1 Dr . Susana Garcia de

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Arriba. Scientific . & Medical Expert . - Regulatory . Affairs. Track 1: . Toxicology Approaches. Human Safety of free Hydroquinone . derived . from . Herbal medicinal . products . containing . Uvae. ID: 741034 Download Presentation

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Slide1

1

Dr. Susana Garcia de ArribaScientific & Medical Expert - Regulatory Affairs

Track 1: Toxicology Approaches

Human Safety of free Hydroquinone

derived

from

Herbal medicinal

products

containing

Uvae

ursi

folium

Slide2

Herbal

Medicinal

Product:HMPC – MonographArctostaphylos uva-ursi folium Pharmacokinetics – EliminationHuman Exposure to Free HQExposure limits for use in risk identification Permitted Daily Exposure of free HQAssessment Genotoxic Potential of Free HQAMESMicronucleous in mice7. Safety Data – Cystinol akut8. Conclusions

Content

Slide3

3

National markets for

Herbal medicines

annual sales per capita consumption Germany -- $2.2 billion --- $36.55 millionDenmark -- $ 30.2 million -- $ 26.6 million The Global Herbal Medicine Market is estimated* to be US$ 60 billion, which is poised to grow to $5 trillon by the year 2050EUJapanAsia + AustraliaUSARest EUOthers*World Health Organization (WHO) / Secretariat of the convention on Biological Diversity (2008)28 US$11 US$10 US$7 US$(US$ billion)

Increase in herbal medicine sales 10 % per Year

Herbal Medicine Market

Global Herbal Medicine Market

Slide4

4

Herbal Medicinal

Products (HMPs)

*

HMPs are defined as any medicinal product, exclusively containing as active ingredients one or more herbal substances or one or more herbal preparations, or one or more such herbal substances in combination with one or more such herbal preparations.HERBAL SUBSTANCE (HS) - dry /fresh: whole or parts of plants, algae, fungi and lichens. HERBAL PREPARATION (HP)- obtained by subjecting HS to treatments such as extraction, distillation,expression, fractionation, purification, concentrationor fermentation.Require a Marketing Authorization or Registration before placement on the market In the EU communityDrug Law on pharmaceutical products for human use - - -> *Directive 2001/83/EC applies to HMP Protect public health Harmonized authorization across the EUFacilitate the authorization/ registration procedure of HMP

Slide5

5

http://ec.europa.eu/health/human-use/herbal-medicines/index_en.htmhttp://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000208.jsp&mid=WC0b01ac05800240cf

1.1 Market access for HMP in the EUunder European medicines legislation,

HMPs must

fall within one of the following 3 categories to reach the market:1. Traditional use registration (simplified registration procedure) Directive 2004/24/EC amending Directive 2001/83/EC. - HMP with sufficient evidence of medical use >30 years, >15 years within EU. Sufficient safety data and plausible efficacy. THMP with Traditional medicinal use based upon long-standing use. 2. Marketing authorisation (Bibliographic application)  HMP with well-established medicinal use on basis of recognized efficacy, at least one controlled clinical study of good quality is required to substantiate efficacy, and an acceptable level of safety. 3. Marketing authorisation – (full/ mixed application)  a product with safety and efficacy data from the company’s own development (‘stand alone’) or a combination of own studies and bibliographic data (‘mixed application’). As a result the product is granted a marketing authorisation.Herbal Medicinal Products

Slide6

6

http://ec.europa.eu/health/human-use/herbal-medicines/index_en.htm

EMAMedical Products human use

Committee

on Herbal Medicinal Products HMPC HMPC MonographRegulation (EC) No 726/2004 and Directive 2004/24/ECResponsible for Agency's opinions on herbal medicines Experts in the field of herbal medicines. Advisers28 EU Member States (+ Iceland and Norway)Scientific evaluation of non-clinical and clinical data; safety and efficacy data; quality of the herbal substance /preparations intended for medicinal use and license Evaluation of long-standing medicinal use in the EUHS/HP complies qualitative - quantitative declaration, therapeutic indication and daily recommended dose  efficacy and safety of the HS or HP are ensured Marketing AutorisationRegistrationSept. 2004European Medical Agency1.2 EMA, HMPC and HMPsHerbal Medicinal ProductsListing!!!**list of herbal substances, preparations and combinations thereof for use in traditional herbal medicinal products

