Discovery and ADME profiling of CNS-active natural products

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List of Abbreviations

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Summary

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Zusammenfassung

Xenopus

Valeriana officinalis Magnolia officinalis

Xenopus

Searsia pyroides

Xenopus

(14)

Xenopus

(15)

1. Aim of the Work

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Xenopus

(18)

Xenopus via

in silico in vivo

via

(19)

References:

Apocynum venetum

(20)

(21)

2. Introduction

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2.1. Natural products in CNS-active drug discovery

Challenges and opportunities

(24)

in silico

(25)

Discovery of novel leads

(26)

Neuroactive leads

(27)

Galanthus nivalis Galanthus woronowii Narcissus Leucojum aestivum

Physostigma venenosum

Huperzia serrata

Fig. 2.1-1:

(28)

Cortinarius infractus

Claviceps purpurea

References:

(29)

in vivo

(30)

Cortinarius infractus

(31)

2.2. The GABA

A

receptors Structure and pharmacology

via

(32)

In vitro GABA

A

receptor modulation models

Figure 2.1-1:

A B

(33)

Xenopus laevis

Xenopus

(34)

In vivo GABAergic activity models

Caenorhabditis elegans Drosophila melanogaster Danio rerio

Danio rerio

(35)

Promising NP-based GABA

A

receptor modulators

(36)

References:

(37)

Xenopus

(38)

Xenopus

Morus alba

Haloxylon scoparium

Boswellia thurifera

Pholidota chinensis

Curcuma kwangsiensis

Kadsura longipedunculata

Acorus calamus

(39)

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

Figure 2.3-1:

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I) Cleavage period.

II) Blastula period.

III) Gastrula period.

IV) Segmentation period.

V) Pharingula period.

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VI) Hatching period.

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2.3.2. Advantages of zebrafish as a model organism

Rapid life cycle and low-cost maintenance

Transparent embryos and larvae

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In vivo

Genetic homology to humans

Multi-organ system similar to humans

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Listeria monocytogenes Streptococcus Mycobacterium marinum Edwardsiella tarda

in vivo

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2.3.3. Zebrafish in drug discovery and development

in vitro

in vivo

Hit discovery and lead development

in vitro

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in vitro

in vivo

Target identification

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in vivo

ADME analysis

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Toxicology studies

(51)

2.3.4. Rationale for neuroactive drug discovery with zebrafish

in vitro

Caenorhabditis elegans Drosophila melanogaster Locusta

migratoria

Complex brain

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Functional blood-brain barrier

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ADME principles

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2.3.5. GABA

A

signaling system in zebrafish

via

(55)

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2.3.6. Behavior-based assays with zebrafish

Photomotor response (PMR)

Figure 2.3-2:

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Locomotor activity

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Escape and avoidance behavior

Figure 2.3-3:

Figure 2.3-4:

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Habituation

Figure 2.3-5:

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References:

in vivo

Brachydanio

rerio

(61)

(62)

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

K

2.4.2. Druggability assessment models

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C P

in silico in vitro

in vivo In silico in vitro

in vivo

in silico in vitro

2.4.2.1. In silico prediction models In silico

C P

(67)

BB K

hsa

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

Figure 2.4-1.

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Cell-based intestinal barrier model

in situ in vitro

in vitro

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S

P

in vitro

Figure 2.4-2:

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i

ii

iii

Cell-based blood-brain barrier model

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in vivo

Figure 2.4-3:

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References:

In silico

in vitro

in-silico

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in silico

In vitro

(74)

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

Sample preparation

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LC-MS/MS instrument and detection optimization

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2.5.2. Bioanalytical method validation

Figure 2.5-1:

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Selectivity and specificity

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Calibration curve

Repeatability and reproducibility

Stability

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Accuracy

References:

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in vitro

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3. Result and Discussion

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3.1. HPLC-based activity profiling for GABA

A

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 GABA

_A

-receptor modulators via

HPLC-based activity profiling of extracts

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n

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Figure S3.

Magnolia officinalis

Figure S4.

a f Magnolia officinalis

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Figure S5.

Valeriana officinalis

Figure S6.

Valeriana officinalis

b d

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3.2. HPLC-based activity profiling for GABA

A

receptor 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

phytochemicals, writing the manuscript draft, and preparation of figures and tables.

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Downloaded by: Universität Basel. Copyrighted material.

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Supplementary data Planta Medica

HPLC-based activity profiling for GABA

_A

receptor modulators from Searsia pyroides leaves using a

validated larval zebrafish locomotor assay

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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

(113)

Table S2. d 1 6

No 1â 2^b 3â 4â 5â 6â

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

Figure S2. 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

Figure S4

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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:

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:

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

Mean

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Table S8:

Run

No. Replicate

2 3 4

Mean

Table S9:

Run

No. Replicate

2 3

Mean

Table S10:

Run

No. Replicate

2 3

Mean

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Table S11:

KMF 4-HPAA

RHB HBSS RHB HBSS Mean concentration (ng/ml)

S.D.

CV%

RE%

Table S12:

Analyte QCL QCM QCH IS

Nominal concentration (ng/mL) 60.0 1000 1600 206.9 Absolute recovery (%)

CV%

SD

Table S13:

CV%

SD

Table S14:

CV % SD

Table S15:

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 Donor

HB Accpt

HB LogPo/

w LogBB

Human Oral Absorption

(%)

PSA

[Å²] LogPo/

w PSA[Å²]

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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4. Conclusion and Outlook

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vs

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Sophora flavescens Morus alba

Boswellia thurifera Biota orientalis

in silico

Magnolia officinalis Valeriana officinalis

Xenopus

Xenopus in vitro

S. pyroides

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Xenopus S. pyroides

Xenopus

S. pyroides

in vitro in vivo

in vitro in vitro

in vivo

(144)

in vitro

in vitro in vivo

via in vitro

in vivo

in vitro

(145)

References:

Sophora flavescens

Morus alba

Boswellia thurifera

Biota orientalis

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Discovery and ADME profiling of CNS-active natural products

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 GABA

receptor

Structure and pharmacology

In vitro GABA

receptor modulation models In vivo GABAergic activity models

Promising NP-based GABA

receptor 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

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 GABA

receptor modulators in extracts validation of an approach utilizing a larval zebrafish locomotor assay

3.2. HPLC-based activity profiling for GABA

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

4. CONCLUSION AND OUTLOOK

List of Abbreviations

Summary

Zusammenfassung

Xenopus

Xenopus

Valeriana officinalis Magnolia officinalis

Xenopus

Searsia pyroides

Xenopus

Xenopus

1. Aim of the Work

Xenopus

Xenopus

Xenopus via

in silico in vivo

via

References:

Apocynum venetum

2. Introduction

2.1. Natural products in CNS-active drug discovery

Challenges and opportunities

in silico

Discovery of novel leads

Neuroactive leads

Galanthus nivalis Galanthus woronowii Narcissus Leucojum aestivum

Physostigma venenosum

Huperzia serrata

Cortinarius infractus

Claviceps purpurea

References:

in vivo

Cortinarius infractus

2.2. The GABA

receptors Structure and pharmacology

via