Prof. Dr. Niraldo Paulino

1. Aspectos históricos, legais e éticos. Desafios e Oportunidades

na Fitoterapia Médica

Um exemplo super-interessante de

estudo científico de Fitoterápico

Uma abordagem completa do estudo

farmacocinético de um produto farmacêutico

derivado de planta medicinal em humanos.

Parte 3

APRESENTAÇÃO DO PRODUTO

HELIORAL

DESENVOLVIMENTO TECNOLÓGICO:

VALIDAÇÃO BOTÂNICA

FERNBLOCK

FERNBLOCK (EPL): descrição botânica

• Polypodium leucotomos

• Familia Polypodiaceae

• Gênero Phebodium – Crescem naturalmente como plantas epífitas em

diversas palmeiras

– América Central • “Calaguala”

• Medicina popular – Antiinflamatorio (psoriasis, dermatitis atópica)

Reprodução

Por meio de esporos

– Que são lançados ao vento e quando encontram condições favoráveis, germinan dando lugar a novas plantas.

Planta adulta de Phlebodium

Germinação controlada

Rosetas e esporócitos sob germinação controlada

.

Plantas desenvolvidas

FERNBLOCK (EPL): cultivo

o Plantação localizada em no Lago YOJOA HONDURAS em Olancho.

o Padronização das condições de cultivo

o Sem uso de defensivos agrícolas.

HELIORAL

DESENVOLVIMENTO TECNOLÓGICO:

VALIDAÇÃO QUÍMICA

FERNBLOCK

FERNBLOCK (EPL): composição

• Extrato aquoso constituido por: – POLIFENÓIS: 3-10 % (p/p)

• Ácido Ferúlico

• Ácido Cafeico

• Ácido Cinámico

– MONOSACÁRIDEOS: 1-12 % (p/p) • Fructose, manose e glicose

– FLAVONÓIDES

– EPL é una fonte natural de substâncias antioxidantes, principalmente

rico em derivados fenólicos

FERNBLOCK (EPL): Laudo de análise

GARANTIA DE QUALIDADE

Reprodutibilidade na produção do fitomedicamento!!

In summary, the presence of both free and conjugated phenolics, such as caffeic and chlorogenic acids,

and the presence of different chlorogenic isomers point toward the relevance of the shikimik pathway in

ferns, and the importance of phenolic compounds as secondary metabolites related to plant

photoprotection (28). In addition to photoisomerization of cinnamic and related phenolic acids, the in

vivo photocyclodimerization has been described and related to cell wall stabilization (29). The presence

of cyclodimers of the phenolic acids herein described, either in Fernblock or as a consequence of UV

irradiation of the extract, is currently under study in our laboratories.

3,4-Dihydroxybenzoic acid Chlorogenic acid

4-Hydroxybenzoic acid Caffeic acid Vanillic acid

4-hydroxycinnamic acid

Neochlorogenic acid

Ferulic acid

COMPARATIVE ANALYSIS BETWEEN IFC Polypodium leucotomos extract

(batch P-07) and VIAFARMA (HUZHOU) Polypodium leucotomos extract

(batch 20070418) (Internal code IFCE 1195):

ATENÇÃO!! “Nem tudo que se parece é igual...”

IFC (PLE)

Advantages

VIAFARMA (PLE)

Disadvantages

1.In vivo photoprotector

2.Natural water based extraction

process

3.Phenol content and in vitro

photoprotective activity the same if not

better

4. Demonstrated anti-oxidant activity

5. Over 12 clinical trials In leading

institution like Harvard and Sloane

Kettering demonstrating;

Anti-oxidant activity

Anti-aging activity

Photo-Inmuno Protection

Photo protection

6. Over 15 years farmacovigilance and

safety data.

7.Registered and commercialed in over

42 countries

8. Registered with USFDA as ODI and

Self Affirmed GRAS

10. On-going pre-clinical and clinical

research.

11. Certified GLP/GMP extraction

processes assuring consistent quality

and stability.

12. Certified QA/QC processes

assuring consistent quality and

stability.

1. No photoprotective activity in vivo

2.Chemical extraction process

Industrial Farmacéutica Cantabria, S.A. Pirita, 6 28850 Torrejón de Ardoz MADRID (España) Tel.: (34) 91 676 90 82 Fax: (34) 91 677 20 41 ifc@ifc.es

HELIORAL

DESENVOLVIMENTO TECNOLÓGICO:

VALIDAÇÃO BIOLÓGICA

FERNBLOCK

In summary, these results underline the antioxidant and photoimmunoprotective properties of the PL extract

orally given in an in vivo model. PL-treated animals are less affected by UVR irradiation, chiefly noted in

decreased GSSG levels and a more favourable GSH⁄ GSSG ratio, and inhibition of UVR-mediated LC

depletion. These and other results strongly suggest the need to extend the concept of photoprotection,

currently restricted mainly to erythema development, which is the most extended criteria to evaluate the

efficacy of photoprotective measures. Oral administration of such antioxidant, photoprotective substances

would provide an excellent, hassle-free complement to the recommended use of topical sunscreens.