Slide7

7

http://ec.europa.eu/health/human-use/herbal-medicines/index_en.htmhttp://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000208.jsp&mid=WC0b01ac05800240cf

All HMPs in the EU market have to fulfill the demands of HMPC and competent national authorities

EU legislation on pharmaceutical products for human use

Pharmaceutical quality – Eur. Pharmacopoeia/NfGStandardized production process: GMP (GDP, GACP) Drug Safety: Preclinical: Genotoxic/Mutagenic potential (GLP)Pharmacovigilance (GVP)Proved Well Established/ Traditional long-standing medicinal use in the EU1.3 HMPs in the EU marketHerbal Medicinal Product

Slide8

8

http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000208.jsp&mid=WC0b01ac05800240cf1.4 EMA - HMPC

Herbal

Medicinal

Product

Slide9

9

http://www.ema.europa.eu/docs/en_GB/document_library/Other/2009/12/WC500017724.pdf1.5 Committee on Herbal Medicinal Products

Herbal

Medicinal

Product

Slide10

10

http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/landing/herbal_medicines_search_landing_page.jsp&mid=

1.6 HMPC Monograph

Herbal

Medicinal ProductLatin name of the genusArctostaphylosLatin name of herbal substanceUvae ursi foliumBotanical name of plantArctostaphylos uva-ursi (L.) Spreng.English common name of herbal substanceBearberry LeafStatusF: Assessment finalisedDate added to the inventory23/11/2005Date added to priority list23/11/2005Outcome of European assessmentCommunity herbal monograph 

Slide11

11

1.6 HMPC Monograph

Herbal Medicinal

ProductFinal Assessment reportFinal community herbal monograph

Slide12

12

1.6 HMPC Monograph

Herbal

Medicinal

Product

Slide13

13

1.7 Assessment Report - HMPC MonographHerbal

Medicinal

Product

Slide14

HMP -

Herbal Preparation  ethanolic (60% v/v) dry extract of Arctostaphylos

uva-ursi folium

Composition

: 1 Tablet 238.7-297.5 mg ethanolic (60% v/v) dry extract Arctostaphylos uva-ursi folium (DER 3.5 – 5.5:1)  corresponding to 70 mg arbutin anhydrousPosology adults and children over 12 years Take 2 coated tablets 3 times dailyRecommended Daily Dose (RDD): ≈ 1.6 g Extract = 420 mg arbutinTraditionally used to treat symptoms of lower urinary tract infections2. Arctostaphylos uva-ursi folium (Cystinol® akut) Cystinol® akut

Slide15

15

 COMPLIANT with the

qualitative and quantitative composition, posology

as well as with the

therapeutic area of application 2.1

Slide16

16

Information about HMP on the market in the Member States - Regulatory status overview

Country

Date – Regulatory status

Formulation1 DoseDaily Dose, RDD Powdered herbal substance in solid dosage formsGermany>1976 MAcoated tablets500 mg 6 tablets 4 times - 12 gSpain >1991 Rcapsule270 mg1 capsule 3 times - 810 mgFrance>1982 Rhard capsules350 mg2 capsules 2 times - 1.4 gGermany, Poland, Spain, Estonia, SloveniaR/MAHerbal Tea1.5-3 g/150ml 3 - 6 times - 4.5 - 18 gHerbal preparations - Dry extractsGermany> 1976 MA (39 y)coated tablets238.7 – 297.5 mg2 tablets 3 times - 1.6 g

Poland

> 2000 R

film-coated tablets

215 mg of

4 tablets 3 times -

2.6 g

France

> 1992 R

hard capsules

200 mg

1 capsule 2 times -

400 mg

MA - Marketing

authorisation

R - Registration

http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_HMPC_assessment_report/2011/07/WC500108750.pdf

2.1 HMPC monograph

Arctostaphylos

uva-ursi

folium

Slide17

17

1. high

pharmaceutical

quality2. widely documented drug safety3. Medicinal use proven by HMPC /National-International Monographs

Quality

of

the

extract

Good

Laboratory

Practice“

GLP

No

systemic

toxic

effects

Medicinal

use

Therapeutical

use

Safety Data

-

preclinical

-

Safety Data

- Human -

Medicinal

use

Therapeutical

experience

Ethanolic

U

vae

ursi

folium

extract

GMP,

GDP,

GACP

Eur.

