Pharmaceutical Drug Development Consulting

Transcription

1 Dr. Julia Eva Diederichs Pharmaceutical Drug Development Consulting Preclinical investigations and animal studies: for comprehensive characterisation what is scientifically possible and meaningful

2 A Case Study Development of a liposomal formulation with a NCE for Phase I long term application chronic disease no oncological indication no orphan drug

3 Characterisation of the API CMC Very poorly soluble < 1 µg/ml High moleculare weight: ~ 750 g/mol Highly lipophilic High plasma protein binding > 99.9 % Very low permeability Chemically instable storage at -20 C Sensitive against base and acid purpose of the liposomal formulation: solubilisation and stabilisation

4 Liposomal formulation CMC Two component approach Empty Liposomes Drug loaded transfer medium Purpose of this formulation Immediate preparation and use due to instable drug Final formulation only stable over 48 hours

5 Preparation of the liposomes CMC Transfer medium by mixing 50mg/ml API PEG300 & Ethanol (9:1) Empty liposomes by high pressure homogenisation 96.2 mg/ml phospholipids (mainly soy PC) 21.6 mg/ml Glycerol 2.5 ml Transfer medium ml liposomal Dispersion oscillation shaker for 20 min In-situ loading by instant solubilisation technique

6 CMC Characterisation of the final formulation Batch to batch conformity? Composition 1.5 mg/ml API 93.3 mg/ml Phospholipid 28.8 mg/ml PEG mg/ml Ethanol 20.9 mg/ml Glycerol Physico-chemical characterisation Particle size: nm / PI (Spec. < 100nm / PI < 0.6) Zetapotential: around - 25 mv (Spec. Report value) Drug to Lipid ratio: 0.016

7 Partical size determination CMC PCS Mean diameter nm / PI Bimodal distribution curve seen for volume and number distribution PI decreases with higher dilution of the sample CyroTEM Particle size: 20 nm Different liposome types were detected

8 Partical size determination CMC CryoTEM picture Determination of lamellarity recommended

9 Application route and dosing Intended treatment for Phase I i.v. influsion over 60 min Application volume: 200ml single escalation dose: mg API in 7 dosage steps Composition of highest dose 0.2 g API g Phospholipid 3.8 g PEG g Ethanol 2.8 mg Glycerol 67 ml 5% Glucose as diluent High amount of excipients needed to solubilise desired amount of API

10 Potential toxicity of excipients The following i.v. applied maximal doses are considered acceptable Ethanol: 4.8 mg/kg Glycerol: 42.1 mg/kg Phospholipid: recommended dose for parenteral nutrition 2g fat/kg BW/day however: adverse effects can not be excluded Reversible increase of liver enzymes are reported PEG300: 3.6 g/patient (oral application) however: for i.v. application of PEG400 nephrotoxicity is reported

11 Pharmacology In vitro primary Pharmacodynamics Mode of action: Inhibition of a peptidase IC50 was tested in various cellular assays ( nm) and in blood (20-40µg/ml due to high plasma protein binding) Cave: All in vitro assays were performed with API DMSO solution Therapeutic effect EC50 was determined in an cellular assay with API DMSO solution: 800nM Interaction of liposomes with cells was not considered

12 In vivo Pharmacodynamics Pharmacology No satisfactory animal model exists Elucidation of pharmacodynamic properties is limited to in vitro studies To monitor proposed in vivo effect inhibition of the peptidase was quantified in various PK and Tox studies as surrogate parameter in plasma and/or liver

13 Pharmacology In vitro secondary Pharmacodynamics Cytotoxic effect CC50: 5.2µM ~ 50% apoptotic and necrotic cells were observed at a concentration >5.5µM Tox Ratio CC50/EC50: 6.4 Cytotoxicity assays were performed with DMSO solution and liposomal formulation Liposomal formulation showed higher toxicity Cytotoxic effects could be observed as with the empty liposomes

14 Pharmacology Safety pharmacoloigy according to ICH S7A No critical findings in the intended dosage range In vivo studies were performed with the liposomal formulation In vitro studies were performed with DMSO solution Different formulations for in vitro and in vivo

15 In vitro metabolism Pharmacokinetic and PK/PD relation Inhibition of CYPs 3A4, 2C9, 2C19 and 2D6 Lowest metabolic stability in cynomolgus Low metabolic stability in human and mice High metabolic stability in dog and rat Cave: study was not performed with liposomal formulation Pivotal preclinical studies were performed in rat and dog

16 Pharmacokinetic and PK/PD relation Preliminary studies with aqueous formulation- rat 10% DMSO, 20% Cremophor RH40 Single dose, i.v. bolus dose Inhibititon on Cmax toxicity 15mg/Kg % No clinical signs 30mg/Kg 71 % No clinical signs 45mg/Kg 90 % MTD, 2/5 died Cmax 3h post administration Good dose/exposure correlation

