single p.o. 0, 4, 8.5, 18 mg/kg No other signs of toxicity were observed Hemodynamic andelectrocardiographic parameters in anesthetized domestic pigsBolus injection: 0.3, 1, 3 mg/kg Infusion 20 mins: 10, 30 mg/kgNo significantly changes. Respiratory Safety Phar-macology StudyRatsSingle p.o. 0, 10, 30, 100 mg/kg No observation.Respiratory parameters in con-scious rats Single p.o. 0, 4, 8.5, 18 mg/kgNo change in respiratory parameters Gastrointestinal Safety Pharmacology StudyGastric emptying study in ratsSingle p.o.0, 30, 100, 300 mg/kg Dose dependent decrease in the rate of gastric emptying with 23% at 30 mg/kg; 196% at 100 mg/kg and 430% at 300 mg/kg respectively. Gastrointestinal transit in rats Single p.o.0, 30, 100, 300 mg/kgA dose-dependent inhibition of gastrointestinal transit (~66% decrease at 300 mg/kg ) Gastric secretion in ratsSingle i.d.Reduced gastric secretion at 300 mg/kg (>10×。