Pharmacopoeia

2.2

Cystinol

®

akut

-

HMP

Documentation -

Quality, DER, HP

HMPC Monograph

No

genotoxic

-

mutagenic

Risk

Pharmacovigilance

Data

HMPC

monograph

WHO, ESCOP,

Comm

. E Monographs

Slide18

18

National and International herbal monographs on Uvae

ursi folium

Commission

E, 1994; WHO, 20021; ESCOP2, 2012 and HMPC, 20123HMPC Monograph on Arctostaphylos uva-ursi (L.) Spreng, folium There is sufficient evidence of medicinal use throughout a period of at least 30 years, including at least 15 years in European CommunityUvae ursi folium extract - Cystinol® akut long-standing medicinal use and experience  testified by bibliographic and expert evidence 1World Health Organization. Folium Uvae ursi. Monografie 2 (2002): 342-3512European Scientific Cooperative on Phytotherapy (ESCOP) Uvae ursi folium, bearberry leaf. (2012), online series. www.escop.com3HMPC Community herbal

monograph

on

Arctostaphylos

uva-ursi

(L.) Spreng.,

folium

(28.02.2012)

http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_Community_herbal_monograph/2011/04/WC500105350.pdf

2.3

The therapeutic efficacy and

safety

Slide19

19

Commission

E

1994

WHO (World Health Organisation)2002ESCOP (European Scientific Cooperative on Phytotherapy) 2012HMPC (Herbal Medicinal Products Committee of EMA) 2012Indication

Inflammatory diseases of the lower urinary tract

Moderate inflammatory conditions of the urinary tract and bladder, such as cystitis, urethritis and dysuria

Uncomplicated

infections of the lower urinary tract, when antibiotic treatment is not considered essential

Treatment of symptoms of mild recurrent lower urinary tract infections (traditional use)

Dosage

3 g drug or

400 - 840 mg

hydroquinone

derivates

, calculated as

arbutin

, up to 4x daily

3 g drug or 400 - 850 mg hydroquinone

derivates

, calculated as

arbutin

, up to 4x daily

400-800 mg of

arbutin

/day,

equivalent preparations

Single dose corresponding to 100-210 mg hydroquinone

derivates

calculated as anhydrous

arbutin

, 2-4x daily

Duration of use

Without medical advice: up to 5x per year, not longer than 1 week in each case

No long-term use, patients with persistent symptoms should consult a physician

Until complete disappearance of symptoms (up to a maximum of 1 week)

Not

longer

than

1

week

Contra-indictions

Pregnancy, lactation, children < 12 years

Pregnancy, lactation, children, patients with kidney disorders

Pregnancy, lactation, children < 12 years

Hypersensitivity

,

kidney

disorders

Interactions

Urinary acidifying substances*

Urinary acidifying substances

None reported

None

reported

Side

effects

Nausea and vomiting possible in persons with a sensitive stomach

Nausea and vomiting may occur due to stomach irritation from the high tannin content

Nausea and vomiting may occur due to stomach irritation from the high tannin content

Nausea,

vomiting

,

stomach-ache

2.4 International /national

Monographs

Positive

Benefit-Risk

Profile

officially

approved

Slide20

20

Extract = active principle

Hydroquinon (HQ)

derivatives

: arbutin to 23.5 - 29.3% free HQ < 0.3%Phenolic acidsgallic acid, Ellagic acidPolyphenols (tannins) gallotannins, ellagitannins Flavonoids hyperosideTriterpenes ursolic acid, uvaeolArbutinHQ

Gallic

acid

Ellagic

acid

2.5 Relevant Constituents : Qualitative -Quantitative

Slide21

Siegers et al. 1997

213.

