UNIVERSIDADE FEDERAL DE SERGIPE

PRÓ-REITORIA DE PÓS-GRADUAÇÃO E PESQUISA

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

MAKSON GLEYDSON BRITO DE OLIVEIRA

EFEITO DO COMPLEXO DE INCLUSÃO CONTENDO α-TERPINEOL E β-

CICLODEXTRINA NA HIPERALGESIA NÃO INFLAMATÓRIA EM ROEDORES

ARACAJU-SE

2015

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2015

MAKSON GLEYDSON BRITO DE OLIVEIRA

EFEITO DO COMPLEXO DE INCLUSÃO CONTENDO α-TERPINEOL E β-

CICLODEXTRINA NA HIPERALGESIA NÃO INFLAMATÓRIA EM ROEDORES

Tese apresentada ao Programa de Pós-Graduação em

Ciências da Saúde da Universidade Federal de Sergipe

como requisito à obtenção do grau de Doutor em Ciências

da Saúde.

Orientador: Prof. Dr. Lucindo José Quintans Júnior

Co-orientadora: Profa. Dra. Jullyana de Souza Siqueira Quintans

ARACAJU-SE

2015

4

FICHA CATALOGRÁFICA ELABORADA PELA BIBLIOTECA DA SAÚDE UNIVERSIDADE FEDERAL DE SERGIPE

O48e

Oliveira, Makson Gleydson Brito de Efeito do complexo de inclusão contendo α-terpineol e β-ciclodextrina na hiperalgesia não inflamatória em roedores / Makson Gleydson Brito de Oliveira ; orientador Lucindo José Quintans Júnior. - Aracaju, 2015. 107 f. il. Tese (doutorado em Ciências da Saúde) - Universidade Federal de Sergipe, 2015. 1. Medicamentos - Interações. 2. Dor crônica. 3. Fibromialgia. 4. Opióides. 5. Analgésicos. I. Quintans Júnior, Lucindo José, orient. II. Título.

CDU 615.038

5

MAKSON GLEYDSON BRITO DE OLIVEIRA

EFEITO DO COMPLEXO DE INCLUSÃO CONTENDO α-TERPINEOL E β-

CICLODEXTRINA NA HIPERALGESIA NÃO INFLAMATÓRIA EM ROEDORES

Tese apresentada ao Programa de Pós-Graduação em

Ciências da Saúde da Universidade Federal de Sergipe

como requisito à obtenção do grau de Doutor em Ciências

da Saúde.

Aprovada em 14 de Dezembro de 2015.

____________________________________________________

Orientador: Prof. Dr. Lucindo José Quintans Júnior

____________________________________________________

Co-orientadora: Profa. Dra. Jullyana de Souza Siqueira Quintans

____________________________________________________

1º Examinador: Profa. Dra. Mairim Russo Serafini

____________________________________________________

2º Examinador: Prof. Dr. Marcelo Mendonça Mota

____________________________________________________

3º Examinador: Profa. Dra. Rosana de Souza Siqueira Barreto

____________________________________________________

4º Examinador: Profa. Dra. Sandra Lauton Santos

PARECER

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

_______________________________________________________________________

6

Dedico este trabalho à minha mãe, Aparecida

Brito, e à minha irmã, Lylyan Gleyze.

Obrigado pelo amor e pela incansável

dedicação. Amo vocês!

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AGRADECIMENTOS

A Deus, por tudo! Sem Ele nada teria sentido. Toda honra e toda glória sejam dadas a Ti, Senhor.

A minha mãe, por sempre me colocar em primeiro plano na sua vida, preferindo os meus sonhos

em detrimentos os dela. Essa vitória é nossa. Amo-te incondicionalmente!

A minha irmã, pela confiança inabalável. Por sempre acreditar em mim quando nem eu mesmo

acho que sou capaz. Amo você!

A Gustavo Ribeiro, pelo apoio nesse último ano com palavras de otimismo, sempre dizendo que

tudo daria certo.

Aos meus familiares e amigos, pelo apoio e incenstivo ao longo dessa jornada.

A Universidade Federal de Sergipe e todo corpo docente por formaram o profissional que sou hoje.

Ao meu orientador, Prof. Dr. Lucindo José Quintans Júnior, meu agradecimento especial pela

paciência, confiança, incentivo e ensinamentos a mim passados. O senhor foi fundamental para

minha chegada até aqui. Obrigado por tudo!

A todos que fazem parte do Laboratório de Neurociências e Ensaios Farmacológicos, em especial a

Profa. Dra. Adriana Gibara, Profa. Dra. Jullyana Siqueira, Heitor Araújo, Renan Guedes, Priscila

Laise, Silvia Sandes e Marlange Almeida.

Um obrigado supre especial a duas companheiras que a vida acadêmica minha presenteou, a

Profa. Dra. Mônica Melo e Profa. Me. Simone Nascimento, cuja pureza de coração e nobreza de

caráter me foram revigorantes durante toda jornada, principalmente nos momentos difíceis dessa

caminhada.

A minha amiga Profa. Me. Marília Trindade de Santana e o Prof. Me. José Rodrigo, pelo

companheirismo e apoio sempre que precisei. Marcela Ávila, por todo empenho nos momentos de

diversão. Eles foram necessários para renovar as energias.

Ao Prof. Dr. Adriano Antunes, Prof. Dr. Waldeccy de Lucca e Paula Menezes, pela parceria

firmada que foi imprescindível para realização dos protocolos experimentais.

Ao CNPq, FAPITEC e CAPES, pelo auxílio financeiro.

De coração, Makson Oliveira.

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RESUMO

Oliveira, Makson Gleydson Brito de. EFEITO DO COMPLEXO DE INCLUSÃO CONTENDO

α-TERPINEOL E β-CICLODEXTRINA NA HIPERALGESIA NÃO INFLAMATÓRIA EM

ROEDORES. Tese de Doutorado em Ciências da Saúde, Universidade Federal de Sergipe,

Aracaju, 2015.

O α-terpineol (TPN) é um monoterpeno alcoólico presente no óleo essencial de orégano e tomilho

com propriedades anticonvulsivantes, antinociceptiva e anti-inflamatória. As propriedades

analgésicas do TPN estão associadas a suas ações no sistema nervoso central (SNC), sendo

sugestivo seu efeito sobre dores crônicas conhecidas como disfuncionais, tais como a fibromialgia

(FM). A FM é uma doença reumatológica crônica de fisiopatologia relacionada a alterações em

sistemas de neurotransmissão e sua terapia atual é caracterizada principalmente pela conduta

farmacoterapêutica, contudo, com significativa farmacoresistência terapêutica. Dessa forma, o

objetivo do trabalho foi avaliar a possível efeito anti-hiperalgésico do complexo de inclusão

contendo TPN e β-ciclodextrina (βCD) no modelo de dor muscular crônica não inflamatória

(considerado ser um modelo experimental de FM) em roedores. O complexo TPN-βCD foi

preparado e caracterizado por termogravimetria (TG), espectroscopia de absorção na região do

infravermelho com transformada de Fourier (FTIR) e microscopia eletrônica de varredura (MEV).

Foram utilizados camundongos Swiss albinos machos pesando de 20 a 30 g. O modelo de

hiperalgesia muscular crônica não inflamatória foi induzida por duas injeções de solução salina (pH

4,0 - 20 µL) no gastrocnêmio esquerdo, sendo a primeira no dia 0 e a segunda no dia 5. Após a

indução da hiperalgesia, os animais foram tratados com αTPN-βCD (25, 50 ou 100 mg/kg; v.o.),

veículo (salina 0.9%, v.o.) ou Tramadol (4 mg/kg; i.p.) durante 10 dias consecutivos. Uma hora

após analise da hiperalgesia mecânica, avaliou-se o desempenho motor no teste do Rota-Rod e a

força muscular no Grip Strength Meter. Além disso, foi testado o possível antagonismo da ação

analgésica pela administração de naloxona e ondansetrona (antagonistas de receptores opióides e

serotoninergico, respectivamente). Os resultados foram expressos como média ± erro padrão da

média e as diferenças entre os grupos foram analisadas por meio do teste de variância ANOVA,

seguido pós-teste de Tukey. Após incorporação do αTPN na βCD, os complexos foram

caracterizados fisico-quimcamente por diferentes métodos: TG, FTIR e MEV, e o conjunto dos

resultados obtidos sugerem a formação do complexo αTPN-βCD. O tratamento oral com αTPN-

βCD, em todas as doses testadas, produziu uma redução estatisticamente significativa (p<0,001), na

hiperalgesia mecânica, sem causar alterações na coordenação motora e aumentando a força

muscular na maior dose testada. A duração do efeito analgésico nos animais tratados com complexo

αTPN-βCD foi quarto horas superior ao tempo de analgesia causada pela αTPN livre, diferença

estatisticamente significativa quanto a comparação desses dois grupos (p<0,01). O efeito analgésico

foi revertido pela administração sistêmica de naloxona ou ondansetrona. Corroborando com esses

resultados, o estudo de “docking” confirmou a possível interação do αTPN com receptores Opióides

(MU, Kappa, Delta) e Serotonina. Assim, pode-se concluir que o complexo αTPN-βCD reduziu a

hiperalgesia mecânica no modelo de nocicepção muscular crônica, provavelmente por ativação de

áreas do SNC, agindo, possivelmente, nos receptores opióides e serotoninérgicos.

Palavras-chave: α-Terpineol, β-ciclodextrina, Fibromialgia, Dor, Dor Crônica, Sistema Opióide.

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ABSTRACT

Oliveira, Makson Gleydson Brito. EFFECT OF AN INCLUSION COMPLEX CONTAINING

α-TERPINEOL AND β-CYCLODEXTRIN IN THE NON-INFLAMMATORY

HYPERALGESIA IN RODENTS. Doctoral Thesis in Health Sciences, Federal University of

Sergipe, Aracaju, 2015.

α-Terpineol (TPN) is an alcoholic monoterpene present in the essential oil of oregano and thyme,

having anticonvulsant, antinociceptive and anti-inflammatory properties. The TPN analgesic

activity is associated with its actions in the central nervous system (CNS), what can suggest that

TPN can act on dysfunctional chronic pain, such as fibromyalgia (FM). The FM is a chronic

rheumatic disease with pathophysiology related to alterations in neurotransmission systems. The

current therapy for FM is mainly characterized by pharmacotherapeutic conduct; however, there is

significant drug resistance. Thus, the aim of this study was to evaluate the possible anti-

hyperalgesic effect of an inclusion complex containing TPN and β-cyclodextrin (βCD) in the non-

inflammatory chronic muscle pain model (considered to be a FM model) in rodents. The TPN-βCD

complex was prepared and characterized by thermogravimetry (TG), absorption spectroscopy in the

infrared Fourier transform (FTIR) and scanning electron microscopy (SEM). Albino Swiss mice

were used, weighing between 20 and 30 g. The non-inflammatory chronic muscle pain model was

induced by two injections of acid saline (pH 4.0 - 20 uL) into the left gastrocnemius, 5 days apart.

After induction, the animals were treated with TPN-βCD (25, 50 or 100 mg/kg, p.o.), vehicle (0.9%

saline, p.o.) or tramadol (5 mg/kg; i.p.) for 10 consecutive days. An hour after the treatment, it was

measured the mechanical hyperalgesia through digital analgesimeter, the motor performance

through the Rota-Rod and muscle strength through the Grip Strength Meter. In addition, it was

tested the action of the administration of ondansetron and naloxone (opioid and serotonin

antagonists, respectively) in the TPN analgesic action. The results were expressed as mean ±

standard error and the differences between groups were analyzed using ANOVA followed by

Tukey's test. After incorporation of TPN in βCD, the complexes were characterized physical

chemically by different methods: TG, FTIR and SEM. These results together suggested the

formation of TPN-βCD complex. The oral treatment with TPN-βCD, in all doses, produced a

significant reduction (p <0.001) in the mechanical hyperalgesia without causing any changes in

motor coordination. The muscle strength increased after the administration of the highest dose. The

analgesic time effect in animals treated with TPN-βCD complex was over four hours to the free

αTPN, being these difference statistically significant (p <0.01). The analgesic effect observed was

reversed by systemic administration of naloxone or ondansetron. Corroborating these findings, the

"docking" study confirmed the possible interaction of αTPN with opioid (mu, kappa, delta) and

serotonin receptors. Thus, it can be concluded that the TPN-βCD complex reduced the mechanical

hyperalgesia in the chronic muscle pain model, probably due to activation of CNS areas, possibly

acting on opioid and serotonin receptors.

