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Ge Lin Department of Pharmacology

An integrated approach for the globalization of Chinese medicines with known active ingredients and verified oral absorbability. Ge Lin Department of Pharmacology. Toxicology. Final formulation. IND application. Toxicology. Toxicology. Our novel integrated approach. CM herb.

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Ge Lin Department of Pharmacology

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  1. An integrated approach for the globalization of Chinese medicines with known active ingredients and verified oral absorbability Ge Lin Department of Pharmacology

  2. Toxicology Final formulation IND application Toxicology Toxicology Our novel integrated approach CM herb Herbal Formulations with Known Absorbable and Active Ingredients 1. Extraction Fractions 2. absorption Absorbable Ingredients 6. Formulation Optimization 3. Analysis 4. Pharmacokinetics 5. Pharmacology Absorbable Active Ingredients

  3. Our goal Development of CM herbal products • Known active ingredients • Proved pharmacological effects • Verified bioavailability Example: Chuanxiong

  4. Chemical base -- misleading TMP FA C O O H H C N C H 3 3 N C H H C 3 3 H O OCH3 Background • Chuanxiong (Ligusticum chuanxiong Hort.), commonly used for the protection and treatment of cardiovascular disorders • Lack of sound studies to provide scientific evidences supporting its therapeutic value tetramethylpyrazine川芎嗪 0.1 g/g dried herb (1977, CTHD) Ferulic acid 300 g/g dried herb Also fund in >100 herbs with diversified activities

  5. Herbal source GAP-base in Sichuan. The plant specie was authenticated according to China pharmacopoeia and also confirmed by DNA sequential analysis Rhizoma Chuanxiong

  6. 1750 1500 B2 B12 B13 1250 B14 B15 1000 B7 B5 Average weight of constituents (mg/one rhizome) B16 750 B18 500 250 0 Oct 8, 2002 Jan 2, 2003 Dec 2, 2002 Oct 30, 2002 Feb 11, 2003 May 20, 2003 April 11, 2003 March 9, 2003 Collection date Time-course accumulation of the major ingredients

  7. 1. Herbal extraction • Decoction • 95% EtOH extract • Crude extract by Supercritical Fluid Extraction (SFE) using CO2

  8. Decoction FA B12 B7 B14 B13 Decoction EtOH B7 TMP B15 B14 B18 IS B19 B17 FA B12 B16 B13 SFE B7 B14 B18 B15 B17 B19 IS FA B12 B16 B13 Comparison of 3 extractions HPLC-UV-MS analysis

  9. B7 B14 B18 B17 B15 B12 B19 FA B16 mAU mAU B5 B13 100 100 80 80 Chromatogram of extract before absorption 60 60 40 40 B7 20 20 B14 0 0 10 10 20 20 30 30 40 40 min min B12 FA? B5 B13 Chromatogram of extract after absorption 2. Intestinal absorption using Caco-2 cell Lumen (Donor compartment) Intestinal cells Blood (Receiver compartment)

  10. Permeability in Caco-2 model Compound Permeability Predicted (10-6 cm/s) Absorption B5 29.3  6.2 Medium-High* (80%-90%) B7 33.3  0.9 High (>98%) B12 48.5  8.3 High (>98%) B13 46.5  8.0 High (>98%) B14 53.4  1.4 High (>98%) *13% of B5 was biotransformed to metabolite (M1)

  11. lumen 150 accumulated in plasma cell 100 in mesenteric blood (nmole/cm2) Plumen Cumulative amount of B5 and M1 Pblood 50 blood 0 Perfusate in Perfusate out 0 10 20 30 40 50 60 70 Blood collection Time (min) B5 (10-2 M) D D D D D D M1 M1 M1 M1 ER: 37% In situ single pass rat intestinal perfusion (SPIP) Extraction ratio (ER) – ability to convert D to M when D passes through the organ Caco-2 may overestimate absorbability if a drug undergoes extensive metabolism in the gut

  12. 3. Analysis of main ingredients • 17 components have been identified in Chuanxiong (TMP was not found) • 10 main ingredients in the herb were quantified • 5 were found to be orally absorbable

  13. 4. Pharmacokinetics Oral bioavailability • Oral absorption of the parent drug • First-pass effect in gut and liver • In vivo kinetic model determines PK fates and bioavailability • In vitro metabolicmodels,usingsub-cellular fractions (S9, cytosol, microsome), investigate intestinal and hepatic metabolism

