Discovery and ADME profiling of CNS-active natural products

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Table of Contents

LIST OF ABREVIATIONS SUMMARY

ZUSAMMENFASSUNG 1. AIM OF THE WORK 2. INTRODUCTION

2.1. Natural products in CNS-active drug discovery Challenges and opportunities

Discovery of novel leads Neuroactive leads 2.2. The GABAAreceptor

Structure and pharmacology

In vitro GABAAreceptor modulation models In vivo GABAergic activity models

Promising NP-based GABAAreceptor modulators

2.3. Zebrafish as a model organism in drug discovery and development 2.3.1.

2.3.2.

Rapid life cycle and low-cost maintenance Transparent embryos and larvae

Genetic homology to humans

Multi-organ system similar to humans 2.3.3.

Hit discovery and lead development Target identification

ADME analysis Toxicology studies 2.3.4.

Complex brain

Functional blood-brain barrier ADME principles

2.3.5. 2.3.6.

Photomotor response (PMR) Locomotor activity

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Escape and avoidance behavior Habituation

2.4. ADME profiling in neuroactive drug discovery 2.4.1.

2.4.2.

2.4.2.1. In silico 2.4.2.2. In vitro

Cell-based intestinal barrier model Cell-based blood-brain barrier model 2.5. Bioanalysis

2.5.1.

Sample preparation

LC-MS/MS instrument and detection optimization 2.5.2.

Selectivity and Specificity Calibration model

Repeatability (precision) and Reproducibility Stability

Accuracy

3. RESULTS AND DISCUSSION

3.1. HPLC-based activity profiling for GABAA receptor modulators in extracts validation of an approach utilizing a larval zebrafish locomotor assay

3.2. HPLC-based activity profiling for GABAA receptor modulators in Searsia pyroides using a larval zebrafish locomotor assay

3.3. Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro blood-brain barrier and intestinal dru

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Zusammenfassung

Xenopus

Xenopus

Valeriana officinalis Magnolia officinalis

Xenopus

Searsia pyroides

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Xenopus

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Xenopus via

in silico in vivo

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References:

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2.1. Natural products in CNS-active drug discovery Challenges and opportunities

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Galanthus nivalis Galanthus woronowii Narcissus Leucojum aestivum

Physostigma venenosum

Huperzia serrata

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Cortinarius infractus

Claviceps purpurea

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2.2. The GABAAreceptors Structure and pharmacology

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In vitro GABAAreceptor modulation models

Figure 2.1-1:

A B

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Xenopus laevis Xenopus

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In vivo GABAergic activity models

Caenorhabditis elegans Drosophila melanogaster Danio rerio

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Xenopus Xenopus

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Xenopus Morus alba Haloxylon scoparium Boswellia thurifera Pholidota chinensis Curcuma kwangsiensis Kadsura longipedunculata Acorus calamus

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Angelica pubescens Xenopus Biota orientalis Sophora flavescens in vitro in vivo In vitro

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2.3. Zebrafish as a model organism in drug discovery and development Danio rerio

2.3.1. Zebrafish development and life cycle

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I) Cleavage period.

II) Blastula period.

III) Gastrula period.

IV) Segmentation period.

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2.3.2. Advantages of zebrafish as a model organism

Rapid life cycle and low-cost maintenance

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In vivo

Genetic homology to humans

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Listeria monocytogenes Streptococcus Mycobacterium marinum Edwardsiella tarda

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2.3.3. Zebrafish in drug discovery and development

in vitro

in vivo

Hit discovery and lead development

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in vitro

in vivo

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in vivo

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2.3.4. Rationale for neuroactive drug discovery with zebrafish

in vitro

Caenorhabditis elegans Drosophila melanogaster Locusta

migratoria

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2.3.5. GABAAsignaling system in zebrafish

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2.3.6. Behavior-based assays with zebrafish

Photomotor response (PMR)

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Escape and avoidance behavior

Figure 2.3-3:

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Habituation

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References:

in vivo

Brachydanio rerio

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In vivo

In vivo

Astragali Radix

Salvia Miltiorrhiza

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Danio rerio

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2.4. ADME profiling in neuroactive drug discovery 2.4.1. Drug-like properties