Keywords: α-terpineol, β-cyclodextrin, Fibromyalgia, Pain, Chronic Pain, Opioid System.

10

LISTA DE FIGURAS

Capítulo 1: Cyclodextrins: improving the therapeutic response of analgesic drugs: a patent

review

Figure 1. Schematic representation of the chemical structure of β-CD and HP-β-CD……... 44

Capítulo 2: α-Terpineol, a monoterpene alcohol, complexed with β-cyclodextrin exerts

antihyperalgesic effect in animal model for fibromyalgia aided with docking study

Figure 1. TG/DTG curves of α-terpineol (αTPN), β-cyclodextrin (βCD), physical mixture

(PM) and slurry complex (SC) obtained at nytrogen atmosphere...........................................

76

Figure 2. FTIR spectra of α-terpineol (αTPN), β-cyclodextrin (βCD), physical mixture

(PM) and slurry complex (SC).................................................................................................

77

Figure 3. Photographs SEM of β-cyclodextrin (A1 and A2), physical mixture (B1 and B2)

and slurry complex (C1 and C2) in the magnifications of 100 and 250x…………………....

78

Figure 4. A) Effect of acute administration of vehicle, α-terpineol and β-cyclodextrin

(αTPN-βCD 100 mg/kg, p.o.) or α-terpineol (αTPN 100 mg/kg, p.o.) on the mechanical

hyperalgesia induced by acid saline. B) Effect of chronic administration of vehicle, α-

terpineol and β-cyclodextrin (αTPN-βCD 25, 50 and 100 mg/kg, p.o.) or Tramadol

(TRAM, 4 mg/kg) on mechanical hyperalgesia induced by acid saline..………….………...

79

Figure 5. A) Effect of vehicle, α-terpineol and β-cyclodextrin (αTPN-βCD 25, 50 and 100

mg/kg, p.o.) or Tramadol (TRAM, 4 mg/kg) on the rota-rod test in mice. Values are the

mean ± SEM (n = 8, per group). B) Effect of chronic administration of vehicle, α-terpineol

and β-cyclodextrin (αTPN-βCD 25, 50 and 100 mg/kg, p.o.) or Tramadol (TRAM, 4

mg/kg) on grip strength test, fore-/hindlimb (4 paws)……………………............................................................

80

Figure 6. A) Effect of the pharmacological antagonists naloxone (NAL) and B)

Ondansetron (ONDAN) in formalin-induced nociception tests after treatment with αTPN

(i.p.), vehicle (i.p.) or morphine (MORF)…………………………………............................

81

Figure 7. Docking results in αTPN A) for 5HT receptor, interactions of two hydrogen

bonds with ASP129 and CYS133; sterics with the residues LLE130 and PHE330; B) for

Delta receptor, only one hydrogen bond with ASP128; C) for Kappa receptor, two

hydrogen bonds with LEU67 and VAL63 of the chain B; steric with VAL55 of the chain A

and D) for MU receptor, only one hydrogen bond with the residue

ASP147.………………………………………………………………………………………

82

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LISTA DE TABELAS

Capítulo 1: Cyclodextrins: improving the therapeutic response of analgesic drugs: a

patent review

Table 1. Pharmaceutical formulations of analgesics complexed in β-CD, their

indications and development status……………………………………………………...

45

Table 2. Pharmaceutical formulations of analgesics complexed in HP-β-CD, their

indications and development status ………………………...……………………….......

Capítulo 2: α-Terpineol, a monoterpene alcohol, complexed with β-cyclodextrin

exerts antihyperalgesic effect in animal model for fibromyalgia aided with docking

study

Table 1. Percentage of weight loss obtained from TG curves and water content

obtained by Titration of Karl Fischer……………………………………………………

47

83

12

SUMÁRIO

1 INTRODUÇÃO............................................................................................................................ 12

2 OBJETIVOS................................................................................................................................. 16

3 DESENVOLVIMENTO.............................................................................................................. 18

3.1 Capítulo 1: Cyclodextrins: improving the therapeutic response of analgesic drugs: a patent

review….……………..…………………………………………………….................................... 19

3.2 Capítulo 2: α-Terpineol, a monoterpene alcohol, complexed with β-cyclodextrin exerts

antihyperalgesic effect in animal model for fibromyalgia aided with docking

study………………………………………………………………………...…………………..…. 50

4 CONCLUSÕES........................................................................................................................... 84

REFERÊNCIAS.............................................................................................................................. 86

ANEXOS.......................................................................................................................................... 92

ANEXO A: Protocolo de aprovação do Comitê de Ética em Pesquisa com Animal da Universidade

Federal de Sergipe (CEPAUFS)....................................................................................................... 93

ANEXO B: Normas da Phytomedicine para submissão do artigo: α-Terpineol, a monoterpene

alcohol, complexed with β-cyclodextrin exerts antihyperalgesic effect in animal model for

fibromyalgia aided with docking study.......................................................................................... 94

ANEXO C: Comprovante de submissão do artigo “α-Terpineol, a monoterpene alcohol, complexed

with β-cyclodextrin exerts antihyperalgesic effect in animal model for fibromyalgia aided with

docking study” na Phytomedicine - Journal of Phytotherapy and

Phytopharmacology....................................................................................................................... 107

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INTRODUÇÃO

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1. INTRODUÇÃO

A fibromialgia (FM) é uma doença reumatológica caracterizada pela presença

de dor musculoesquelética generalizada e crônica, associada a outras manifestações

como fadiga, alterações do humor, distúrbios do sono e depressão. Mesmo sua

patogênese ainda não estando totalmente compreendida, uma das possíveis causas é

explicada por um desequilíbrio neuroquímico, interferindo na modulação da dor pelo

sistema nervoso central (SNC), o que desencadeia o aumento da percepção dolorosa

(SLUKA et al., 2002, GRACELY et al., 2002; ARNOLD et al., 2004; NEBEL et al.,

2009).

A farmacoterapia é um dos recursos utilizados para o seu tratamento e, dentre os

medicamentos mais prescritos descam-se os fármacos neuromoduladores, como

antidepressivos tricíclicos, bloqueadores seletivos de captação de serotonina, hipnóticos,

opióides, relaxantes musculares dentre outros (CROFFORD, APPLETON, 2001;

SUMPTON, MOULIN, 2008). No entanto, vale ressaltar que uma parcela significativa

dos pacientes não respondem bem à farmacoterapia, quer seja por respostas analgésicas

insatisfatórias, quer seja pela intolerância aos efeitos colaterais (PILLEMER et al.,

2005, CHONG, NG, 2009).

A pregabalina, duloxetina e milnaciprano foram os três medicamentos

aprovados pelo Foods and Drugs Administration (FDA) para o tratamento da FM.

Porém, metade dos usuários apresentam melhora de apenas 30% nos sintomas, sendo

necessário terapias complementares (DUSSIAS et al., 2010). Nesse contexto, uma

alternativa interessante para o tratamento da FM está nos produtos naturais, incluindo as

plantas medicinais e seus derivados metabólicos, os quais apresentam diferentes tipos

de benefícios terapêuticos que podem auxiliar no controle da FM, com melhora

significativa de sintomas tais como dor, insônia e ansiedade, melhotando a qualidade de

vida (LISTER, 2002; SKRABEK et al., 2008; WARE et al., 2010; EDDOUKS et al.,

2012; CASANUEVA et al., 2012).

Os óleos essenciais, extraídos das plantas medicinais, são de natureza volátil e

lipofílica, podem ser encontrados em diferentes partes das plantas. Esses óleos são

utilizados pelas mesmas como forma de defesa ou atração de polinizadores. As

substâncias presentes nos óleos são ativas biologicamente e apresentam potenciais

efeitos terapêuticos, como os monoterpenos que representam cerca de 90% dos

constituintes dos óleos essenciais (BAKKALI et al., 2008). À esses metabólitos

secundários estão associados efeitos calmantes, hipnóticos, antioxidantes, anti-

15

cancerígenos, anticonvulsivantes, dentre outros (HE et al., 1997; ZEYTINOGLU et al.,

2003; DE SOUSA et al., 2006). Trabalhos publicados têm demonstrado efeitos

antinociceptivos e anti-inflamatórios de vários monoterpenos que sugerem sua aplicação

no desenvolvimento de novas drogas para este fim (GUIMARÃES et al., 2010; MELO

et al., 2010; QUINTANS-JÚNIOR et al., 2011; QUINTANS et al., 2013;

NASCIMENTO et al., 2014).

O α-terpineol (αTPN) é um monoterpeno alcoólico e componente majoritário de

óleos essenciais de diversas espécies de plantas tais como Ravensara aromatica

(„Ravensara‟), Melaleuca qinquenervia („Niaouli‟), Myrtus communis („myrtle‟),

Laurus nobilis („laurel‟) (DAGNE et al., 2000; GOLSHANI et al., 2004; RAINA et al.,

2004). Vários trabalhos descrevem suas propriedades antimicrobianas, anti-

espasmódico, anticonvulsivantes, antinociceptiva e imuno-estimulante. Descrevem

também o aumento a permeabilidade da pele para compostos solúveis agindo por

mecanismos de inibiçao do NF-kappaB (WILLIAMS, BARRY 1991; FRANCHOME et

al., 1995; LEE et al., 1997; DE SOUSA et al., 2007; HASSAN et al., 2010). Os efeitos

anti-inflamatórios e anti-nociceptivos centrais deste composto sugerem seu possível

emprego em nosologias crônicas, tais como a fibromialgia (DE SOUSA et al., 2007;

QUINTANS-JÚNIOR et al., 2011; OLIVEIRA et al., 2012).

Em meio aos benefícios promissores supracitados desses derivados naturais,

aproximadamente 40% deles apresentam fraca hidrossolubilidade, representando um

desafio para a sua utilização terapêutica (MENEZES et al., 2012; SHARMA, 2012;

RAJESH, 2013). Uma possibilidade de minimizar este problema é a incorporação de

óleos essenciais e/ou seus derivados em ciclodextrinas (CDs). Essa prática vem sendo

aplicada pela indústria farmacêutica com a intenção de proteger esses compostos da

degradação pela temperatura, oxidação, luz, evaporação e umidade (SZEJTLI, 1998;

UEKAMA et al., 1998; HIRAYAMA, UEKAMA, 1999; CORTÉS et al., 2001). Vale

ainda pontuar que trabalhos recentes, publicados pelo nosso grupo de pesquisa,

relataram que a incorporação de substâncias à CDs provocam melhoria na eficiência

analgésica de diferentes tipos de drogas (OLIVEIRA et al., 2015; BRITO et al., 2015).

As CDs apresentam um papel importante para contornar os problemas dos

fármacos de baixa solubilidade em água, devido à sua propriedade de formar complexos

de inclusão, podendo atuar como carreadores hidrofílicos de fármacos. Essa é umas das

aplicações na indústria farmacêutica, podendo ainda aumentar a além da estabilidade, a

segurança e a biodisponibilidade de fármacos. As CDs naturais são oligossacarídeos

16

cíclicos, com seis a oito unidades de α-D-glicopiranose, classificadas em αCD, βCD ou

γCD, respectivamente, adquirindo uma conformação na forma de anel cuja superfície

externa apresenta solubilidade em água e a interior constitui uma cavidade central

hidrofóbica. Dessa maneira, estas moléculas apresentam a capacidade de acomodar uma

grande variedade de fármacos e outras substâncias de carater lipofílicas. A mais

utilizada é a βCD, por exibir cavidade apropriada para acomodar uma grande variedade

de fármacos e seu baixo custo (BREWSTER, LOFTSSON, 2007; RASHEED et al.,

2008).

Neste trabalho, utilizou-se o αTPN complexado a βCD, e foi avaliada sua

ação terapêutica sobre a dor muscular crônica não inflamatória, considerado um modelo

experimental de FM em roedores. Os modelos animais de doenças humanas, são de

grande valia para o desenvolvimento de novos tratamentos, bem como para investigar

os mecanismos subjacentes (SLUKA, 2009). O modelo utilizado nesse estudo foi

desenvolvido por Sluka et al. (2001), sendo um modelo de dor muscular que apresenta

três particularidades: 1. Instalação da hiperalgesia bilateral mecânica de longa duração

na pata, músculo e vísceras; 2. não há danos nos tecidos periféricos, 3. a hiperalgesia é

mantida por alterações no SNC (SLUKA et al., 2001; SKYBA et al., 2002; SKYBA et

al., 2005; YOKOYAMA et al., 2007; TILLU et al., 2008).