  14. B7 B14 IS B15 B12 B18 B5 B13 B16 B2 IS B7 M-4 B14 M-6 M-1 Endogenous M-5 B5 M-3 Endogenous Pharmacokinetics in rats Extract Before dosing B5, B7, B14, M-1, M-2, M-3 and M-4 were considered as the main active components contributing to the in vivo activities of Chuanxiong In plasma after dosing

  15. Pharmacokinetics Oral administration of individual B in rats PK parameterB5B7 B14 Dose (mg/kg) 100 500 100 Tmax (h) 0.460.09 0.360.19 0.210.08 t1/2 (h) 0.780.23 3.431.01 0.520.03 Vd (L/kg) 27.514.81 42.693.16 5.450.37 Cl (L/h.kg) 28.404.20 10.683.79 7.500.95 MRT (h) 1.490.22 5.141.56 0.780.07 F (%) 22.7 2.6 7.9 First-pass effect Intestine B5 (35.6%) B7 (50.1%) B14 (67.9%) Liver B5 (41.7%) B7 (47.3%) B14 (24.2%)

  16. Pharmacokinetics Kinetic interaction in rats via administration of SFE extract PK parameterB5B7 B14 t1/2 (h) 0.820.17 0.220.07 0.690.31 Vd (L/kg) 5.760.67** 5.621.19 7.122.16 Cl (L/h.kg) 5.661.22**20.353.05** 9.171.81 MRT (h) 0.870.22 0.190.03 0.570.14 Relative F (%) 448.0190.0**45.611.0** 88.429.0

  17. In vitro intestinal metabolism Instability in GI tract Compound Vmax/km (µl/min/kg) ER (%) %Degradation (30 min) stomach intestine B5 11.55  0.42 37 0 0 B7 0 0 12 2 B14 0 0 37 37 In vitro hepatic metabolism Compound Vmax/km (µl/min/kg) ER (%) B5 47.72  2.40 48 B7 37.10  2.85 47 B14 9.96  0.38 23 Hepatic microsomal metabolism: B7 B5 Oxidation [P450] First-pass effect in intestine and liver

  18. Effects of pure constituents against U46619-induced contraction in the pig coronary artery 5. Pharmacological studies i. Vasodilatory effects in tissue preparations

  19. ii. Anti-platelet aggregation Effect (%) on the platelet aggregation Sample Collagen U-46619 ADP B1 (300 M) 100±3 91±6 97±2 B2 (300 M) 100±5 70±21 102±7 B5 (300 M) 26±16*** 56±20 93±8 B7 (145 M) -3±2*** -9±2*** 39±13*** B10 (300 M) 93±4 95±4 105±7 B14 (300 M) 48±16* 65±21 97±6 Ext (300 g/mL) 23±16*** 42±15* 89±4 Cica (10 nM) 5±1*** 3±2*** 3±3*** Cica: Cicaprost as a positive control

  20. iii. Anti-thrombotic effect Sample Dose Pretreated time Mortality Saline 10 mL/kg 30 min 100% Extract 3 g/kg, po 30 min 63% 4 hr 38%* 1 g/kg, po 30 min 60% 4 hr 70% B5 100 mg/kg, po 30 min 100% 4 hr 50%* B7 100 mg/kg, po 30 min 80% 4 hr 70% B14 100 mg/kg, po 30 min 33%** 4 hr 40%** Mice pulmonary embolus model induced by collagen (1.14 mg/kg) and epinephrine (0.13 mg/kg) via a tail vein injection

  21. 6. Pre-formulation oral vs not oral? • Solid droplets extract--cyclodextrin inclusion complex • Soft gel capsules Self-emulsifying drug delivery system • Sublingual drug delivery --fast delivery • Buccal drug delivery --control release

  22. Summary Using our integrated approach • Have developed herbal formulations with: • identified active chemical ingredients • verified pharmacokinetic profiles of the active chemical ingredients • proved pharmacological activities 4 formulations for oral/non-oral routes Mainly 3 in vivo active compounds Low oral bioavailability / short duration Vasodilatation, antiplatelet aggregation and antithrombotic effects

  23. Acknowledgements ITF fund from ITC HKSAR Government

  24. Thanks

  25. What are the problems? • In general, CM-based herbal products have not been reaching the international standards in terms of: • known active components • defined quantities of active ingredients • proved pharmacological effects • verified bioavailability & PK fates • evidence-based therapeutic outcomes

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