17 Studies with liposomal formulation- rat Pharmacokinetic and PK/PD relation Single dose, i.v. bolus, 30mg/Kg Cmax Max. Inhibition solution 8.8 µg/ml 71 % after 3 hours liposomes µg/ml 63 % after 8 hours Toxicity comparable Recovery delated (48 vs 24 hours) Influence of liposomal formulation on pharmacokinatic

18 Pharmacokinetic and PK/PD relation i.v. bolus, single dose 30mg/Kg, rat - PK/PD relation Blue: plasma concentration Red: inhibition

19 Studies with liposomal formulation- rat Pharmacokinetic and PK/PD relation Single and repeat dose, 30 min i.v. infusion every second day, 15mg/Kg Repeated dose caused mild clinical findings PD effect in plasma: up to 60% inhibition but high fluctuation Clear and more pronounced PD effect in the liver For better evaluation: Organ distribution study liposomal vs. nonliposomal formulation

20 Studies with liposomal formulation- rat Pharmacokinetic and PK/PD relation Two weeks repeat dose study, 60 min i.v. infusion every third day 5% glucose, empty liposomes, 5, 10, 15mg/Kg Toxicity: NOAEL: 5mg/ml MTD: 15mg/ml Target organ: liver PD effect could not be assessed due to technical issues

21 Pharmacokinetic and PK/PD relation NonlinearPK, significantlylower exposureafter thelast dose, no dose correlation For better evaluation: Organ distribution study liposomal vs nonliposomal formulation?

22 Studies with liposomal formulation- dog Pharmacokinetic and PK/PD relation Single dose, i.v. bolus vehicle, 2, 4 and 7.5 mg/kg Toxicity: no clinical finding No relevant PD effect dosage too low Gender specific difference in pharmacokinatik for high dose group

23 Studies with liposomal formulation- dog Pharmacokinetic and PK/PD relation mg/kg male 4 mg/kg male 7.5 mg/kg male 2 mg/kg female 4 mg/kg female 7.5 mg/kg female Concentration [ng/ml] time profile time (h)

24 Studies with liposomal formulation- dog Pharmacokinetic and PK/PD relation Gender specific pharmacokinetic in high dose Dose [mg/k g] Non-compartmental analysis [µg/ml Sex ] C max AUC last AUC inf t max [h] # [h.µg. /ml] [h.µg t t 1/2 (z) V z /ml] [h] [ml/kg] Cl [ml/h/k g] 2 M F M F M F Formulation related effect as not seen with DMSO/surfactant formulation?

25 Pharmacokinetic and PK/PD relation Studies with liposomal formulation dose range finder in dogs Part 1: single escalation dose, i.v. 30 min infusion vehicle, 10, 15 and 22.5 mg/kg Results: No dose dependent plasma level Toxicity - liver and spleen were identified as target organ No gender specific pharmacokinatic No specific PD effect was observed

26 Pharmacokinetic and PK/PD relation Studies with liposomal formulation dose range finder in dogs Part 2: multiple dose, i.v. 30 min infusion, 22.5 mg/kg every second day, 3 applications 3rd dose, female rats Cmax: µg/ml AUCinf: h.µg/ml Results: No difference in PK profile after three applications 3rd dose, female dogs Cmax: µg/ml AUCinf: h.µg/ml No gender specific pharmacokinatic No specific PD effect was observed Dosage still too low? Wrong dosage schedule? Already toxicity observed!

27 Studies with liposomal formulation- dog Pharmacokinetic and PK/PD relation Two weeks repeat dose study, 60 min i.v. infusion every third day saline, empty liposomes, 3, 10, 20mg/Kg Toxicity: NOAEL: 3mg/ml MTD: 20mg/ml Target organ: liver PD effect could not be assessed due to technical issues

28 Pharmacokinetic and PK/PD relation Nonlinear PK, plasma level-time profiles comparable after firstand last administration Half-life increased with phospholipid dose

29 Pharmacokinetic and PK/PD relation Exposure in female animals was higher in high dose group Dose [mg /kg] Non-compartmental analysis [µg/ Sex ml] C max AUC last AUC inf V z t max [h.µg./ [h.µg t 1/2 (z) [ml/ [ml/h [h] # ml] /ml] [h] kg] /kg] R 1 st administration 3 M N/A F N/A M/F N/A 10 M N/A F N/A M/F N/A 20 M N/A F N/A M/F N/A Cl For better evaluation: Comparasion with non-liposomal formulation

30 Repeat dose toxstudy rat Toxicity i.v. 30 min infusion every second day Vehicle, 8, 12, 15, 22.5 and 30 mg/kg Male animals reacted more sensitive than female animals m: single dose 30mg/Kg 3/3 died 20mg/Kg 3/5 died f: after three applications 20mg/Kg 1/5 had to be killed Comparable body weight loss in vehicle and treatment groups

31 Repeat dose toxstudy rat Toxicity Target organ: liver White spots Pale-brown discoloration Moderate elevated liver enzymes: ASAT, ALAT in 20mg/Kg group Increased organ weight Infusion was better tolerated than bolus injection of the same dosage