Considerando que a FM apresenta por substancial incidência no que se

refere a dor musculoesquelética crônica não-inflamatória e como o processo de

desenvolvimento e instalação desta síndrome pelos pacientes fibromiálgicos, com

relevantes repercussões na qualidade de vida relacionada à saúde, a descoberta de

tratamentos farmacológicos eficazes para o alívio da dor na fibromialgia pode

contribuir, de sobremaneira, para a redução dos gastos com consultas com diferentes

especialistas da área de saúde, internações e/ou tratamentos e reduzir as taxas de

morbidade nesses indivíduos. Sendo assim, o presente estudo buscou avaliar o possível

efeito anti-hiperalgésico do complexo de inclusão contendo αTPN em βCD, frente à

nocicepção fibromiálgica em roedores, podendo contribuir de forma efetiva com a

comunidade científica, no que se refere à descoberta de molécula promissora para o

desenvolvimento de novas propostas terapêuticas para o controle da dor.

17

OBJETIVOS

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2 OBJETIVOS

2.1 OBJETIVO GERAL

- Avaliar o possível efeito anti-hiperalgésico do complexo de inclusão contendo

α-terpineol e β-ciclodextrina em roedores.

2.2 OBJETIVOS ESPECÍFICOS

- Realizar o levantamento de patentes, buscando a elaboração de uma revisão

sobre o aumento no efeito analgésico com o uso da ciclodextrina;

- Preparar complexo αTPN-βCD e realizar caracterização físico-química;

- Avaliar o possível efeito anti-hiperalgésico do complexo αTPN-βCD;

₋ Investigar o envolvimento do sistema opióide e serotoninérgico na possível

ação do complexo αTPN-βCD;

- Analisar a possível interação entre o αTPN com os receptores MU, Kappa,

Delta e 5HT;

₋ Verificar possíveis alterações motoras na força muscular induzida pela

administração do complexo αTPN-βCD.

19

DESENVOLVIMENTO

20

3.1 Capítulo 1

Cyclodextrins: improving the therapeutic response of analgesic drugs: a patent

review.

Artigo publicado em Expert Opinion On Therapeutic Patents (Fator de Impacto –

JCR 2014: 4,297)

Makson GB de Oliveira, Adriana G Guimarães, Adriano AS Araujo, Jullyana SS

Quintans, Márcio RV Santos & Lucindo J Quintans-Júnior†

†Federal University of Sergipe, Department of Physiology, Avenue Marechal Rondom,

São Cristóvão, Sergipe, Brazil

21

Abstract

Introduction: Cyclodextrins (CDs) are cyclic oligosaccharides which have recently

been recognized as useful tools for optimizing the delivery of such problematic drugs.

CDs can be found in at least 35 pharmaceutical products, such as anticancer agents,

analgesic and anti-inflammatory drugs. Besides, several studies have demonstrated that

CD-complexed drugs could provide benefits in solubility, stability and also improve

pharmacological response when compared with the drug alone.

Areas covered: The patent quest was conducted in the databases WIPO, Espacenet,

USPTO, Derwent and INPI, using the keywords: cyclodextrin, pain and its related terms

(analgesia, hyperalgesia, hypernociception, nociception, antinociception,

antinociceptive). We found 442 patents. Criteria as the complexation of analgesic agents

and evidence of improvement of the therapeutic effect were indispensable for the

inclusion of the patent. So, 18 patents were selected.

Expert opinion: We noticed that some patents are related to the complexation of

opioids, nonsteroidal anti-inflammatory, as well as natural products, in different types

of CDs. The use of CDs creates the prospect of developing new therapeutic options for

the most effective treatment of painful conditions, allowing a reduction of dosage of

analgesic drugs and the occurrence of side effects. Thus, CDs can be an important tool

to improve the efficacy and pharmacological profile of analgesic drugs.

Keywords: Analgesia, cyclodextrin, drugs, nociception, pain, inflammation.

22

Article highlights.

Cyclodextrins (CDs) are cyclic oligosaccharides that have been used as vehicles of a

wide variety of hydrophobic guest molecules.

Analgesic drugs have been widely used to control acute and chronic pain. However,

there are side effects related to high doses that may limit their use.

The incorporation of analgesic drugs with the cyclodextrin molecules increases the

solubility of the analgesic, improves bioavailability and may reduce side effects.

The pharmaceutical industry adds β-cyclodextrin and its synthetic derivative,

hydroxypropyl-β-cyclodextrin, in the formulations to improve the therapeutic properties

of the different analgesic compounds such as opioid and non-steroidal anti-

inflammatories.

The use of cyclodextrins is a useful tool to enhance the pharmacological effect of

analgesic drugs and also to reduce side effects.

23

LIST OF ABBREVIATIONS

A61K: Preparations for medical purposes, dental or hygienic

A61P: Therapeutic activity specific chemicals or preparations medical

BR: Brazil

C07C: Acyclic or carbocyclic compounds

C07D: Heterocyclic compounds

C08B: Polysaccharides; derivatives thereof

CDs: Cyclodextrins

Derwent: Derwent Innovations Index®

DK: Denmark

ED50: Effective dose 50%

Espacenet: European Patent office

FDA: Food and Drug Administration

FR: France

GRAS: Generally Recognized as Safe of FDA

HP: Hydroxypropyl

HPLC: High-performance liquid chromatography

HP-β-CD: Hydroxypropyl-β-CD

IN: India

INPI: Instituto Nacional de Propriedade Industrial

IT: Italy

KR: Republic of Korea

Me: Methyl

MX: Mexico

NSAIDs: Nonsteroidal Anti-Inflammatory Drugs

24

SBE: Sulfobutylether

SI: Slovenia

TMX: Tomoxiprole

US: United States of America

USPTO: United States Patent and Trademark Office

WIPO: World Intellectual Property Organization

α-CD: Alpha cyclodextrin

β-CD: Beta cyclodextrin

γ-CD: Gamma cyclodextrin

25

1. Introduction

Cyclodextrins (CDs) are oligomers synthesized from the union of glucose

monomers (glucopyranose), by making part of the family of closed-chain

oligosaccharides [1]. Depending on the number of glucopyranose units, the natural

occurrence of CDs can be classified as alpha (6 glucose units), beta (7 glucose units)

and gamma (8 glucose units) CD [2]. Changes in the molecule of CDs originate

derivatives with new features. This has enabled the use of CDs by the pharmaceutical

industry as an excipient for the formulation of drugs, being the hydroxypropyl (HP),

methyl (M) and sulfobutylether (SBE) the derivatives that excel at such a purpose [3].

CD cyclic structure has a cavity and host hydrophobic molecules forming

inclusion complexes. Therefore, in aqueous solution, CDs in the form of a truncated

cone, by arrangement of the glucose unit, keep one or two molecules saved. This is

because the accommodation of the CH2 groups within the cavity, conferring

hydrophobic characteristics, since on the surface, the presence of hydroxyl groups

imparts hydrophilic characteristics [4, 5].

Therefore, molecules hosted by the CDs can have their physico-chemical

characteristics modified, enhancing the stability against environmental agents,

controlling the volatility and sublimation properties, masking potentially adverse flavors

and odors, increasing solubility in body fluids and also allowing the physical isolation

of incompatible compounds. These excipients may modulate the delivery speed of

encapsulated compounds (i.e., prolonging or delaying it), improve the amount of

bioavailable drug through the stabilization of the drug molecules on the surface of the

membrane and reduce the dose required to produce a biological effect and prolong the

validity of the formulation. In addition, CDs also reduce the toxicological / irritant

26

effects of the active agents. All these benefits of using CDs have recently been widely

reported in several reviews [1–7].

The cyclodextrins discovery took more than 100 years, but they were rapidy

accepted as pharmaceutical excipients, with monographs available in both the US

Pharmacopoeia/National Formulary and the European Pharmacopoeia, besides being

described in the Handbook of Pharmaceutical Excipients [4]. Use of CDs in the

pharmaceutical formulation development is extremely attractive due to their low

toxicity and low immunogenicity, as recently reviewed [6, 8,9].

Some studies were conducted with cyclodextrins aiming evaluate your toxicity

and no significant results was found [4]. Although some CDs can be found in the

market as parenteral formulations, the administration of α-CD, β-CD and some of their

synthetic derivatives by this route can result in renal toxicity; such use is restricted to

very low concentrations or contra-indicated. Another good characteristics of CDs are

your high biocompatibily and your use approval by FDA, which make them a good drug

delivery system for humans [7].

Another great advantage of CDs is the high versatility of pharmaceutical forms

that can be employed. Liquid, semi-solid and solid formulations using CDs can be used

by oral, sublingual, nasal, pulmonary, ocular, rectal and dermal delivery, as well as

parenteral [1,2]. Another contributing factor to the use of CDs in pharmaceutical

industry is the high availability of these excipients, since their price and production

costs have declined in recent years [8].

Besides, CDs can complex with large group of molecules from straight or branch

aliphatic chains to polar compounds [10], and ranging from small molecules, ions,

proteins, oligonucleotides and natural products [2,7]. Analgesics, nonsteroidal anti-

inflammatories, antibacterial, anticancer, antipsychotic and antifungal are some

27

pharmacological classes of drugs that have been marketed in preparation with CDs

which are available worldwide [1,5].

The search for new medicines for pain control has increased significantly over

the past 50 years. However, even increasing our knowledge of the mechanisms of pain,

drugs with analgesic activity still have a limited improvement. That can be confirmed

by the high load adverse effect and relative inefficiency of the majority of analgesics,

which in some cases do not control the pain in users [11,12]. Thus, Woolf (2010) [11],

by critically assessing the development path of new analgesics, suggests changes as

increasing innovation, specificity, delivery and targeting for the generation of more

effective and safer analgesic drugs.

In this review, the advancing in technological development of therapeutical

patents targeting the employment of the inclusion complex of cyclodextrin(s) and

analgesic drugs for a more effective pain relief is demonstrated. The specialized

databases like DERWENT, ESPACENET, INPI, USPTO and WIPO were used for

patent research in June 2014, using the term cyclodextrin (s) combined with pain,

nociception and analgesia. As the aim of this review was to demonstrate the

pharmacological advantage of CDs associated with analgesic drugs, only patents that

performed in vivo or in vitro studies demonstrating the benefit of the complexation with

CDs were included in this review. Thus, eighteen patents were selected, out of which

seven reported the β-cyclodextrin use and eleven demonstrated the effect of its synthetic

derivative, hydroxypropyl-β-cyclodextrin, on the improvement of the pharmacological

effects of different analgesic compounds.

2. β-Cyclodextrin

Pharmaceutical preparations containing CDs are available worldwide, and β-

form (FIGURE 1) [13] is the most used to encapsulate drugs. This fact occurs at low

28

value of β-Cyclodextrin (β-CD), where more than 10,000 tons are produced per year,

with an average price around 5 US$ per kg [5,7,8,14 ]. Besides, β-CD presents an ideal

cavity diameter to host drugs with molecular weight between 200 and 800 g/mol, acting

also as a flavor carrier and protectant, standing on the GRAS list since 1998 [15]. The

formation of microencapsulation by β-CD and the drug form a molecular complex

between the two entities, united by non-covalent binding. That interaction occurs both

in aqueous solution and in solid phase [16].

INSERT FIGURE 1

This section of the review reports the use of β-CD in the complex described in 7

patents (Table 1). The first patent registering the pharmacological benefits of the

complexation of analgesics compounds in CDs dates back to 1985. Chiesi and Servadio

(1985) [17] deposited a patent (publication number EP 0153998) use of β-CD molecules

to host piroxicam. The complex (10 mg/kg) was tested in a preclinical study to prove

the benefits of the complexation in solution form. Rodents were treated orally and

submitted to two animal models: abdominal writhing induced by phenylquinone and

paw edema induced by carrageenan. The results demonstrated a better pharmacological

effect in the complexed piroxicam compared to piroxicam-free β-CD. Also in this

patent, a bioavailability study with rabbits and dogs confirmed a higher plasma

concentration of piroxicam for the formulation using β-CD.

