Drug Photostability: The Science and

Application of Photo-Produced

Reactive Oxygen Species

Steven W. Baertschi, Ph.D.

Baertschi Consulting, LLC

Email: Steve@BaertschiConsulting.com

Website: http://baertschiconsulting.com

October 14-15

Science of Stability Conference

Amsterdam, Netherlands

Introduction

Photostability of Drugs: Background

Photochemistry Basics

– Terminology, Definitions, Light sources and measurements, Jablonski Diagram, etc.

Photosafety Context

Direct and Indirect Photochemistry

Methods for Measurement of ROS

Conclusions / Summary

Drug stability and the environment

http://www.stovesonline.co.uk/wood_burning_stoves/Morso-Squirrel-Stove.html http://www.graphisoft.com/

http://www.hazemsakeek.com/QandA/Phloresent.htm

Direct

Sunlight

Window-filtered

Sunlight

Light and the hospital environment

http://www.onmeda.de/arztbesuch/behandlung/chemotherapie.html

http://www.bannerhealth.com/resources/infusion-225px.jpg

http://www.apollolighting.co.uk/articles/partnership.php

Through a windowLight/radiation below 310

nm cannot penetrate a

window pane

Indoor lighting Direct

Sunlight

Photostability Issues in Marketed Products

Overall impact of photostability is evident from an examination of the USP 27 (2004) Reference Table “Containers for Dispensing Capsules and Tablets”– 743 pharmaceutical products listed

– 248 (33%) require light resistant packaging

Developing an improved understanding is desirable– To effectively control the quality of the product

– Address the specific requirements of each product

Presented by Robert A. Reed, AAPS Workshop on Stress Testing and

Degradation Chemistry, Nov 10, 2007, San Diego, CA

The Spectrum of Electromagnetic Radiation

Lehninger, Principles of

Biochemistry, 3rd ed., p. 693.

UV-Vis Wavelength Ranges

UV-C: <290 nm (<280 nm, CIE)

UV-B: 290-320 nm (280- 315 nm, CIE)

UV-A: 320-400 nm (315-400, CIE)

Visible: 400-800 nm

First Law of Photochemistry:Grotthuss (1817) – Draper (1843)

“Only radiation that is absorbed by a system

can be effective in producing a photochemical

reaction.”

The “system” is not just the API.

For a formulation, one must consider all

components in the formulation

Photochemistry Basics

The interaction of matter

with electromagnetic radiation

So(singlet ground

state – almost all

organic molecules)

hn(absorption)

S1(First excited

singlet state)T1(First excited

triplet state)

Energy

hn’(fluorescence)

hn’(phosphorescence)

Vibrational

relaxationMultiplicity = 2s+1, where s=(±½)

ISC

Light Absorption by a Molecule –What can a molecule do with the energy?

The Jablonski Diagram

The molecule will typically get rid of the excess energy

based on a combination of kinetic/thermodynamic factors

slide provided courtesy of Mark Kleinmann, GSK, USA

heat

(internal conversion)

heat

(internal

conversion)

(Inter-System

Crossing)

10

Measuring Light: Radiometric vs

Photometric Units

Courtesy of Allen Zielnik, Atlas Material Testing Technology, LLC.

Mixing Terminology

ICH Terminology for light measurements

For confirmatory studies samples should be exposed to [Visible] light providing an overall illumination of not less than 1.2 million Lux-hours

and

an integrated near-ultraviolet energy of 200 watt-

hours/square-meter to allow direct comparisons to be

made between the drug substance (API) and the drug

product (FPP).

A confusing issue with the Guideline is that it mixes radiometric

and photometric light measurements . . .

Rational for Confirmatory Photostability

Exposure Recommended by ICH

•3 months of continuous exposure to visible light without protective packaging in the pharmacy, warehouse, or home. (~400 – 800 nm, 500 lux x 24 h x 100 days = 1.2 x 106 lux•hrs, visible radiation).

•Over the same 3 months, the product could be exposed to solar radiation behind windowed glass, which was [somewhat arbitrarily] defined as 200 watt•hrs/m2 of integrated near UV radiation (~300 -400 nm). This is roughly equivalent to 1-2 days of “windowsill” exposure.

Thatcher, S.R.,* Mansfield, R.K., Miller, R.B., Davis, C.W., and Baertschi, S.W.* “Pharmaceutical Photostability: A Technical and Practical Interpretation of the ICH Guideline and Its Application to

Pharmaceutical Stability: Part I”, Pharmaceutical Technology, 25:3, pp. 98-110, March (2001).