Pharmacokinetics12 healthy volunteers

(6 males - 6 females)

Dose 3 x 2 Tablets Cystinol® akut 1.6 g Extract/day 420 mg Arbutin (169 mg HQ)Urine samples collected after 36hFree and Total HQ quantified in individual urine samples – HPLC STUDY 1 - PharmacokineticsEliminationFree HQ  hydroquinoneTotal HQ = Free HQ + conjugated HQ

Slide22

22

3.1 Single Data Elimination

Individual urine samples n=12

n=11

Free HQ (mg)Total HQ (mg)HMP intakeFree HQ (mg)Time (hours)Subject 3 had an extreme high level of free HQ before HMP intake.Total HQ = Free HQ + HQ-ConjugateElimination of Free HQ and Total HQ Time-dependent and gender independent6/12 eliminate Free HQ3/6 high levels free HQ at t=0High Free HQ elimination no related To increase of Metabolism/elimination 0.99109.4

Slide23

23

N= 11 0.6% Arbutin Dose as Free HQ =

0.99 mg Free HQ70% Arbutin Dose as conjugate HQ

to

glucuronic and sulfuronic acid = 109.4 mg total HQ3.2 Quantitative Elimination of Free and Total HQMean values ± SDParallel and progressive eliminationSTUDY 1 - RESULTSTime (h)

Slide24

24

Pharmacokinetic

profile HQ; Hydroquinone

HQ-

Glu: HQ-glucuronide; HQ-Sulf: HQ-sulphate3.3 Metabolism - EliminationMajor elimination: HQ-glucuronide >> HQ-sulphate (≈ 70% of total conj.)0.6% Free HQ70% Total HQ

Slide25

25

3.4 Comparison with Bibliographic Data

Subjects, urine collection (h)

Arbutin

Daily Dose% Arbutin Dose Free HQTotal HQSiegers et al. 1997n=11, 36 420 mg0.6%70%Paper et al. 1993 n=5, 24100 mg085%Schindler et al. 2002n=16, 36210 mg0.1%66.7%Quintus et al. 2005n=3, 24150 mg0.6%75%* Detection limit (1µg/ml)Arbutin MW 272.25HQ MW 110.11

Slide26

26

20fold

0.6% HQ

>70%

3.5 Bacterial deconjugation4 healthy volunteers (2 males - 2 females)Dose 3 x 2 Tablets Cystinol® akut 420 mg ArbutinUrine samples collected after 24hFree and Total HQ quantified STUDY 2 – Bacterial deconjugation24h 37°C100 µl/ml Percloric acidHPLC quantificationHQ; HydroquinoneHQ-Glu: HQ-glucuronide; HQ-Sulf

:

HQ-

sulphate

Siegers

et al. 2003; Garcia et al. 2010; Garcia

et al.

2013

Slide27

27

3.6 Summary Elimination /deconjugation

Garcia et al. Int J Toxicol. 2013

The

elimination of arbutin metabolites after oral intake of 420 mg arbutinOnly 0.6 % of the administered arbutin-dose was excreted as free HQ in the urine In 6 out of 12 volunteers free HQ was not detected 70% of the administered arbutin-dose was eliminated as HQ-conjugates (HQ-glucuronide; HQ-sulphate) Free HQ deconjugation/accumulation inside of the bacteria Uropathogenic bacteria such as E. coli have the ability to deconjugate the HQ-conjugates eliminated in the human urine to a high extent20fold higher amount of free HQ was detected in bacteria sediments as compared to the supernatant HQ-conjugates found in urine are ingested by the uropathogenic bacteria and transformed into free HQ inside of the bacteria Intracellularly formed free HQ is responsible for the bactericide effect

Slide28

28

0.99 mg Free HQ420 mg Arbutin

3.7

Summary Elimination process

Recovered in Urine

Slide29

29

Elimination of Free HQ in 36 h: 0.99 mgLevel of Free HQ in urine after 36 h:

0.6 – 0.5 µg/mlCorresponding to Human exposure level: 11 µg/Kg

b.w

. day ** 60 kg body weight4. Human exposure to Free HQ*Garcia et al. Int J Toxicol. 2013

Slide30

30

4.1 Food sources of Free HQ

Product

Arbutin

(µg/kg)Free Hydroquinone (HQ)

Per day

LEVEL OF EXPOSURE

µg free HQ/kg

b.w

./day

*

Coffee and Tea

20 - 90 µg /glass

1

2-3 glass/day

0.66 - 4.5

Red wine

562 µg / glass

1,2

1 glass/ day

9.3

non-filter cigarettes

110 - 300 µg /cigar.