INSERT TABLE 1

29

In 1986, Chiesi Farmaceutici SpA [18], the same proprietary industry of

previous patents, registered a new patent with piroxicam complexed with β-CD. The

innovation occurred in new types of formulation, now tablets, effervescent tablets, bags

and suppositories, with 20 mg of NSAID in 191.2 mg of β-CD. These formulations

were also evaluated using the abdominal writhing test induced by phenylquinone.

Effervescent tablets yielded a faster rate of absorption. The gain through the use of β-

CD was increased bioavailability by increasing the analgesic and anti-inflammatory

activity and reducing side effects.

Ibuproxam was also used to form complexes with β-CD in 1995 by Zmitek et al

[19]. Suspensions of complexed ibuproxam and β-CD-free (100 mg/kg, p.o.) were

evaluated in a preclinical study through writhing test induced by the

phenylbenzoquinone. Best effects were achieved by the inclusion complexes. ED50

(effective dose 50%), calculated based on the concentration of the drug, was three times

smaller to ibuproxam-β-CD than the ibuproxam alone. In this case, β-CD formulations

improved the pharmacological effect with a reduced dose of the drug.

The pharmacological properties of opioid drugs can also be improved by the

association with β-CD. The patent EP 1174152 [20] reports a brief description of the

incorporation of L-Tyrosyl-D-alanyl-glycyl-N-methyphenylalanyl-glycyl-

isopropylamide in β-CD, obtaining a liquid formulation, tablets and transdermal tape

with just over 10 mg/kg of the analgesic drug. Dwivedi et al. have shown the benefits of

the complexation on tail-flick test in rats treated orally or subcutaneously, enhancing the

analgesic effect of the drug by the presence of β-CD in the formulation. Improved anti-

inflammatory effect was also achieved when the formulation was evaluated in the paw

edema induced by carrageenan in rats.

30

Quintans-Junior et al. (2013) [21] demonstrated the benefit of the complexation

of natural products in β-CD. This patent registered the improvement of analgesic effect

of the essential oil from the leaf of Ocimum basilicum complexed with β-CD. Complex

was initially tested in rodents that received administrations of acid saline (pH 4.0) in the

gastrocnemius muscle (a fibromyalgia animal model), unilaterally, producing diffuse

bilateral long-lasting hyperalgesia, motor deficits with no significant or tissue damage.

The animals were evaluated as mechanical hyperalgesia, motor performance and muscle

strength during 27 days. All doses of complex (25, 50 or 100 mg/kg, p.o.) significantly

improved the analgesic effect of the essential oil when comparing the use thereof alone.

That same year, Quintans-Junior et al. (2013) [22] placed a new patent in INPI

reporting now the benefit of the essential oil of Lippia grata with β-CD on orofacial

pain. The improvement of analgesic and anti-inflammatory properties of the essential oil

were confirmed through orofacial pain induced by different nociceptive agents. Mice

were pretreated orally with the essential oil from the leaf of L. grata complexed to β-CD

in doses of 25, 50 or 100 mg/kg and after received formalin, glutamate or capsaicin

(subcutaneously) as pain induction agent in the right upper lip (paranasal region). The

inventors reported that the complex essential oil with β-CD could be a potential

formulation to treat diseases related to the central nervous system, pain and

inflammation (acute or chronic).

Secondary metabolites extracted from the essential oils of plants, such as

monoterpenes, are also complexed with β-CD to improve pharmacological properties.

()-Linalool, with analgesic activity already described, was complexed to β-CD and

tested (25, 50 or 100 mg/kg, by gavage) in a preclinical study on mechanical

hyperalgesia protocols installed by the injection of double of acid saline (pH 4.0) in the

gastrocnemius muscle. Analgesic profile of ()-linalool complexed with β-CD was

31

better when compared to ()-linalool alone, showing the benefit of using CD as a host

molecule (Quintans-Junior et al. 2013) [23]. Inventors report that this invent may be a

formulation used for the treatment of chronic painful conditions, such as fibromyalgia.

3. Hydroxypropyl-β-cyclodextrin

Although β-CD is rather used in the pharmaceutical industry for the

encapsulation of drugs, this excipient presents some drawbacks, as relatively poor

aqueous solubility and consequently parenteral administration contra-indicated. Thus,

natural CD derivatives, such as hydroxypropyl-β-CD (HP-β-CD), were developed

through the substitution of multiple β-CD hydroxyls on both rims of the molecule with

crystallinity reduction, resulting in a notably improved aqueous solubility and lower

toxicological profiles in comparison to their parent CDs [14], consisting of 7 cyclo-α-

(1,4)-anhydroglucose units with hydroxypropyl groups [24]. These features make the

HP-β-CD one of the CDs derivatives mostly used in the pharmaceutical industry,

especially in the development of formulations for oral and parenteral application [8].

Here, we reported eleven patents about the HP-β-CD use in the improvement of

pharmacological effect of analgesics (Table 2). Cohen and Dubois (1991) [25] were the

first to register a patent in employing the HP-β-CD in pharmaceutical formulations

containing tiaprofenic acid, a non-selective cyclo-oxygenase enzyme inhibitor. Sachets

(50 mg of complexed drug) and tablets (100 mg drug alone) were produced and both

formulations were tested in 12 volunteers treated orally, in one randomized clinical

study (Phase I). Pharmacokinetic parameters were evaluated through blood samples to

compare the bioavailability of the formulations developed. Biochemical dosage

demonstrated higher plasma peaks of tiaprofenic acid complexed to the formulation

with less time to achieve higher blood concentration. Thus, the optimization of the

32

analgesic and anti-rheumatic effects of this non-steroidal anti-inflammatory drug

(NSAID) complexed in HP-β-CD was demonstrated.

INSERT TABLE 2

In order to enhance the analgesic effect of opioid drugs, Yaksh et al. (1992) [26]

have developed formulations using hydroxypropyl-β-cyclodextrin to incorporate this

drug class. The inclusion complexes were obtained for morphine, sufentanil, alfentanil

and lofentanil, which were administered by intrathecal route; according to the inventors,

the best way to use the drugs by this route occurs from complexed form rather than the

free formulation of HP-β-CD. A preclinical study in rodents tested formulations at

concentrations of 3 and 0.1 μg/ml in the hot-plate test (a central analgesic animal

model). The beneficial complexation occurred by increasing the potency of drugs

reducing the ED50 and also optimizing the duration of the effect on pain control. In this

same patent, the inventors complexed Alfentanil (400 μg/ml) with HP-β-CD 2% and

evaluated the pharmacological activity in dogs in the protocol of thermal nociception

(skin twitch), which confirmed the prolonged effect of the complexation.

Three years later, a new patent was filed at the World Intellectual Property

Organization also using opioid analgesics complexed in HP-β-CD. The complexes

containing different concentrations of morphine (0.02, 0.2 and 2%) were dissolved in

water and administered intramuscularly or subcutaneously in rabbits, an experimental

trial using electrical stimulation to induce nociception. Thus, Borgbjerg (1995) [27]

developed a systemic treatment for pain control with higher duration of analgesic

actions and minor side effects.

33

Kim et al. 2003 [28] used the method of forming complexes with HP-β-CD to

host molecular 1-(4-t-Butylbenzyl)-3-(3-fluoro-4-methanesulfonylaminobenzyl)

thiourea, a modulator for vanilloid receptors. Various formulations have been

developed: tablet, liquid formulation, injection formulation and transdermal gel.

However, only the liquid formulation was used for the evaluations of pharmacokinetic

parameters, demonstrating the benefit of the complexation. A preclinical study was

conducted with rats treated orally with 10 mg/kg of analgesics associated or free of HP-

β-CD drug, and blood samples were collected and evaluated in HPLC to quantify the

plasma concentrations of the drug. As expected, the addition of HP-β-CD increased

about three times the bioavailability of the drug, enabling an increase in its analgesic

effect when purchased using the free thiourea of HP-β-CD.

A clinical study with healthy volunteers was conducted by Milano et al. (2005)

[29] to confirm the best analgesic and anti-inflammatory effect of diclofenac sodium

after the complexation with HP-β-CD. A pharmaceutical formulation containing 75 mg

of NSAID complexed was administered to humans by subcutaneous or intramuscular

injection to demonstrate the benefit of the technique of inclusion of lipophilic drugs in

the cavity of cyclodextrin molecules. After 16 hours of treatment, blood samples were

collected from the participants and the dosage thereof showed a high concentration of

diclofenac sodium in plasma, suggesting improved anti-inflammatory and analgesic

effects of the drug.

Clinical study performed by Wright et al. (2009) [30], also confirmed the

improvement in the pharmacological effect of the complex of diclofenac sodium. Study

double-blind with 336 patients treated diclofenac sodium complexed with HP-β-CD

(3.75, 9.4, 18.75, 37.5 or 75 mg) by single intravenous administration of 2 ml bolus

injection. Patients used a visual analog scale to express the pain intensity after treatment

34

at different times. Significant differences were found for pain intensity expressed in

patients treated with the drug complexed, compared with patients treated with

diclofenac sodium-free HP-β-CD. Inventors observed that the complex improved the

solubility and stability of NSAID and increased effectiveness of the analgesic effects of

this drug. This invention was subsequently protected in the US (2011 0218247) [31], EP

(2012 2522344) [32] and US (2014 0107209) [33].

Diclofenac sodium was used again as the drug patent filed by Lacouture et al.

(2013) [34] to incorporate the HP-β-CD. DylojectTM

, at doses of 3.75 to 75 mg, was

tested in a clinical study of over 1,579 patients with moderate to severe acute pain

following orthopedic surgery, pelvic surgery, including the elderly, obese, and those

with cancer. The complexed drug was administered by intravenous bolus injection, and

showed an efficacy of 100% when compared to the control, thus reducing drug toxicity

and increasing efficiency by up to five times. The formulation with HP-β-CD was up to

50% more efficient than the clinically recommended doses.

Milanese et al. 2013 [35] showed that tomoxiprole (TMX), an imidazole

derivative of NSAIDs clinically used for pain, inflammation and arthritis, was

complexed with HP-β-CD and administered orally (5 mg/kg) in rats. It was

demonstrated that the TXM concentration reached its maximum in 90 minutes, while

the TXM incorporated with HP-β-CD took only 40 minutes to reach maximum

concentration, which in mg/L was 0.22 and 0.36, respectively. For these parameters,

blood sample were analyzed by HPLC (high-performance liquid chromatography)

(TXM in the dosing time of 0.3, 0.6, 1, 1.5, 2 and 6 hours after treatment). TXM

presented in the first hour twice the peak in plasma-complexed formulation, which

shows that the formulation improves the bioavailability of the complex.

35

4. Expert opinion

Through the initial patent search about the use of CDs in the pharmaceutical

formulations, the development of analgesic drugs was found in 442 patents. Repeated

patents were excluded, leaving 139 patents. Afterwards, 84 patents written in other

languages rather than English, Spanish or Portuguese were excluded, along with 33

patents that did not demonstrate the efficiency of the complexation by preclinical or

clinical protocols, and 4 patents that were not available. At the end, respecting the

criteria for inclusion and exclusion, 18 patents reporting the benefits of complexation

for analgesic drugs on CDs were included.

We observed a wide use of β-cyclodextrin (7 patents) and its synthetic

derivatives, hydroxypropyl-β-cyclodextrin (11 patents). In fact, CDs can now be found

in over 35 commercially available drug products [31] and β-form is the most used in

approximately 54.8% the marketed preparations of CDs worldwide, according to

Kurkov and Loftsson (2013) [8]. Among the synthetic CDs, HP-β-CD also has achieved

prominent role in the pharmaceutical industry [32]. However, over the years, there has

been an evolution in the technological development of analgesic formulations

containing HP-β-CD, whereas for β-CD a decline was observed between 1995 and

2005. This fact probably occurred because the full potential of the formulation power of

HP-β-CD is becoming apparent; it is applicable in dosage forms for all the main

administration routes, but it is of particular interest for injectable products with

improved efficiency [8].