Overall, the effects of P. leucotomos predominated with or without exposure to UVA or UVB radiation in both keratinocytes and fibroblasts, respectively. PL improved membrane integrity, inhibited lipid peroxidation, stimulated elastin expression and inhibited MMP-1 expression in both fibroblasts and keratinocytes. The only exception to the predominant effects of P. leucotomos was in the inhibition of MMP-1 expression only in combination with UV radiation, particularly UVB, in fibroblasts

REGULAÇÃO BIOQUIMICA NA CÉLULA

O sistema bioquímico

complexo que produz

espontaneamente

formas radicalares de

oxigênio reativo (ROS)

a cada movimento de

respiração celular.

Esses radicias livres

são consumidos pelos

nossos sistemas de

defesa antioxidante:

SOD, CAT, GSHpx, etc...

REGULAÇÃO DA RESPOSTA ANTIOXIDANTE

Além disso, fatores ambientais: inflamação, radiação, agentes químicos, lesão de reperfusão, podem induzir radicais livres

adicionais nas células levando a danos celulares.

Geração de radicais livres

Oxidação de proteínas

Radical

superóxido

Radical

peroxil Radical

Alcoxil

Radical

Hidroxil

Outros

Controle da

transcrição gênica

NFKapaB e AP1

Controle da

diferenciação

celular

Mudança estrutural das proteínas

de regeneração celular

P. leucotomos e seus derivados

fenólicos e flavonóides apresentam

forte atividade ANTIOXIDANTE

e SEQUESTRANTE DE

RADICAIS LIVRES

Polypodium leucotomos

P. leucotomos e seus derivados

fenólicos e flavonóides apresentam

forte atividade INIBITÓRIA

SOBRE OS FATORES DE

TRANSCRIÇÃO NUCLEAR

Polypodium leucotomos

Efeito do P. leucotomos na reação oxidativa

induzida por radiação solar

In summary, P. leucotomos demonstrated dual protective eVects on the ECM for anti-skin aging via the inhibition of the ECM proteolytic enzymes and the stimulation of TIMPs, structural ECM collagens (types I, III, V), and TGF- in Wbroblasts. P. leucotomos’s anti-cancer eVects encompass the inhibition of MMPs and TGF- and stimulation of TIMPs in melanoma cells. In addition, P. leucotomos anti-aging and anti-cancer mechanism lies in its direct inhibition of MMPs. The eVects of P. leucotomos on Wbroblasts and melanoma cells may be partly via its cell-speciWc regulation of TGF- expression and partly via its antioxidant property. Polypodium leucotomos is used for the treatment of psoriasis (Difur®) and for photoprotection (Fernblock®) [18, 20]. Its topical or oral administration is tolerated without toxicity; for protection against UV radiation or PUVA (Psoralen § UVA) mediated phototoxicity, depletion of langerhans cells, erythema and skin damage [18, 20, 21, 23, 35]. P. leucotomos is readily absorbed and metabolized cellularly [18]. We therefore infer an in vivo potential of P. leucotomos for antiskin aging and anti-cancer, respectively, via speciWc beneWcial regulation of ECM remodeling.

Epiderme Epiderme ERITEMA

Estímulo inflamatório

O2-

Vaso

Macrófagos PMN

ROS

Apoptose ou Necrose

PGs AA

COX

EDEMA

NO Citocinas

PGE2

Linfócitos

Carrageina

Inflamação

Efeito do P. leucotomos na reação inflamatória tópica

induzida por radiação solar

Estímulos TLR

Myd-88 IRAK

TRAF6

NIK

p50 p65

p50 p65

IKK IKK

IB

TNF-

iNOS

COX-2

IL-12

NO

PG

IL1

Redução da inflamação e da angiogênese associada ao P. leucotomos

Quimioterápicos, LPS

Ativação de

receptores tipo TLR

Desencadeia uma

cascata de fosforilação

Ativação da

quinase do

IkappaB

Fosforilação do

inibidor do NFkB

Liberação do Fator

de Transcrição

Nuclear NFkB

Translocação

nuclear para a

ativação da

expressão gênica Expressão de

inúmeros genes

pró-inflamatórios Expressão de

genes das citocinas

IL1, 12 e TNF-

Expressão de

genes da

cicloxigenase 2

Aumento da

produção de PGE2,

PGF2 ...

Inflamação:

aumento da

permeabilidade

vascular,

vasodilatação,

migração celular,

etc…

Condições ideiais

para o crescimento

do tumor e

metástase

Specifically, PL has the capacity to neutralize free radicals, membrane-lipid peroxidation, and various ROS

(19, 20). It is possible that PL’s ability to prevent UVB radiation-induced immunosuppression and skin

ancer in mice is accomplished through its antioxidant properties as other antioxidants have been shown to

inhibit UVB-induced immune suppression in mouse models (21–24). ROS are thought to be involved in the

in the decrease in Langerhans cell numbers and the inhibition of CHS observed after exposure of murine

skin to UVB radiation (21) and ROS-induced lipid peroxidation of membrane lipids may impair antigen

presenting function in the remaining Langerhans cell. In addition, ROS increase TNF-a production (25), a

mediator important in CHS suppression in UVB-irradiated mouse skin (26). In this regard, PL has been

shown to inhibit TNF-a release stimulated by exposure of human keratinocytes to solar simulator radiation (27). Furthermore, PL inhibitsthe photoisomerization of trans-urocanic acid to cis-urocanic acid, another mediator implicated in UVB-induced immune suppression (28).