P K

P K

K

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C P in silico in vitro in vivo In silico in vitro in vivo in silico in vitro in silico in vitro

2.4.2.1. In silico prediction models In silico

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BB Khsa

S P

BB

in silico in vitro in vivo

in silico

in vitro in vivo

2.4.2.2. In vitro membrane permeability studies

In vitro

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Cell-based intestinal barrier model

in situ in vitro

in vitro

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S

P

in vitro

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i

ii

iii

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in vivo

in vivo Figure 2.4-3:

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References:

In silico

in vitro

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in silico

In vitro

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In vitro in vitro In vitro in vivo in vitro in vivo in vitro

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2.5. Bioanalysis

2.5.1. Bioanalytical Method development

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2.5.2. Bioanalytical method validation Figure 2.5-1:

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Calibration curve

Repeatability and reproducibility

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Accuracy

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3.1. HPLC-based activity profiling for GABAA receptor modulators in extracts validation of an approach utilizing a larval zebrafish locomotor assay

Journal of Natural Products, DOI:

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-Supporting Information Journal of Natural Products

Validation of a larval zebrafish locomotor assay for discovery of GABAA-receptor modulators via

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Figure S3.

Magnolia officinalis

Figure S4.

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Figure S5.

Valeriana officinalis

Figure S6.

Valeriana officinalis

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3.2. HPLC-based activity profiling for GABAAreceptor modulators in Searsia pyroides using a larval zebrafish locomotor assay

Planta Medica DOI:

Searsia pyroides S. pyroides Xenopus 1 3 I Xenopus

My contributions to this publication: Activity assessments with the zebrafish larvae locomotor assay, HPLC-based activity profiling of the active extract, purification and identification of the

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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D ow n lo a de d by : U n iv er si tä t B as e l. C op yr ig ht ed m at er ia l.

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Supplementary data Planta Medica

HPLC-based activity profiling for GABAAreceptor modulators from Searsia pyroides leaves using a

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Table S1. d 1 - 6 No 1 2 3 4 5 6 3 d d d d d d 4 dd dd dd dd dd dd 5 d d d d d d 6 7 m m m m m m 8 m m m m m m 9 m m m m m m 10 11 12 13 14 m 15 m m 16 m m m m 17 m m m m 18 m m m m m 19 m m m m m 20 m m m m m 21 t t t m m m 22 m m m 23 t t t OH

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Table S2. d 1 6 No 1a 2b 3a 4a 5a 6a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 COOH Table S3. 1 6

compound acc. mass found [M-H]

-acc. mass calculated [M-H] -calcd formula 1 2 3 4 5 6

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Figure S1. E Z 1 d

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3.3. Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to blood-brain barrier and intestinal drug permeability studies

Journal of Pharmaceutical and Biomedical Analysis DOI:

in silico

My contributions to this publication: development and validation of the UPLC-MS/MS method in the RHB and , performing the intestinal transport studies with Caco-2 cells and the BBB transport experiments with the mono-culture HBMEC model, sample preparation and analysis, writing the manuscript draft, and preparation of figures and tables.

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Supporting Information

Journal of Pharmaceutical and Biomedical Analysis

Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro blood-brain barrier and intestinal drug permeability studies

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Figure S1

Figure S2

Figure S3

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Table S1:

Run No. Nominal concentration (ng/mL) Regression parameters 20.0 50.0 100 250 500 1000 2000 A B C R2 1 2 3 4 Mean S.D. CV% RE% Table S2:

Run No. Nominal concentration (ng/mL) Regression parameters 20.0 50.0 100 250 500 1000 2000 A B C R2 1 2 3 Mean S.D. CV% RE%

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Table S3:

Run No. Nominal concentration (ng/mL) Regression parameters 20.0 50.0 125 250 500 1000 2000 A B C R2 1 2 3 Mean S.D. CV% RE% Table S4:

Run No. Nominal Concentration (ng/mL) Regression parameters 20.0 50.0 100 250 500 1000 2000 A B C R2 1 2 3 Mean S.D. CV% RE%