Within the patents analyzed, solid pharmaceutical forms mostly used were

tablets and capsules. The most prominent liquid presentation was the solution of the

complexed drug, which was tested by the oral and parenteral routes, the latter especially

for preparations containing HP-β-CD. The oral route, among other routes, presents more

36

confort and better acceptance by the patient and the ease in administration. It is of easy

intake and a non-invasive route wherein the drug has greater stability [37]. The natural

CDs and their derivatives have been used in the pharmaceutical industry to improve the

plasmatic bioavailability of drugs. The benefit is the increase in solubility of a

significant number of drugs that express low aqueous solubility [5].

Low water solubility drugs marketed in injectable formulations have a need for

different excipient in the parenteral formulation, which is a barrier to use injections for

inflammation and pain, with a possibility of drug precipitation [38]. Under these

circumstances, parenteral formulations containing hydrophilic CDs derivatives, such as

2-hydroxypropyl-β-cyclodextrin, are increasingly being used in studies of new

pharmacologically active compounds, to be free of toxicity, unlike what happens with

organic solvents and surfactant formulations [1]. However, due to β-CD nephrotoxicity,

it cannot be used in parenteral formulations.

Pain can be divided in acute and chronic, being the duration of the second one

more than 3 months. The chronic pain can also be associated with long medication

exposure, which can increase some adverse events [39]. Thus, technological strategies

for the development of safer and effective drugs are still needed. Hence, CDs are an

interesting alternative to improve the therapeutic response of analgesics and adherence

to treatment of pain, since these excipients may enhance solubility and bioavailability of

poorly soluble drugs, using hydrophilic CDs as HP-β-CD, or still prolonging or

modifying the release of the drug through the hydrophobic CDs, such as ethylated and

acylated CDs [5].

The encapsulation efficiency of analgesic agents in β-CD or HP-β-CD was

demonstrated largely through preclinical studies of acute and chronic pain, as

fibromyalgia or cancer pain. Only formulations with HP-β-CD were administered in

37

humans, in clinical trials of phase I, II and III to study pharmacokinetic parameters and

pharmacological effects. However, on a cursory analysis of scientific papers published

on this subject, it is possible to realize a high interest in this field of research in the

various status of drug development, as demonstrated by Scarpignato (2013) [40],

Daniels et al. (2013) [41], Quintans et al. (2013) [42] and Quintans-Júnior et al. (2013)

[43].

In selected patents, the pharmaceutical class mostly used for the development of

inclusion complexes with CDs was NSAIDs (53.3%) and opioid analgesics (20.0%).

These drugs have been the mainstay of pain treatment for a very long time. Their

adverse effects and insufficient effectiveness in many types of pain were the main

driving forces in the development of new analgesics [12]. In fact, several formulations

in CDs complexed with painkillers are already marketed worldwide, such as piroxicam

(Brexin®), meloxicam (Mobitil

®), tiaprofenic acid (Surgamyl

®), nimesulide (Nimedex

®)

[5,8]. In the pharmaceutical industry, CDs have mainly been used as complexing agents

to increase aqueous solubility of poorly soluble drugs and to increase their

bioavailability and stability, and to reduce side effects as gastrointestinal irritation, as

well as to prevent drug-drug and drug-excipient interactions [1,3].

Diclofenac sodium administered orally and parenterally presents significant

toxicity, adverse cardiovascular, renal and hepatic effect, gastrointestinal discomfort and

allergy [30]. In this review, we found a patent family of diclofenac sodium

(DylojectTM

), which was evaluated in clinical trials of phase I, II and III. Complexation

of this NSAID in HP-β-CD increased plasma half-life, reduced side effects and

improved its pharmacologic efficiency. Even though this formulation developed by

Javelin Pharmaceuticals Inc. has not yet been approved by the FDA, there are several

38

evidence that HP-β-CD-diclofenac is safe and well tolerated following parenteral

administration [41,44,45].

Recently, naturally obtained compounds (20.0%), such as essential oil (13.3%)

and monoterpenes (6.6%), have also been subjected to CDs complexation. Furthermore,

these pure compounds represent interesting analgesic agents for the development of new

therapeutic options for pain control, as recently reviewed by Guimarães et al. (2013,

2014) [46,47], Quintans et al. (2014) [48] and Siqueira-Lima et al. (2014)

[49].Therefore, these therapeutic effects can be enhanced by CDs complexation of these

bioactive agents, as demonstrated in various studies and patents cited herein

[7,42,43,49,50].

From the above, it is noticed that the use of CDs in the formulation of analgesics

is an interesting alternative to the constant problems related to poor pain management.

The reduced number of patents shows that this is still a vast research field to be

explored, with great prospects to provide more effective and safe treatment to patients

with painful syndromes, ensuring them a better quality of life.

Acknowledgement

This work was supported by grants from FAPITEC-SE/Brazil and CNPq/Brazil. We

thank Abilio Borghi for the grammar review of the manuscript.

Declaration of interest

The authors report no conflict of interest and have received no payment in preparation

of this manuscript.

39

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46

FIGURE

Figure 1: Chemical structure of β-cyclodextrin and hydroxypropyl-β-cyclodextrin [13].

47

48

49

50

51

3.2 Capítulo 2

α-Terpineol, a monoterpene alcohol, complexed with β-cyclodextrin exerts antihyperalgesic

effect in animal model for fibromyalgia aided with docking study

Artigo submetido à Phytomedicine (Fator de Impacto-JCR 2014: 3,126)

Makson G.B. Oliveira1; Renan G. Brito

1; Priscila L. Santos

1; Heitor G. Araújo-Filho

1; Jullyana S.S.

Quintans1, Paula P. Menezes

2; Mairim R. Serafini

2, Yasmim M.B.G. Carvalho

2; Shanmugam

Saravanan2; Juliane C. Silva

3; Jackson R.G.S. Almeida

3; Luciana Scotti

4; Marcus T. Scotti

4;

Adriano A.S. Araújo2; Lucindo J. Quintans-Júnior

1*

1Department of Physiology,

2Department of Pharmacy. Federal University of Sergipe, Av. Tancredo

Neves, S/N, Rosa Elza, CEP: 49.000-100, São Cristóvão, Sergipe, CEP 49.100-000, Brazil;

3Department of Pharmacy, Federal University of San Francisco Valley, Petrolina, Pernambuco,

CEP 56.304-917, Brazil.

4Centre of Applied Sciences and Education, Federal University of Paraíba, Rio Tinto, Paraíba, CEP

58.297-000, Brazil.

*Author for correspondence: L.J. Quintans-Júnior, Department of Physiology, Laboratory of

Neuroscience and Pharmacological Assay. Federal University of Sergipe, Av. Marechal Rondon,

s/n, São Cristóvão, Sergipe, Brazil (fax +55-79-2105-6640, Email: lucindojr@gmail.com;

lucindo@pq.cnpq.br).

52

Abstract

The anti-hyperalgesic effect of the complex containing α-terpineol (αTPN) and β-cyclodextrin

(βCD) was analyzed in a non-inflammatory chronic muscle pain model, as well as its mechanism of

action through docking study for a possible interaction with receptors. The αTPN-βCD complex

was prepared and characterized through the thermogravimetry/derivate thermogravimetry

(TG/DTG), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope

(SEM). The model of chronic muscle pain was induced by two injections of pH 4.0 saline (20 μl)

into the left gastrocnemius 5 days apart. After confirming hyperalgesia, male mice were treated with

αTPN-βCD (25, 50 or 100 mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 10 days. 1 h after the

mechanical hyperalgesia, motor performance was evaluated. In addition, the systemic

administration of naloxone and ondansetron tested the analgesic effect on the active opioid and

serotonin receptors, respectively. The characterization tests indicated that αTPN was efficiently

incorporated into βCD. The oral treatment with αTPN-βCD, at all doses tested, produced a

significant (p<0.001) decrease in the mechanical hyperalgesia, without causing any alteration in the

force and in motor performance. This analgesic effect was reversed by the systemic administration

of naloxone or ondansetron. These findings are corroborated by the docking study described in the

present study, which verified a possible interaction of αTPN-βCD with opioid (MU, Kappa, Delta)

and 5-HT receptors. Thus, it can be concluded that αTPN-βCD reduced the hyperalgesia followed

by the chronic muscle pain model, probably evoked by the descending inhibitory pain system,

specifically by opioid and serotoninergic receptors.

Key-words: α-Terpineol, β-cyclodextrin, fibromyalgia, Chronic pain, pain.

53

Abbreviations

HPβCD - 2-hydroxypropyl-β-cyclodextrin

5-HT - Serotonine

CNS - Central Nervous System

CDs - Cyclodextrins

FDA - Food and Drug Administration

FM - Fibromyalgia

FTIR - Fourier transform infrared spectroscopy

NSAIDs - Nonsteroidal anti-inflammatory drug

SEM - Scanning electron microscope

TG/DTG - Thermogravimetry/derivate thermogravimetry

αTPN - α-terpineol

βCD - β-cyclodextrin

54

1. Introduction

Fibromyalgia (FM) is a chronic musculoskeletal disorder characterized by widespread pain

of neurogenic origin, caused by neurochemical imbalances in the central nervous system (CNS)

triggering the central amplification in the perception of pain characterized by allodynia and

hyperalgesia (Sluka et al., 2002). FM can be associated with other symptoms including persistent

fatigue, morning stiffness, sleep disorders and depression. (Gracely et al., 2002; Arnold et al., 2004;

Nebel et al., 2009). It is a persistent disorder that weakens the individual, affecting the quality of

life, interfering in the work, in daily activities and interpersonal relationships (Arnold et al., 2008).

Pharmacological treatment for FM includes medications that have a neuromodulator function like

antidepressants, uptake inhibitors of serotonin and serotonin/norepinephrine, central analgesics,

hypnotics, calcium channel modulators and muscle relaxants (Crofford and Appleton, 2001;

Sumpton and Moulin, 2008). In spite of that, the development of new drugs for the treatment of FM

or the management of the main symptoms remains as a challenge for the pharmaceutical área

(Nagakura, 2015).

In recent years, the search for new drugs for pain control has increased significantly.

However, although our knowledge of pain mechanisms has increased, drugs with analgesic activity

still suffer from limited improvement. A number of adverse effects and the relative inefficiency of

most analgesics, which in some cases do not control the pain of users, prove this limitation. Thus, it

is necessary to invest in the development of new drugs to control pain, especially chronic pain such

as “dysfunctional pain”, such as FM (Nagakura, 2015), with greater specificity and better

bioavailability, generating more effective and safer analgesic drugs (Arun et al., 2008; Woolf, 2010;

Nascimento et al., 2013; Quintans et al., 2014).

In this context, cyclodextrins (CDs) are used by the pharmaceutical industry as excipients to

increase the availability of analgesics, NSAIDs, antibacterial, anticancer, antifungal and

55

antipsychotic drugs presenting low price and production costs, as well as FDA approval due to the

high biocompatibility (Loftsson et al., 2005; Arima et al., 2011; Khan and Durakshan, 2013; Pinho

et al., 2014). CDs are formed from the union of glucose monomers (glucopyranose), part of the

family of closed chain oligosaccharides (Loftsson et al., 2005), and can complex with large group of

molecules from straight or branched aliphatic chains to polar compounds (Arun et al., 2008), and

ranging from small molecules, ions, proteins, oligonucleotides and natural products (Zhang and Ma,

2013; Pinho et al., 2014).

The search for new medicines based on natural products and/or their secondary metabolites

is gaining notoriety in the modulation of painful conditions, especially when they are associated

with the CDs that improve their analgesic effects and increase plasma availability with possible

dose reduction (Li and Vederas, 2009; Guimarães et al., 2014; 2015; Menezes et al., 2015; Santos et

al., 2015; Nascimento et al., 2015; Oliveira et al., 2015; Brito et al., 2015). In this case, we highlight

the use of monoterpenes, a chemical entity in the essential oils with significant pharmacological

activity in controlling pain, which has added enough scientific value (Quintans et al., 2013;

Nascimento et al., 2014; Oliveira et al., 2015).

α-Terpineol (αTPN) is a volatile monoterpene alcohol, a major component of the essential

oils of various plant species, such as Ravensara aromatica (“Ravensara”), Melaleuca qinquenervia

(“Niaouli”), Croton sonderianus (“Marmeleiro black”, in northeastern Brazil) and Eucalyptus

globulus (“Eucalyptus”), which are widely used in folk medicine and aromatherapy, also used in

perfumery, in cosmetic industries and in soap and household products (Craveiro et al., 1981;

Franchome et al., 1995; De Sousa, 2011). Several studies have reported its antimicrobial,

antispasmodic, anticonvulsant and antinociceptive immunostimulant properties, in addition to

increasing the permeability of the skin to soluble compounds also acting in mechanisms of

inhibition of NF-κB (Williams & Barry 1991; Franchome et al., 1995; Lee et al., 1997; De Sousa et

al., 2007; Hassan et al., 2010). The anti-inflammatory and antinociceptive central effects of this

56

compound have been described recently and are suggested to be possibly used in chronic pain, such

as FM (De Sousa et al, 2007; Quintans-Júnior et al., 2011; Oliveira et al., 2012).