Photodermatology, Photoimmunology & Photomedicine 24, 134–141 2007

In conclusion, this study shows that anapsos is able to modulate the immune

response after surgery, by expressing an inhibitory effect on Th2 pathway

activation with no effect on Th1 pathway activation. This might be very important

in trauma and clinical surgery with regard to modulator treatment of the

postoperative immune response, since anapsos might prevent the shifting

Th1/Th2 balance and, in addition, preserve inflammationfollowing tissue repair

processes, and avoid “hyperinflammation” consecutive to immunosuppression

induced by surgery.

Carcinogenic stimulus

Rac2 Cdc42

GTP

Rac2 Cdc42

GDP

P

+

MKK3MKK6

p38

TLR

Myd-88 IRAK

TRAF6

NIK IKK

IKK

IB

p50 p65

p50 p65 TNF-

IL-1

iNOS

COX-2

IL-12

NO

PG

Mecanismos imunomodulador que justifica a melhoria da

resposta imune após o uso de P. leucotomos

Carcinogenic stimulus

Estímulos carcinogenicos

externos

Ativação de proteinas da

classe Ras (GTPase)

Prenilação pós-

translacional

Ativação de proteinas

quinases ativadas por

mitógenos

Ativação da p38-MAPK

Ativação do fator de

modulação imunologica

Aumento da

resposta

imune celular

P. leucotomos

P. leucotomos

P. leucotomos

In addition, PL downregulates TNF-a and iNOS expression induced by SSR. UV radiation induces TNF-a

expression (42). Interestingly, UV irradiation also induces expression of TNF-a receptors (43), which

suggests that TNF-a mediates the inflammatory reaction in skin induced by UV irradiation. On the other

hand, iNOS is also induced by UV irradiation in HaCaT cells (44). Increased iNOS expression causes

elevated levels of NO, which is one of the causal factors of erythema formation. The inhibitory effect of PL

on NO production and iNOS expression strongly suggests that NO increase is caused by de novo

synthesized iNOS. Finally, TNF-a and iNOS up-regulation are linked to increased apoptosis [reviewed in

Refs. (45,46)], thus inhibition of UV-mediated TNF-a and iNOS expression by PL is a likely cause for the

observed decrease in apoptosis. In summary, these findings greatly extend previous observations on the

photoprotective effect of PL, and represent a rigorous approach to explain its molecular basis, providing a

rationale for increased cell survival, decreased inflammation and changes in pigmentation observed both in

vivo and in vitro.

Conclusions

Originally described as a natural alternative for the treatment of inflammatory skin disorders such as psoriasis, P. leucotomos is a

more powerful tool than previously thought. It combines extremely low toxicity with proven beneficial effects, even with oral

administration. These include antioxidant properties, the capability to inhibit t-UCA photoisomerization (which makes it a very

valuable additive in sunscreen formulations), and the ability to block UV-induced apoptosis and DNA photodamage

andimmunosuppression. It seems clear that P. leucotomos is not an alternative to traditional sunscreen use, but it provides a very

effective complement for sensitive skin phototypes and adds extra protec- S. Gonzalez, J.L. Alonso-Lebrero, R. Del Rio and P.

Jaen 7 Table II: Beneficial effects of P. leucotomos. On... Molecular effects Short-term effects Long-term effects References

Generation of free Inhibits ROS Relieves photoaging, (32, 62, 75) radicals generation prevents tumor formation Generation of

other Inhibits t-UCA (Predicted) (64) deleterious photoisomerization, Relieves immunometabolites photodecomposition

suppression and tumor formation Inflammation Inhibits cell death and Reduces erythema, Relieves photoaging, (35, 45, 50, 51,

53) apoptosis sunburn* prevents tumor formation DNA damage Decreases the Reduces SOS (Predicted) Inhibits (53) formation of

response tumorigenesis pyrimidine dimers Immunosuppression Prevents depletion (Predicted) Reduces (Predicted) Inhibits (51,

53, 73, 74) of eLC recurrent infections chronic inflammation and cancer *Oral administration of P. leucotomos (1,080 mg)

increases skin tolerance to sunlight by a factor of 2.81 (48). ROS = reactive oxygen species; UCA = urocanic acid; SOS = the

SOS response: the depression of multiple genes encoding DNA repair proteins, leading to more rapid DNA repair and enhanced

bacterial survival; eLC = epidermal Langerhans cells. tion in cases in which exposure to UV radiation cannot be avoided, such as

those in UVB phototherapy and psoralens-UVA treatments.

HELIORAL

DESENVOLVIMENTO TECNOLÓGICO:

INOVAÇÃO EM FITOTERAPIA

FERNBLOCK

Recentes trabalhos científicos (2012) sobre P. leucotomos