ICH Q1B Confirmatory

Exposure in Perspective

UV (295-400 nm)

• The average daily UV irradiance is:

– 358 W-h/m2 in Miami, FL, USA

– 154 W-h/m2 in Oslo, Norway

• VISIBLE (400-800 nm)

14

--from Baertschi, SW et al., J.

Pharm. Sci. (2013) ); 102:11,

3888-3899.

--from Baertschi, SW et al., J.

Pharm. Sci. (2013) ); 102:11,

3888-3899.

For Reference…

Sun intensity in Miami on a clear summer day…

• from the Eppley total UV radiometer in Miami,* (July 24, 1996)

– the “total” UV dose for the day

– 473 W-h/m2 (295-400 nm)

– the “total” UV dose between 10 am and 2 pm...

– 245 W-h/m2 (295-400 nm)

*Courtesy of Norma Searle, Ph.D., consultant, and

Atlas Electric Devices, Chicago, IL

Measuring Light: Photometry

The photometric system (e.g., lux) defines light in terms of how it is perceived by the human eye

Slide courtesy of Allen Zielnik, Atlas Material Testing Technology, LLC.

(Night Vision)

(Day Vision)

Lux-hours

Lux X time (hours) = Exposure in Lux-Hours

1 lux = 0.0929 ft-candles

1 ft-candle = 10.76 lux

1 ft-candle = 1 lumen/ft2

meter candle = 1 lux = 1 lumen/m2

The

Human

Eye

Direct: Drug molecules absorb UV or visible radiation resulting in

transformation into a photoproduct

Indirect (sensitized): The primary excited species mediates the

formation of secondary reactive species which subsequently lead to

the formation of the photoproduct .

Direct and Indirect Photoreactivity

Drughn

Drug*

Photoproduct(s)

Direct: Drug molecules absorb UV or visible radiation resulting in transformation into a photoproduct

Indirect (sensitized): The primary excited species mediates the formation of secondary reactive species which subsequently lead to

the formation of the photoproduct .

– Type I(simplified view)

– Type II

Direct and Indirect Photoreactivity

Drug

hn

Drug* 3O2

Drug Reactive oxygen species+

HOO , O2 , HO(or othercomponent)

(or othercomponent)

Drug

Photoproduct(s)

Groundstate

Excitedstate

Radical orrelated species

(or othercomponent)

Drug

hn

Drug* 3O2

(or othercomponent)

(or othercomponent)

Drug +

(or othercomponent)

1O2

singlet oxygen

Drug

Photoproduct(s)

Potential

Photosafety

Concerns

Summary of Potential

Photochemical Processes

Direct

Indirect

Indirect

Potential

Photosafety

Concerns

Photosafety

➢ ICH S10 Photosafety Evaluation of

Pharmaceuticals

➢Applies to new APIs, new excipients, new

dermal application formulations,

photodynamic therapy products

➢Depends on:

➢Photochemical properties (UV-VIS absorption,

photostability, and ROS production)

➢Phototoxic potential of related compounds

➢Tissue Distribution

➢Clinical / non-clinical findings indicative of

phototoxicology

Prediction of Potential Phototoxicity

using Chemistry

Interrogate:

– UV-VIS absorption in 290-800 nm region

(MEC >1000 M-1cm-1 )

– Photoexposure of API in solution (to simulated

sunlight) for a total exposure in the UV region

of 5 J/cm2 = ~14 W-h/m2 (290-400 nm)

➢Measure total photodegradation

➢Measure formation of superoxide radical anion

(Type I)

➢Measure formation of singlet oxygen (Type II)

Experimental Filters for In-Vitro

Phototoxicity Predictions

Chemistry of Reactive Oxygen

Species: Hydroperoxyl Radical

HOO • + R-H HOOH + R •

H-atom transfer reaction

Exothermic by 5 to 20 kcal/molReaction occurs at the rate of diffusion

+ O2 OO

tBu radical tBu peroxyl radical

HOO-H R-H

Bond Dissociation Enthalpies (BDEs) as a Guide to Free Radical Reactivity

BDE ~88 kcal/mol BDE ~variable with structure, 70-105 kcal/mol

If R-H BDE is less than 88 kcal/mol, H-atom transferreaction will likely occur.