The monoterpenes in general have lipophilic characteristics that can pose challenges to their

biological administration (Pinho et al., 2014). Furthermore, CDs have the ability to host this type of

non-polar compound, protecting against oxidation, heat and light degradation, evaporation and

moisture, and turning liquid monoterpenes into water-dispersible and easy-to-handle powders

(Marreto et al., 2008; Menezes et al., 2012). The complexation of lipophilic compounds with CDs

has improved the analgesic effects of them (Quintans et al., 2013; Oliveira et al., 2015; Brito et al.,

2015). Thus, we evaluated the antihyperalgesic effect of αTPN complexed with βCD on animal

models for non-inflammatory muscle pain, considered to be an animal model of FM, also evaluating

the mechanism of action through docking study for a possible interaction with receptors.

Material and Methods

Chemicals

α-Terpineol (≥96% purity), Ondansetron, Naloxone and β-cyclodextrin (98% purity) were

purchased from Sigma (USA). Tramadol, in free form without additives, was purchased from

Teuto/Pfizer (Anápolis-GO, Brazil, lot 00710/2012). Ketamin and Xylazin were purchased from

Cristália (Itabira-SP, Brazil).

Preparation of the inclusion complex

The samples were obtained through slurry complex (SC) and compared with physical

mixture (PM) (Menezes et al, 2012.). 154 mg of αTPN was added to 1.135 mg of βCD followed by

mechanical mixture (PM). The SC was obtained by adding 20 ml of distilled water containing 1.135

mg of βCD to 154 mg (αTPN) with subsequent magnetic stirring for a 36-hour step. All the samples

were subjected to drying in a desiccator glass.

57

Characterization of the inclusion complex

Thermogravimetry/derivative thermogravimetry (TG/DTG)

TG/DTG curves were obtained with a TGA 60 (Shimadzu) thermobalance in the

temperature range of 25-900°C, performed with platinum crucibles containing approximately 3 mg

of the samples, under dynamic nitrogen atmosphere (100 mL.min-1

) and heating rate of 10°C.min-1

.

The TG/DTG was calibrated with calcium oxalate monohydrate, according to the ASTM standard.

Titration of Karl Fisher

The moisture contents of the physical mixture and slurry complexes were determined

through the Karl Fischer Titrino Plus KF 870 method (Metrohm) and methanol (Fluka) as titrating

solution. The analyses were carried out in triplicate.

Fourier transform infrared spectroscopy

Infrared spectra of αTPN, βCD and the inclusion complex were recorded on an IRTracer-

100 (Shimadzu) Fourier Transform Infrared Spectrophotometer, at room temperature. Spectrum

infrared absorption was obtained in the range of 4000–500 cm−1

in KBr pellets.

Scanning electron microscopy (SEM)

The dried products were mounted and visualized with a JEOL JSM-7410-F model scanning

electron microscope, at an accelerated voltage of 1 kV.

Animals

Experimental protocols were performed using male Swiss mice (25-30g) obtained from the

Animal Facilities of the Federal University of Sergipe (UFS). Mice were housed in controlled-

temperature rooms (22-25°C), under a 12/12 h light-dark cycle, with access to water and food ad

libitum until use. All behavioral protocols were performed between 8:00 a.m. and 2:00 p.m.

58

Experimental protocols were approved by the Animal Care and Use Committee at UFS/Brazil

(08/11). All behavior experiments were performed with the examiner blinded to the group. All

effort was sought to minimize the number of mice used and any discomfort.

Acid saline-induced chronic muscle pain

The experimental procedure to induce chronic widespread non-inflammatory muscle pain

(an animal model for Fibromyalgia) followed that described by Sluka et al. (2001) and reviewed by

DeSantana et al. (2013). Mice were anesthetized with Ketamine (80 mg/kg) and Xylazine (10

mg/kg); 20 μL of saline (pH 4.0) was injected in the left gastrocnemius muscle. This procedure was

performed again 5 days after the first injection. This model produces a long-lasting bilateral

mechanical hyperalgesia lasting for 4 weeks after the second injection as described by Sluka et al.

(2001).

Behavior Testing

Paw sensitivity to mechanical stimulation

Mechanical hyperalgesia was tested in mice as reported by Cunha et al. (2004), with

adaptations by Guimarães et al. (2012). This method consisted of evoking a hind paw flexion reflex

with a hand-held force transducer (electronic analgesimeter; Model EFF 301, Insight®, Ribeirão

Preto-SP, Brazil) adapted with a polypropylene tip. The investigator was trained to apply the tip

perpendicularly to the central area of the hind paw with a gradual increase in pressure. The end

point was characterized by the withdrawal of the paw followed by clear flinching movements. The

intensity of the stimulus was obtained by averaging five measurements taken with minimal intervals

of three minutes.

Assessment of the motor performance

59

To assess whether the treatments with αTPN-βCD could harm the motor coordination

activity, mice (n=8, per group) were pretreated as described above and were evaluated through the

grip strength meter (Insight, Brazil) and the rota-rod apparatus (AVS®, Brazil). The vehicle and

experiment group were treated 24 hours before the test and the positive control group 30 minutes

before.

To perform the rota-rod test, mice were selected 24 h previously by eliminating those mice

which did not remain on the bar for two consecutive periods of 180 s. All mice received the same

pharmacological treatment as previously described in chronic muscle pain induced by acidic saline.

The latency to falling was measured up to 180 s. The results are expressed as the average time(s)

the animals remained on the rota-rod in each group (De Santana and Sluka, 2008).

The hindpaw grip strength was tested before and at the end of the experiment as previously

described (Burnes et al., 2008). The mice were familiarized to the apparatus twice a day for 2 days

by performing the grip force task. Mice were pulled by the tail for grip strength of the hindpaw to

be read. An average of three trials was recorded. The results are expressed as the average force

(mN) in each group.

Pharmacological treatments

After confirming hyperalgesia, mice (n=8, per group) were pretreated with αTPN-βCD (25,

50 or 100 mg/kg; per os, p.o.), tramadol (TRM, an opioid pain drug; 4 mg/kg; i.p.) or vehicle (saline

0.9%, p.o.). αTPN-βCD and vehicle were treated daily for 10 days. The mechanical hyperalgesia

was tested daily; the rota rod and grip meter strength were evaluated day in and day out. To analyze

the time effect, the animals were treated once and were evaluated through von frey digital until the

drug had no effect.

Antagonism assessment

60

To assess the possible involvement of the opioid and serotonergic systems, different groups

of animals (n=8, per group) were pretreated, after the pain induction with formalin (1%; 20 μL;

i.pl.) (Hunskaar and Hole, 1987), with αTPN-βCD (p.o.) or vehicle (p.o.), and on the day 2 they

received an injection of naloxone (an opioid antagonist, 5 mg/kg; i.p.) and ondansetron (an 5HT3

antagonist, 0.5 mg/kg; i.p.). The formalin test was chosen because it assesses the pharmacological

antagonism due to the facility to be run and also because it has two phases: neurogenic and

inflammatory (Le Bars et al., 2001).

Docking Study

Molecular modeling: Using the program Hyperchem v. 8.0, the chemical structures of the

Αtpn were drawn, and the geometry was optimized using MM+ force field (Allinger, 1977).

Afterwards, we performed a new geometry optimization based on the semi-empirical method AM1

(Austin Model 1) (Dewar et al., 1985). The optimized structure was subjected to conformational

analysis, and the least energy conformer (EMIN) was saved.

Docking: The αTPN was submitted to docking with the receptors: 5-HT (ID PDB 4IAQ), KAPPA

(ID PDB 4EA3), DELTA (ID PDB 4EJ4) and MU (ID PDB 4DKL). Through the Molegro Virtual

Docker 6.0 program, we created a template with the already-complexed ligands, for the GRID. We

selected the MolDock SE algorithm (Thomsen and Christensen, 2006), with 10 runs for each ligand.

Statistical analysis

Values are expressed as mean ± SEM. The data obtained were evaluated through the oneway

analysis of variance (ANOVA) followed by Tukey‟s test. In all cases, differences were considered

significant if p<0.05. All statistical analyses were performed using the software GraphPad Prism 5.0

(GraphPad Prism Software Inc., San Diego, CA, USA).

Results

61

The TG curve of αTPN showed a step weight loss between 31 and 169ºC (Δm = 98.9%)

characteristic of the volatilization of monoterpene according to the results shown in Table 1 and

Figure 1.

<INSERT TABLE 1 AND FIGURE 1>

Regarding the βCD of the curve, a first step of mass loss was observed, which was attributed

to dehydration molecule with %H2O=13.75% ± 0.39. Subsequent steps are then defined as

decomposition and degradation of the molecule. In the PM curve, the first step had Δm=20.07%,

this value is associated with the release of water (%H2O=12.56 ± 1.12%) and surface oil. By

analyzing the second step, it was observed that the PM showed mass loss similar to αTPN and βCD

frees. On the other hand, the SC method in the second step showed Δm = 5.16%. Thus, it was

possible to infer that there was complexation by the SC method, since the mass loss percentage was

higher compared with other samples. Furthermore, in the first step the weight loss value (12.80%)

was corroborated with Karl Fisher´s titration technique, in which the sample moisture contente

(%H2O) was 11.71 ± 0.64%, showing that there barely was adsorbed oil on the surface.

Figure 2 shows the infrared spectra of pure αTPN, βCD, PM and SC. In all bands related to

αTPN, it was possible to observe in the region of 3488-3270 cm-1

related to a broadband

connection, OH stretch which are characteristic of the compound and which are also present in the

spectrum of βCD. From 1660-1120 cm-1

, it was described by stretching band C=C, which

characterizes the monoterpene. By observing the spectrum of the PM and SC, it was observed in the

extension band that it represents the alkene group indicating the presence of αTPN in βCD

complexation and, therefore, demonstrating this method.

62

<INSERT FIGURE 2>

In the figure 3, the SEM images are described, showing the surface of the samples. In

addition, the SEM results corroborate with previous studies showing that SC was the best method,

since it exhibited morphological change of the sample, not maintaining the rectangular shape of the

βCD crystal, as noted in the PM.

<INSERT FIGURE 3>

The αTPN-βCD had a lasting effect when compared with the free compound (αTPN

isolated) (Figure 4A). Additionally, the figure 4B demonstrates that αTPN-βCD, in all doses,

caused a significant inhibition of the mechanical hyperalgesia (p<0.01) when compared to the

control group.

<INSERT FIGURE 4>

Through the rota-rod and grip strength meter, it is possible to see that the pretreatment with

αTPN-βCD did not cause any alteration in the motor performance and force (Figures 5A and 5B).

<INSERT FIGURE 5>

63

The intraperitoneally naloxone and ondansetorn injection blocked the anti-hyperalgesic

effect of the αTPN-βCD (Figure 6A and 6B), confirming the involvement of the opioid and 5-HT

systems.

<INSERT FIGURE 6>

In docking study for 5HT receptor, the energy obtained was -66000 Kcal/mol. Two

hydrogen bonds with ASP129 and CYS133 were observed: sterics with the residues LLE130 and

PHE330 (Figure 7A). For Delta receptor, the energy calculated was -33400 Kcal/mol and only one

hydrogen bond with ASP128 (Figure 7B); for kappa, the energy obtained was -57800kcal/mol and

two hydrogen bonds were observed with LEU67 and VAL63 of the chain B: steric with VAL55 of

the chain A (Figure 7C); and for MU receptor, the energy was -37900 kcal/mol. The interaction

observed was a hydrogen bond with the residue ASP147 (Figure 7D).

<INSERT FIGURE 7>

Discussion

Musculoskeletal pain syndromes, such as fibromyalgia and myofascial pain, have affected

between 11-24% of the global population, resulting in high economic costs in order of $ 500 billion

per year in the US alone, which it is due to the difficulty of treating pain in these syndromes.