32 Toxicity TwoweeksGLP toxicitystudy rat 60 min i.v. infusion every third day Vehicle (lipid concentration comparable to high dose), 5% glucose, 5, 10 and 15 mg/kg Target organ: Liver Small focal necrosis, single cell necrosis and dissociation of liver cell strains in mid and high dose group moderate degree Effect of vehicle: dissociation of liver cell strains, reduced food consumption, effect in clinical chemistry NOEAL: 5mg/Kg, MTD: 15mg/Kg

33 Toxicity Two weeks GLP toxicity study in rats clinical chemistry Group No. M/F ALAT [U/l] AP [U/l] ASAT [U/l] T-BIL [U/l] TG [mmol/l] M F M F M F M F M F After the last treatment (study day 14) 1 11/ / / / / End of recovery period(study day 35) 1 3/ / / / Group 1: 5% glucose, Group 2: Vehicle, Group 3: 5mg/Kg, Group 4: 10mg/Kg, Group 5: 15mg/Kg

34 Toxicity Extended single dose toxicity study rat 60 min i.v. infusion Vehicle (lipid concentration comparable to high dose), 5% glucose, 1.25, 2.5, 5, and 15 mg/kg Target organ: Liver Dose dependent necrosis No correlation to increase of liver enzymes no reliable monitoring parameter for clinic No correlation between exposure and increase of liver enzymes No recovery more pronounced effect after 15 days Effect of vehicle: slight liver necorsis Liver toxicity related to liposomal formulation? Administered volume of liposomes above NOEAL?

35 Toxicity Extended single dose toxicity study rat Group 1: Vehicle Group 2: 5% glucose, Group 3: 1.25mg/Kg, Group 4: 2.5mg/Kg, Group 5: 5mg/Kg, Group 6: 15mg/Kg Clinical chemistry

36 Toxicity Extended single dose toxicity study rat Group 1: Vehicle Group 2: glucose, Group 3: 1.25mg/Kg, Group 4: 2.5mg/Kg, Group 5: 5mg/Kg, Group 6: 15mg/Kg NOEAL: 1.25mg/Kg, MTD: 15mg/Kg [histopathological MTD: 2.5mg/Kg]

37 Dose range finder in dogs Toxicity Single dose, 30 min i.v. infusion Vehicle, 10, 15 and 22.5 mg/kg 30 min i.v. infusion, every third day Vehicle and 22.5 mg/kg Results: Changes in biochemistry parameter in all groups: Increased ALAT, ASAT, GGT and Bilirubin Yellow colour of serum samples Spleen weight increased yellow colour of fat tissue in all groups Significant effect of vehicle

38 Toxicity TwoweeksGLP toxicitystudy dogs 60 min i.v. infusion every third day Vehicle (lipid concentration comparable to high dose), saline, 3, 10 and 20 mg/kg Changes in hematology comparable haemolytic effect seen in all groups Biochemistry parameter Elevated ALT, AST, ALP, Bilirubin, GGT and cholesterol level in all groups Increase in spleen and liver weight in all groups no liver necrosis Bile duct hyperplasia in mid and low dose NOEAL: 3 mg/kg, MTD: 20 mg/kg

39 Toxicity TwoweeksGLP toxicitystudy dogs ALT AST GGT ALP CHOL LDH Control 1 2 Vehicle Formulation 3 3 mg/kg VL mg/kg VL mg/kg VL-01 M F M F M F M F M F [U/l] x-fold of control [U/l] % of control [U/l] x-fold of control [U/l] x-fold of control [mmol/l] % of control [U/l] % of control Significant influence of vehicle

40 Toxicity TwoweeksGLP toxicitystudy dogs Adverse effects observed in the vehicle formulation treated groups included considerably increased liver enzye levels (ALT, AST, GGT, ALP, LDH, bilirubin and cholesterol) changes in hematology (decreased RBC, HGB, MCHC levels and increased MCV and RET count) and blood coagulation (decreased APTT) parameters.

41 Lessons learned The following studies were not performed, but seemed to be meaningful for better evaluation of the presented data Organ distribution and PK study liposomal versus non-liposomal formulation Non-liposomal formulation included in toxicity studies In vitro release of the API from liposomes Determination of encapsulated versus nonencapsulated API in vivo In vivo integrity test In vitro assay showed very fast disintegration at low plasma concentration, but no disintegration at high plasma concentration

42 Clinical trial application Feed back from authorities and ethic committée No comments or questions with regard to CMC part Mean issue: therapeutic window too small NOEAL in the most sensitive species was below the dose required to get a significant PD effect

43 Controversial discussion Clinical trial application Are liposomes liver toxic? Does the liposomal formulation contributes to the observed liver toxicity due to liver targeting? Are the toxic effects of the vehicle relevant for the clinical situation or species specific?

44 Dr. Julia Eva Diederichs Pharmaceutical Drug Development Consulting Ehrengutstrasse München Telefon +49 (89) Mobil +49 (179) diederichs-pharma-consult.scherkl.com design by scherkl.com copyright 2012 Dr. Julia Eva Diederichs