Chemistry of Reactive Oxygen

Species: Superoxide

The Chemistry of Superoxide radical ion

1. dismutes rapidly in water

2 O2 + 2H+H2O2 + O2

O2 + H+ HOO •, a reactive species2. forms a neutral free radical

3. In organic solvents (DMSO) is stable and a modest nucleophile

O2 + R-X ROO • + X

p

pp

p* p*p

pp

p* p*

p

pp

p* p*

Ground State Oxygen, 3O2

Singlet Oxygen 1st Excited State, 1O2

22.5 kcal/mole higherenergy than ground state

1-g

1g

3+g

37.8 kcal/mole higherenergy than ground state

Singlet Oxygen 2nd Excited State, 1O2

Chemistry of Reactive Oxygen

Species: Singlet Oxygen

1O2

Measuring Singlet Oxygen and

Reactive Oxygen Species

Article describes relatively simple methodology

involving

(1) bleaching of a singlet oxygen acceptor (imidazole) monitoring

440 nm absorbance

(2) the reduction of nitroblue tetrazolium (increase in absorbance

at 560 nm) by superoxide

Measurement of Singlet Oxygen Formation Using

Method of Kraljic and Mohsni

Measurement of Superoxide Radical Anion by

Reduction of Nitroblue Tetrazolium

Reduction of NBT via a

one-electron reduction

(from superoxide radical

anion) forms

monoformazan, which

can be quantitatively

determined by the

absorbance at 560 nm.

Correlation of ROS Production with

Known Phototoxicity

Plot of photo-induced

singlet oxygen and

superoxide generation

for 39 compounds.

Open circles represent

non-phototoxic

compounds

Photochemistry and Photostability:

Useful References

A. Albini and E. Fasini, Editors, “Drugs: Photochemistry and Photostability”, (1998), The Royal Society of Chemistry.

Gilbert and J. Baggott, “Essentials of Molecular Photochemistry - Photostabilityof Drugs and Formulations” (1991) CRC Press.

H.H. Tønneson, Editor, “Photostability of Drugs and Drug Formulations”, 1st

Edition (1996), Taylor & Francis, 2nd Edition (2004) CRC Press

Baertschi / Alsante / Reed, Eds. “Pharmaceutical Stress Testing” (2011) CRC Press.

J.T. Piechocki and K. Thoma, Editors, “Pharmaceutical Photostability and Stabilization Technology”, (2007) Informa Healthcare.

N. J. Turro, V. Ramamurthy, J. C. Scaiano, “Modern Molecular Photochemistry of Organic Molecules, University Science Books, (2010).

Thatcher, S.R., Mansfield, R.K., Miller, R.B., Davis, C.W., and Baertschi, S.W. “Pharmaceutical Photostability: A Technical and Practical Interpretation of the ICH Guideline and Its Application to Pharmaceutical Stability: Parts I and II”, Pharmaceutical Technology, 25:3&4, March-April (2001)

CRC Handbook of Organic Photochemistry and Photobiology, 2nd

Ed, Horspool, Lenci, Editors, CRC Press (2004)

Templeton AC, Xu H, Placek J, Reed RA, “Implications of

Photostability on the Manufacturing, Packaging, Storage, and

Testing of Formulated Pharmaceutical Products”, Pharm Technol,

29:3 (2005) 68-83.

Photobiological Sciences Online

– Excellent online resource for many topics related to photostability,

photochemistry, photobiology, etc.

– http://www.photobiology.info/

B.A. Kerwin, R.L.J. Remmele, “Protect from light:

Photodegradation and protein biologics”, J Pharm Sci 96 (2007)

1468–1479, http://dx.doi.org/10.1002/jps.20815.

32

Photochemistry and Photostability:

Useful References

Summary / Conclusions

➢ Many drugs are associated with photostability issues

➢ A subset of these are associated with photosafety concerns

➢ Photosafety issues are strongly correlated with Type I

(superoxide production) and Type II (singlet oxygen

production) photodegradation processes

➢ Singlet oxygen and superoxide radical anion production

can be readily measured using established procedures

➢ Simulated sunlight exposure, 14 W-h/m2 (290-400 nm) (7% of the

ICH Q1B 200 W-h/m2 confirmatory exposure)

➢1O2 measured by RNO bleaching in presence of imidazole

(decreased absorbance at 440 nm)

➢ Superoxide radical anion measured by 1-electron reduction of

NBT (increased absorbance at 560 nm)

Acknowledgements

FreeThink Technologies, Inc. for hosting

the conference and for the opportunity to

give this presentation!