3

18 cigar./day

**

33 - 90

Wheat products

0.015 - 0.13

0.2 µg/g

1

250 g/day

0.833

Broccoli

0.14 µg/g

1

250 g/day

0.6

Pears

0.037 - 0.18

0.02 - 0.05 µg/g

1

100 g/day

0.033 - 0.083

High-HQ diet

(n=4)

4 mg/kg

1

125 -275

1

Cystinol

RDD

420 mg/day

0.99 mg in urine

5

11

*Body weight of 60 kg;

1

Deisinger et al. 1996;

2

IPCS, 1996; 3NTP, 2009;

4

Carlson and Brewer (1953);

5

Siegers et al.

1997.

**

Mean value in EU.

These foods are consumed several times a day and for a lifetime without causing AE or safety concerns

Garcia et al.

Int

J

Toxicol

. 2013

Slide31

31

5. Exposure limits for

risk identification

Estimates

the dose below which there is a negligible risk to human health *- Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared Facilities EMA/CHMP/ CVMP/ SWP/169430/2012 *- Appendix 3 of ICH Q3C (R4) “Impurities: Guideline for Residual Solvents” Represents a substance-specific dose that is unlikely to cause an adverse effect if an individual is exposed at or below this dose every day for a lifetimePermitted Daily Exposure Definitions*

Slide32

32

5.1 Calculation of PDEToxicological parameters of free

HQ CAS N.: 123-31-9 (IPCS, 1994; IPCS, 1996; OECD/ SIDS, 2002; Williams et al. 2007; NTP, 2009).PDE for

free

HQ = (25 mg/kg b.w./day x 50 kg b.w.) (5 x 10 x 5) PDE free HQ = 5 mg/day or 100 µg/kg b.w./ day

Slide33

33

PDE for Free HQ = 5 mg/day or 100 µg/kg b.w./ dayExposure level of Free HQ derived from HMP = 11 µg/k.g. b.w./day

PDE

level is

9-times higher than the maximum exposure level to free HQ in a “daily usual” scenario after ingestion of a therapeutic RDD (420 mg of arbutin) of a herbal medicinal product containing Uvae ursi foliumPDE level corresponds to 8.3-times RDD of Cystinol HMP (6 tables/day). A patient should take 50 Tablets/day in order to be exposed to the PDE value of free HQ Accordingly, the level of free HQ produced by the administration of a RDD of Uvae ursi folium has a negligible probability of any toxicological risk to human health*5.2 Results - PDA*Garcia et al. Int J Toxicol. 2013

Slide34

34

5.3 Results - PDA

Supporting this low risk human exposure level, the HMPC monograph on

Uvae

ursi folium concluded that concentrations reached in the human body by monographed Uvae ursi folium-products are below the most conservative Thresholds of Toxicological Concern (TTC) (HMPC, 2012).In agreement with the above statement, the benefit-risk ratio of the medicinal use of Uvae ursi folium extract has been considered favourable for the claimed indication and dosage by all national and international current Monographs on Uvae ursi folium.Commission E (1994) WHO monograph (2004).HMPC monograph (2012), ESCOP (2012)

Slide35

35

6. Assessment of genotoxicity

Guidelines for genotoxicity testing of pharmaceuticals have been established by

HMPC, OECD and ICH

Testing of medicinal products involves a battery of genotoxicity tests, in which pro- and eukaryotic systems in in vitro and in vivo experimental setups with and without metabolic activation are employedEMEA/HMPC/107079/2007 into effect 01.12.2008 Guideline on the assessment of genotoxicity of herbal substances / preparations Note for guidance on genotoxicity: guidance on specific aspects of regulatory genotoxicity tests for pharmaceuticals (CPMP/ICH/141/95) = ICH guideline S2 (R1) OECD (1997). OECD Guidelines for the Testing of Chemicals No. 471: Bacterial Reverse Mutation Test [the Ames test]. OECD (1997). OECD Guidelines for the Testing of Chemicals No. 474: Mammalian Erythrocyte Micronucleus Test [in vivo micronucleus test].