64

Therefore, the management of chronic pain remains a challenge to modern medicine (McCain,

1994; Cimmino et al., 2011; Kissin, 2010; Fallon, 2013, Gregory and Sluka, 2014).

Recently, it has been suggested that the use of cyclodextrins (CDs) can improve the efficacy

of analgesic compounds, and they are an important tool in the search for greater analgesic effect

(Brito et al. 2015; Oliveira et al, 2015). Hence, we carried the complexation and characterization of

αTPN into βCD and evaluated if the complexed drug had a better analgesic effect than did the free

drug.

TG/DTG is a thermal analysis technique used to evaluate the weight loss of the sample

controlled by temperature programming. It is also very used to characterize the inclusion complex

formation (Ionashiro et al., 2014). It is important to note that TG cannot distinguish between αTPN

and water mass losses from mechanical mixture or inclusion complexes. Thus, a volumetric water

determination method (Karl Fisher) is very used for the determination of water because this

technique is more accurate than is TG due to the fact that it evaluates only water concentration.

Furthermore, the “surface” and “strong-bonded” water molecules can be determined (Menezes et

al., 2012). Therefore, the water content of cyclodextrin complexes could be an indirect parameter

which indicates the complexation success according to Hădărugă et al. (2012). According to

Hădărugă et al. 2012, classical Karl Fischer water titration is a good tool for the evaluation of water

concentration in cyclodextrins and their micro/nanoparticles used in food industry. Karl Fischer

results are in good agreement with the thermogravimetric results (Hădărugă; Hădărugă; Isengard,

2012). Our results demonstrated a decreased of water content between physical mixture

(12.56±1.12%) and slurry (11.71±0.64%). According to Serafini et al (2011) and Galvão et al.

(2015), this decrease is due to the complex formation, since water molecules originally found in the

cavity of βCD were replaced by guest molecules. Thus, both the TG/DTG curves and Karl titration

suggest complexation of αTPN inside the βCD cavity through the slurry method. Futhermore, in the

second step of TG/DTG curves, a greater amount of αTPN complexed was released between 169-

282ºC (5.16%). Valle (2004) reported that both the stirring and the major amount of water in the

65

complexation process lead to the replacement of water molecules from the cyclodextrin cavity for

molecules of the drug intended to be complexed.

In the infrared, the physical mixture and slurry spectrum broadening was observed in the

band that represents the alkene group indicating the presence of the αTPN inside the βCD

complexation, therefore demonstrating this method. In addition, the SEM results corroborate with

previous showing that slurry was the best method, since it showed changes in shape, not keeping

the βCD rectangular crystal, as observed in the physical mixture. Similar results were obtained by

(Dos Santos; Buera; Mazzobre, 2011), who studied the complexation of αTPN with βCD through

the co-precipitation method and 2-hydroxypropyl-β-cyclodextrin (HPβCD) using freeze-drying

method. The results confirm the complexation of αTPN both in HβCD and βCD.

In the time effect test, it was observed that the αTPN-βCD has a better analgesic profile than

does the free drug, confirming that the complexation was capable to improve the αTPN activity. In

fact, it has been suggested that CDs may increase the bioavailability and the pharmacological

efficacy of liposoluble compounds (Loftsson et al., 2015), including analgesic drugs (Brito et al.,

2015).

The acid saline induced-chronic muscle pain model produces a bilateral and widespread

hypersensitivity, being initiated and maintained by the processes within the CNS. In this model,

there is a significant decrease in mechanical withdrawal threshold of the paw, being interpreted as

secondary hyperalgesia (Sluka et al., 2001; Da Silva et al., 2010). We demonstrated that the

pretreatment with αTPN-βCD was able to increase the threshold sensitivity of mechanical

hyperalgesia.

The antagonism experiments demonstrated that the treatment with naloxone, a nonselective

opioid antagonist, blocked the anti-hyperalgesic effect of the αTPN-βCD, and ondansetron, na

antagonist of 5HT3 receptor, also antagonized this effect. Thus, the analgesic effect of αTPN-βCD

seems to involve the opioid and serotoninergic systems. The involvement of the opiod system was

also observed above by Oliveira et al. (2011), who demonstrated that the analgesic effect of αTPN

66

was antagonized by naloxone in the hot-plate test. These findings were corroborated by docking

study, which verified a possible interaction of αTPN-βCD with opioid (MU, Kappa, Delta) and 5-

HT receptors.

In this analgesic process, a number of neurotransmitters, neuromodulators and receptors are

involved in pain modulation, being the serotonin, noradrenaline and opioids the key mediators

involved in the activation of descending pathways (Crofford and Casey, 1999; Marks et al., 2009).

The opioid system is involved both in the ascending component and in the descending pain

modulation. In the ascending pathway, μ, δ and κ receptors play a key role. Specifically in the PAG

(periaqueductal gray) and RVM (Rostroventromedial medullary), the μ and κ receptors are

primarily responsible for the analgesia resulting from the modulatory effect exerted by tramadol,

which stimulate the release of norepinephrine and serotonin in the spinal cord dorsal horn

(Stamford. 1995), what suggests that αTPN is capable to activate the inhibitory descending

pathway. Similar results were found in another monoterpene, linalool, which showed na

antihyperalgesic effect also in the model of non-inflammatory chronic muscle pain with the

involvement of the opioid system activating descending inhibitory pain pathway (Nascimento et al.,

2014; 2015).

The CNS depression and the non-specific muscle relaxation effects can reduce the response

of motor coordination and might invalidate the behavioral tests (Le Bars et al., 2001; Raboisson and

Dallel, 2004; Passos et al., 2009). In this context, we evaluated the effect of αTPN-βCD on the rota

rod and grip-strength meter and no significant alteration in mice force and motor coordination were

observed. Therefore, the anti-hyperalgesic effect of αTPN-βCD observed in this study is not

entirely due to an inhibitory effect on the CNS or muscle relaxation.

All together, our results can conclude that αTPN-βCD reduced the secondary hyperalgesia

followed by the chronic muscle pain model, probably evoked by the descending inhibitory pain

system, specifically by opioid and serotoninergic receptors. Hence, the inclusion complex

67

containing αTPN-βCD can represent a promising tool for the study and development of a new

therapeutic approach in the management of “dysfunctional pain”, such as FM.

Acknowledgements

The authors were supported by grants from FAPITEC-SE, CAPES, CNPq and FINEP, all

from Brazil. Authors thank Jessica Deise Santos Dias (in memoriam) for technical support. We

thank teacher Abilio Borghi for the grammar review on the manuscript

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Authors Contributions: M.G.B.O., R.G.B., P.L.S., H.G.A.F., J.S.S.Q., J.C.S., J.R.G.S.A.

conducted pharmacological protocols. P.P.M., M.R.S., Y.M.B.G.C., A.A.S.A., L.J.Q.J. performed

the preparation and characterization of αTPN-βCD. L.S. and M.T.S. were responsible for docking

study. M.G.B.O., H.G.A.F., J.S.S.Q., P.P.M., M.R.S., A.A.S.A., L.S., M.T.S and L.J.Q.J. wrote and

corrected the article.

77

Figures and Table

Figure

Figure 1: TG/DTG curves of α-terpineol (αTPN), β-cyclodextrin (βCD), physical mixture (PM) and

slurry complex (SC) obtained at nytrogen atmosphere.

78

Figure 2: FTIR spectra of α-terpineol (αTPN), β-cyclodextrin (βCD), physical mixture (PM) and

slurry complex (SC).

79

Figure 3: Photographs SEM of β-cyclodextrin (A1 and A2), physical mixture (B1 and B2) and

slurry complex (C1 and C2) in the magnifications of 100 and 250x.

80

Figure 4. A) Effect of acute administration of vehicle, α-terpineol and β-cyclodextrin (αTPN-βCD

100 mg/kg, p.o.) or α-terpineol (αTPN 100 mg/kg, p.o.) on the mechanical hyperalgesia induced by

acid saline. B) Effect of chronic administration of vehicle, α-terpineol and β-cyclodextrin (αTPN-

βCD 25, 50 and 100 mg/kg, p.o.) or Tramadol (TRAM, 4 mg/kg) on mechanical hyperalgesia

induced by acid saline. Each point represents the mean ± SEM of the paw withdrawal threshold (in

0 1 2 3 4 5 6 7 8 90

2

4

6

8

Vehicle

TPN (100 mg/kg)

TPN-CD (100 mg/kg)

**

***

**

*** ***

**

** **

** **

Time (h)

Inte

nsit

y o

f sti

mu

lus (

g)

0 1 2 3 4 5 6 7 8 9 100

3

6

9

12 Vehicle

TPN-CD (25 mg/kg)

TPN-CD (50 mg/kg)

TPN-CD (100 mg/kg)

TRAM (4 mg/kg)

***

***

***

***

***

***

*

***

***

***

**

***

******

***

***

***

***

***

***

*****

******

***

**

***

******

***

***

***

***

***

***

Days

Inte

nsit

y o

f sti

mu

lus (

g)

A

B

81

grams) to tactile stimulation of the ipsilateral hind paw. **p < 0.01 and ***p < 0.001 vs. vehicle

group; #p < 0.05 and # #p < 0.01 vs. α-terpineol group (ANOVA followed by Tukey test).

CONCLUSÕES

Figure 5. A) Effect of vehicle, α-terpineol and β-cyclodextrin (αTPN-βCD 25, 50 and 100 mg/kg,

p.o.) or Tramadol (TRAM, 4 mg/kg) on the rota-rod test in mice. Values are the mean ± SEM (n =

A

B

82

8, per group). **p < 0.01 and *** p < 0.001 vs. control group (ANOVA followed by Tukey test). B)

Effect of chronic administration of vehicle, α-terpineol and β-cyclodextrin (αTPN-βCD 25, 50 and

100 mg/kg, p.o.) or Tramadol (TRAM, 4 mg/kg) on grip strength test, fore-/hindlimb (4 paws). Bars

represent the means (SEM) grip strength measurement (in grams of force) averaged across 3 trials.

*** p < 0.001 vs. control group (ANOVA followed by Tukey test).

FIGURE 6. A) Effect of the pharmacological antagonists naloxone (NAL) and B) Ondansetron

(ONDAN) in formalin-induced nociception tests after treatment with αTPN (i.p.), vehicle (i.p.) or

morphine (MORF). Each point represents the mean ± S.E.M. (n = 8, per group). ***p < 0.001 vs.

control group (ANOVA followed by Tukey´s test).

A

B

1stPHASE

1stPHASE

2ndPHASE

2ndPHASE

84

Figure 7. Docking results in αTPN A) for 5HT receptor, interactions of two hydrogen bonds with

ASP129 and CYS133; sterics with the residues LLE130 and PHE330; B) for Delta receptor, only

one hydrogen bond with ASP128; C) for Kappa receptor, two hydrogen bonds with LEU67 and

VAL63 of the chain B; steric with VAL55 of the chain A and D) for MU receptor, only one

hydrogen bond with the residue ASP147.

A

C

B

D

85

Table

Table 1: Percentage of weight loss obtained from TG curves and water content obtained by

Titration of Karl Fischer.

Sample 1st step/%

31-169ºC

2nd

step/%

169-282ºC

3rd

step/%

282-400ºC

4th

step/%

400-900ºC

% H2O

αTPN 98.9 0.35 0.17 0.58 1.55±0.11

β-CD 13.89 0.31 76.45 9.35 13.75±0.39

PM 20.07 0.98 70.65 9.86 12.56±1.12

SC 12.80 5.16 73.56 10.16 11.71±0.64

86

CONCLUSÃO

87

4 CONCLUSÕES

É imprescindível encontrarmos estratégias para melhorar a eficácia de medicamentos

analgésicos, pois mesmo tendo aumentado significativamente nossos conhecimentos sobre os

mecanismos de transmissão dolorosa, ainda existem muitas limitações na farmacoterapia da dor,

com expressiva farmacoresistência terapêutica. Nesse contexto, uma evolução marcante ocoore no

uso das ciclodextrinas, como apresentado aqui no artigo de revisão de patentes. As ciclodextrinas

tem melhorado o efeito terapêutico de diferentes classes de fármacos usados para o controle da dor,

sejam de drogas de origem natural ou sintética sendo, portanto, uma alternativa para minimizar

essas limitações apresentadas pelos medicamentos analgésicos.