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Table S5: Nominal concentration (ng/ml) 20.0 60.0 1000 1600 2000 Within-run Mean S.D. CV% RE% Between-run Mean S.D. CV% RE% Table S6: Nominal concentration (ng/ml) 20.0 60.0 1000 1600 2000 Within-run Mean S.D. CV% RE% Between-run Mean S.D. CV% RE% Table S7: Run No. Replicate

Peak response (counts)

Carry-over (%) Mean Carry-over (%) Blank sample LLOQ

Analyte IS Analyte IS Analyte IS Analyte IS 1

2

3

4

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Table S8:

Run

No. Replicate

Peak response (counts)

Carry-over (%) Mean Carry-over (%) Blank sample LLOQ

Analyte IS Analyte IS Analyte IS Analyte IS 1 2 3 4 Mean Table S9: Run No. Replicate

Peak response (counts)

Carry-over (%) Mean Carry-over (%) Blank sample LLOQ

Analyte IS Analyte IS Analyte IS Analyte IS 1 2 3 Mean Table S10: Run No. Replicate

Peak response (counts)

Carry-over (%) Mean Carry-over (%) Blank sample LLOQ

Analyte IS Analyte IS Analyte IS Analyte IS 1

2

3

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Table S11: KMF 4-HPAA RHB HBSS RHB HBSS Mean concentration (ng/ml) S.D. CV% RE% Table S12: Analyte IS QCL QCM QCH Nominal concentration (ng/mL) 60.0 1000 1600 206.9 Absolute recovery (%) CV% SD Table S13: Analyte IS QCL QCM QCH Nominal concentration (ng/mL) 60.0 1000 1600 206.9 Absolute recovery (%) CV% SD Table S14: Analyte IS QCL QCM QCH Nominal concentration (ng/mL) 60.0 1000 1600 827.6 Absolute recovery (%) CV % SD Table S15: Analyte IS QCL QCM QCH Nominal concentration (ng/mL) 60.0 1000 1600 1655 Absolute recovery (%) CV% SD

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Table S16: RHB HBSS Dilution factor 10X 100X 10X 100X Mean S.D. CV% RE% Table S17: RHB HBSS Dilution factor 10X 100X 10X 100X Mean S.D. CV% RE% Table S18: RHB HBSS Nominal concentration (ng/mL) 60.0 1600 60.0 1600 Table S19: RHB HBSS Nominal concentration (ng/mL) 60.0 1600 60.0 1600

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Table S20:

KMF SS stored below -65°C for 180 days with freshly prepared IS SS

KMF SS freshly prepared with freshly prepared IS SS Mean peak area ratio S.D. CV% Difference% Table S21:

4-HPAA SS stored below -65°C for 35 days

with freshly prepared IS SS 4-HPAA SS freshly prepared with freshly prepared IS SS Mean peak area ratio S.D. CV% Difference% Table S22:

VA SS stored below -65°C for 190 days

with freshly prepared 4-HPAA SS VA SS freshly prepared with freshly prepared 4-HPAA SS Mean peak

area ratio S.D. CV% Difference%

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Table S23: in silico

QikProp descriptors (3D) Chemaxon Marvin (2D)

Compound MW DonorHB AccptHB LogPo/

w LogBB Human Oral Absorption (%) PSA [Å2] LogPo/ w PSA[Å 2] Table S23: in silico

QikProp descriptors (3D) Chemaxon Marvin (2D) Exp. Com-poun d M W Dono r HB Accp t HB LogPo/ w LogB B Human Oral Absorptio n (%) PSA [Å2] LogPo/ w LogD7. 4 PS A [Å2] pKa

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Sophora flavescens Morus alba

Boswellia thurifera Biota orientalis

in silico

Magnolia officinalis Valeriana officinalis

Xenopus

Xenopus in vitro

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Xenopus S. pyroides Xenopus S. pyroides in vitro in vivo in vitro in vitro in vivo

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in vitro in vitro in vivo via in vitro in vivo in vitro in vitro

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References:

Sophora flavescens

Morus alba

Boswellia thurifera

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Figure

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