Slide36

36

6.1 Scheme Mutagenic-Genotoxic Studies

RDD 6 tablets

Cystinol

/ day1.6 g Uvae ursi folium extract =7 mg/kg/day arbutinN= 12 Urine pooltotal HQ: 36.00 µg/mlfree HQ: 0.512 µg/mlIn vitro AMES testRisk???? In vivo Mice Micronucleus testSTUDY 1 - PharmacokineticsSTUDY 3 – AMES testSTUDY 4 – Micronucleus test

Slide37

37

6.2 AMES Test

Salmonella typhimurium LT2 : Four different histidine auxotrophic strains. Identify two basic classes of point mutations:

base

pair substitutions (TA 1535, TA 100) frameshift mutations (TA 1537, TA 98). All strains contain GC base pairs at the site of the histidine mutationFifth strain for detecting point mutations at adenine-thymine (AT) Do not comply with genotoxicity guidelinesInvestigation: Urine samples according to OECD-AMES guideline by two independent experiments: (I) the plate incorporation test (II) the pre-incubation testEvaluation: Mutagenic Potential  it induces a > 2-fold increase in the number of revertant colonies over negative control (spontaneous reversion rate)

Slide38

38

6.2 AMES Test: Preliminary Toxicity Test

Concentrations from 36.00/0.511 µg/ml to 0.240/0.0026 µg/ml of total HQ and free HQBacterial strains TA 98 and TA100

P

late incorporation method with or without addition of S9 fraction. Two independent assays (n=3). 4-nitro-o-phenylene-diamine (4-NOPD, 10 μg/plate) sodium azide (NaN3, 10 μg/plate) 2-aminoanthracene (2-AA, 2.5 μg/plate)control urine sample + 5 µg/plate free HQ

Slide39

39

6.2 AMES Test: Plate Incorporation Test

Plate incorporation test with/without metabolic activation. Two independent assays (n=3).

sodium

azide (NaN3, 10 μg/plate) 2-aminoanthracene (2-AA, 2.5 μg/plate)control urine sample + 5 µg/plate free HQ

Slide40

40

Pre-incubation test with /without metabolic activation. Two independent assays (n=3). Mean±SD

6.2 AMES Test: Pre-incubation Test

sodium

azide (NaN3, 10 μg/plate) 2-aminoanthracene (2-AA, 2.5 μg/plate)control urine sample + 5 µg/plate free HQ

Slide41

41

6.3 Micronucleus Test: in bone marrow cells

4 NMRI mice (2 females and 2 males)

U

nder identical conditions as in the in vivo micronucleus assay concerning test item, animal strain, vehicle, route, frequency and volume of administration. Administration undiluted pooled urine (D1) i.p., 10 ml/kg b.w.Examination of acute toxic symptoms (e.g. death, reduced spontaneous activity, eyelid closure, apathy etc.) at intervals of 1, 6, 24 and 48 hours Preliminary study on acute toxicity RESULTS: No signs of toxic reactions after 1, 6, 24 and 48 h were observed The undiluted urine D1 was considered as a suitable test item

Slide42

42

6.3

Micronucleus Test: in bone marrow cells

Urine

Undiluted D1Urine ½ D1Urine 1/10 D110 NMRI mice /test group ControlUrineControlUrine + free HQControl0.9% NaCl+ ControlCPA**cyclophosphamide (CPA) at 30 mg/kg b.w24 – 48 h(10 ml/kg b.w.) i.p. mice bone marrow micronuclei stained with May-Grünwald-GiemsaCellular target: Polychromatic erythrocytes (PCE) in the bone marrow mouse.Preparation of the marrow and cellsEach group: 5 male/5 female

Slide43

43

2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) were scored per animal for frequency of micronucleated cells

in 1000 erythrocytes the ratio of PCEs/normochromatic erythrocytes (NCE) in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicityA

test item is considered

mutagenic, if it induces either a dose-related increase in the number of micronucleated PCE or a statistically significant positive response for at least one of the test pointsThe cytotoxic effect of the test item due to the treatment was estimated as the ratio between PCE and NCE and reported as the number of NCE per 1000 PCEStatistical significance p< 0.05 was evaluated by means of non-parametric Mann-Whitney test6.3 Micronucleus Test Evaluation:

Slide44

44

Test Item

Dose

(ml/kg

b.w.)Sampling time (h)PCE with micronuclei (%)Rangeratio PCE/NCEControl urine10240.130-41000/726Control urine + HQ (50µg/ml)10240.120-21000/7450.9% NaCl-solution 1024

0.13

0-4

1000/722

Urine 1/10 D1 diluted

10

24

0.14

0-4

1000/736

Urine ½ D1 diluted

10

24

0.10

0-3

1000/747

Urine undiluted (D1)

10

24

0.09

0-3

1000/790

Cyclophosphamide

30 mg/kg b.w.

24

0.97*

4-25

1000/736

Urine 1/10 D1 diluted

10

48

0.11

0-4

1000/718

Urine ½ D1 diluted

10

48

0.09

0-2

1000/738

Urine undiluted (D1)

10

48

0.12

0-4

1000/756

Frequencies

of

micronucleated

PCE in 2000 PCEs per animal

Ratio

PCEs/ NCE in

1000

6.3

Micronucleus Test: DATA -RESULTS

*increase

micronucleus frequency

statistically

significant (p<0.0001, non-parametric Mann-Whitney test

Slide45

45

NO dose-related increase in the number of micronucleated PCE cells were observed NO clear increase in the number of micronucleated

cells in a single dose group at a single sampling time were observedNO Cytotoxicity = No change in the ratio polychromatic erythrocytes (PCEs) /

normochromatic

erythrocytes (NCE) was observed Results :6.3 Micronucleus Test:

Slide46

46

A multicenter survey with 75 registered doctor’s offices located in Germany was conducted in 1992-1993 reporting their clinical experience with Cystinol

mono (Cystinol akut since February 1996) for the treatment of diseases of the lower urinary tractA

total of

186,122 patients, (117,282 women)56 doctors reported experience with a total of 17,430 children and adolescents.Physicians with >30 years of experience (average of 10 years) 34.6%Therapeutic Indications Cystinol akutSafety 7.1 Cystinol acut – Medicinal use

Slide47

47

98.6%

Change

of

Urine pH is not requiered Mean duration Treatment (days)Time to reach Effect (days)Uncomplicated cystitis93Irritable bladder10

4

Urinary tract infection

10

4

Prevention of recurrence bacterial cystitis

28

 

Safety

7.2

Cystinol

akut

– Medical use

Slide48

48

Wide-spread OTC medicinal use  Pharmacovigilance

Data Post-marketing

patient

exposure Cystinol Akut from all over the world Cumulative period of 10 years  ≈ 7,000,000 patients In this period of time  A total of 161 cases (275 AEs) SPC/PIL  Side effects, adverse reactions≥ 1/10,000 to < 1/1,000 gastrointestinal complaints (nausea, stomachache and vomiting) < 1/10,000 allergic reactions

Safety

7.2

Pharmacovigilance

Slide49

Relevant

beneficial Properties 49Antibacterial

/anti-adherent /anti-inflammatory

properties

Broad spectrum of antibacterial activity NO development of antibiotic resistant bacteria Local antibacterial effect  Urine + BladderNO effect on intestinal, vaginal flora/immuno defensesSafe and well-toleratedBenefit – Risk Balance clearly positive

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8. ConclusionsFree und conjugate HQ

were detected and quantified in human urine samples. Human exposure to Free HQ was below its Permitted Daily Exposure

 No risk for human health

exposed at or below this dose every day for a lifetimeIn vitro and in vivo mutagenic and genotoxic studies were negative  Accordingly, urine samples obtained from healthy volunteers receiving a regular recommended dose as proposed for therapeutic use in the HMPC monograph bears no mutagenic risk.Pharmacovigilance Data from more than 30 years do not found AE different as reported in SPC/PIL In agreement with HMPC monograph Uvae ursi folium preparations are safe

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Thank you

for

your

attentionDon’t worry…We can eat him. He took Uva ursi only!!

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