O complexo preparado foi caracterizado e os resultados sugeriram a interação do αTPN com a

βCD. O complexo αTPN-βCD apresentou efeito anti-hiperalgésico no modelo animal de

fibromialgia, com maior tempo de efeito em comparação com o αTPN puro. Essas ações parecem

envolver o sistema opióide e serotoninérgico, sugeridos por meios dos protocolos de antagonismos

farmacológicos e corroborados com os estudos de Docking que comprovaram interações químicas

entre o αTPN com os receptores MU, Kappa, Delta e 5HT. Vale ressaltar ainda que a administração

do complexo não causa alterações motoras e na força muscular.

Portanto, o complexo de inclusão αTPN-βCD pode representar uma alternativa promissora no

desenvolvimento de novas opções terapêuticas para o controle da dor, como a fibromialgia.

88

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ANEXOS

95

ANEXO A: Protocolo de aprovação do Comitê de Ética em Pesquisa com Animal da Universidade

Federal de Sergipe (CEPAUFS)

96

ANEXO B: Normas da Phytomedicine para submissão do artigo: α-Terpineol, a monoterpene

alcohol, complexed with β-cyclodextrin exerts antihyperalgesic effect in animal model for

fibromyalgia aided with docking study

Guide for authors

INTRODUCTION

PHYTOMEDICINE

International Journal of Phytotherapy and Phytopharmacology

Scope

Phytomedicine is primarily a therapy-oriented Journal, which publishes innovative studies on

efficacy, safety, quality and mechanisms of action of specified plant extracts, phytopharmaceuticals

and their purified constituents. This includes clinical and preclinical studies of properly

standardized herbal medicinal products, herbal preparations and isolated compounds, which have

reproducible pharmacological activity.

The journal covers the following sections: Clinical pharmacology and toxicology (randomized,

placebo controlled, double blind, and observational open label studies), Behavioral, mental,

affective, and stress-associated disorders, Age-associated disorders, Neuropharmacology, Endocrine

pharmacology, Metabolic syndrome and obesity, Cancer, Immunopharmacology, inflammation,

Infectious diseases, Pulmonary diseases, Gastrointestinal diseases, Cardiovascular diseases,

Urogenital diseases, Systems biology, Pre-clinical toxicology of herbal preparations, Interaction

with drugs, Pharmacokinetic of natural compounds, Standardization and quality of herbal

preparations, Legislation of botanicals, Invited reviews.

BEFORE YOU BEGIN

Article requirements

Please note the following requirements for consideration of an article, upon submitting your

manuscript:

1. Is your article within the scope of Phytomedicine?

Your article must meet the scope of Phytomedicine (please see above). Articles that are not in the

scope, will be rejected immediately! Articles on the isolation and structure elucidation of novel

bioactive compounds or the development of new analytical methods do not fall into the scope of

Phytomedicine. However, pharmacological and clinical studies of novel natural products, where

new compounds or methods of analysis of active of pharmaceutical ingredients in herbal

preparations and biological fluids and tissues are reported (e.g. in pharmacokinetic studies), are

welcome. Dietary Supplements, "Botanicals" or "Functional Food" are not within the scope of

Phytomedicine unless they are specified/standardized and pharmacologically investigated analogues

to herbal drugs and if the evidence presented is comparable to therapeutic outcomes with a positive

control. Studies on purê compounds are not accepted if their origin is not clearly related to the plant

kingdom. Pharmacological

studies of isolated compounds in various forms (salts, ethers, etc.), which do not exist in nature are

out of scope of Phytomedicine. Screening results of a large number of plant extracts or plant

constituents for pharmacological activities will not be considered unless they are focused on those

plants or constituents which show superior activities in comparison with generally accepted positive

(reference) compounds.

2. Does your article comply with the standard requirements of Phytomedicine?

Your article must meet the criteria assuring reproducible quality and efficacy of herbal preparations.

97

Plant name and herbal substance

Latin binomial name and the author, local name and English name and plant part(s) used must by

specified for all plants used in the study. It should be stated that the plant name has been checked

with http://www.theplantlist.org. The authentication of fresh plants or dried herbal drugs, including

those of formulas, must be carried out by means of macroscopic and/or microscopic, molecular

biological, chemical, chromatographic and/or other suitable pharmacognostic methods. Voucher

specimens of plant materials used for all studies must be deposited and identified with a voucher

number, the dat and location of collection. The plant material may derive from natural origin, from

cultivated plants, or from an herbal drug market. In case of commercially procured material the

source, batch number, and quality control data should be specified. All scientific names of the

plants must be written in italics through the whole manuscript! Herbal medicinal products and

herbal extracts.

Herbal medicinal products or herbal preparations must be declared in accordance to EMA

guidelines (http://www.ema.europa.eu/docs/en_GB/document_library/

Scientific_guideline/2009/09/WC500003272.pdf). In particular, herbal extracts must be clearly and

comprehensively described with respect to the plant part used, the drug extract ratio, type and

concentration of extraction solvent, extraction conditions etc. They must be sufficiently

characterized (e.g. by HPLC fingerprints) and specified for the content of marker compounds to

ensure a consistente quality and reproducible pharmacological activity. The choice of marker must

be justified. The analytical methods have to be validated for selectivity, accuracy and precision and

briefly described, providing the most important information necessary to obtain reproducible

results. Traditional and commercial names of herbal preparations should be mentioned in the

Introduction of the manuscript, but not in the title. Phytomedicine accepts only international

standard terminology binomial Latin names of the plants and their combinations. Herbal

combinations

Studies with herbal drug combinations (e.g. 2-5 plants) will be accepted only if each herbal drug

undergo the same authentication and standardization process as described above, each single herbal

extracts is HPLC fingerprinted and relevant marker constituents are quantified before and after the

extracts are mixed. A 3-D-HPLC-profile of the multiherbal drug combination must be provided,

e.g.: Amagaya S. et al. 2001, Phytomedicine 8: 338-347. http://www.sciencedirect.com/science/

article/pii/S0944711304700495; For TCM-multi-herbal drug combinations (formula) see: Zeng K.-

W. et al. 2012, Phytomedicine 19, 122-129. http://www.sciencedirect.com/science/article/pii/

S0944711311002674. Additionally, we encourage the use other relevant and validated

physiological, biological, or biochemical methods, which ensure reproducible pharmacological

activity of multi-herbal drug combinations.

Chemicals, phytochemicals and other purified compounds For purified compounds, please provide

chemical names using relevant information from the NCBI PubChem which can be found on the

website http://www.ncbi.nlm.nih.gov/pccompound. In studies with purified compounds the

evidences of their purity (13C NMR or HPLC peak purity test) are required.

Gene nomenclature Authors should use approved nomenclature for gene symbols. Please consult

the appropriate nomenclature data bases for correct gene names and symbols. "Entrez Gene" is a

useful resource. Approved human gene symbols are provided by HUGO Gene Nomenclature

committee (HGNC): http://www.gene.ucl.ac.uk/nomenclature Approved Mouse symbols are

provided by The Jackson Laboratory: http://www.informatics.jaxorg/mgihome/nomen

Approved C. elegans symbols are provided by Caenorhabditis Genetics Center: http://

www.cbs.unmn.edu/CGC/Nomenclature/no menguid.htm For approved S. cerevisiae and S. pombe

symbols see http://yeastgenome.org/help/yeastGeneNomenclature.shtml and http://

www.sanger.ac.uk/Projects/S_pombe/SP_Name_FAQ.shtml, respectively Statistical analysis

Statistical hypothesis and methods should be described in detail. Actual P values should be used

unless less than 0.001. Reporting of 95% confidence intervals is encouraged. The choice of

98

appropriate parametric or nonparametric tools has to be justified. Refer to B.S. Evererett. Statistical

Methods for Medica Investigations, Oxford University Press, New York, 1989.

3. Is your article approaching new findings?

Scientific novelty of your study must be clearly demonstrated. The articles limited with a repetition

of well-known data or identification of only well-known ubiquitous compounds with little or no

relation to activity are not acceptable.

4. Is your article relevant to clinical medicine?

Your article must be based on a thorough study, using proper controls and convincing evidences of

therapeutic significance and observations.

Not acceptable are: In vitro studies with concentrations of active compounds, which could not be

implemented in-vivo and that are not appropriate for further pharmaceutical development. In vitro

studies without results on organs, tissues, fluids or cells. In vitro studies without positive control. In

vivo single dose studies or studies with one set of experiments and few animals. Studies on

antimicrobial activity with only single dose, very high concentration, measuring only inhibition

zones without MIC values, without information on type of activity (or growth inhibition) or

microorganisms investigation. Pharmacological studies of pure compounds, which are not

supported by evidences on pharmacological activity of plant extract where it was identified.

5. Does your article meet the requirements to clinical and pharmacological studies?

Your article must comply with the basic criteria for conducting and reporting clinical and

pharmacological studies.

Requirements for clinical studies: Clinical studies must meet the current standards for clinical trials

(GCP = Good Clinical Practice), which are equivalent to those required for synthetic drugs.

http://www.fda.gov/downloads/Drugs/Guidances/ucm073122.pdf http://www.ema.europa.eu/docs/

en_GB/document_library/Scientific_guideline/2009/09/WC500002832.pdf Articles should be in

line with Extensions of the Consolidated Standards of Reporting Trials Statement for Herbal

Medicinal Interventions (CONSORT), particularly when it comes to description of study

medication, which is a strict requirement of acceptance for Phytomedicine. For guidelines and

necessary information, please use the following internet addresses: http://www.consort-

statement.org http://www.consort-statement.org/ extensions?ContentWidgetId=557.

http://www.consort-statement.org/Media/Default/Downloads/ Extensions/CONSORT Extension for

Herbal Interventions.pdf http://www.ema.europa.eu/docs/

en_GB/document_library/Scientific_guideline/2009/09/WC500003370.pdf. Use of a CONSORT

checklist and flow diagram is recommended for illustration of grouping and flow of patients in all

clinical studies, randomized clinical trials as well as other trials. Clinical studies must be approved

by an Institutional Ethics Committee or its equivalent. The Methods section must state that the

study followed the guidelines of the Declaration of Helsinki and Tokyo for humans!

Requirements for pharmacological studies (in vitro, ex vivo or in vivo): Investigations with animals

must state in the Methods section that the research was conducted in accordance with

internationally accepted principles for laboratory animal use and care (e.g. European community

guidelines/ EEC Directive of 1986 or the US guidelines/ NIH publication). The route of drug

administration, different of oral, must be justified. Appropriate and justified statistical methods

must be used.Positive controls (reference standards must be included in study design). Many natural

compounds are known for their polyvalent (pleiotropic) activities and are only of interest if one or

two pharmacological activities are dominant and somehow superior in comparison with generally

accepted reference standards/compounds. Their potential therapeutic application must be justified

for specified indication. Antimicrobial evaluation of plants are of scientific value only if these plant

99

extracts show superior biological activities in comparison with a synthetic or natural antimicrobial

agent standard. It is preferred that in vitro activity (MIC) of an extract in not higher than 100 ìg/ ml.

For the correct determination of MIC values, standardized methodologies such as those of CLSI or

EUCAST are preferred. All articles that are reporting gene expression profiling data (microarray

experiments) should comply with the Minimum Information about Microarray Experiments

(MIAME, http://www.mged.org/Workgroups/MIAME/miame.html). At least two microarrays

should be provided for each experimental condition. Results of selected genes should be validated

by a second method (e.g. RT-PCR) or protein data should be provided. In addition functional test

(animal experiments/ clinical data) undertaken simultaneously are desirable to allow an

appraisement of the biological/ clinical relevance of the data. Alternatively, results of in vivo

experiments with comparable dosages can be discussed. The presentation of a sole data collection is

not acceptable. Biologically relevant information should be presented. We recommend do not

overuse specific names, notions and terms from various theories of traditional medical systems (e.g.

TCM, Ayurveda, etc.). That makes articles difficult for perceptions and understanding. The essence

of these theories should be translated into internationally accepted scientific theories, while

traditional names and terms should be converted to English. Final interpretation of the results of the

study must adhere to conventional scientific theories.

Ethics in publishing

For information on Ethics in publishing and Ethical guidelines for journal publication see

http://www.elsevier.com/publishingethics and http://www.elsevier.com/journal-authors/ethics.

Conflict of interest

All authors are requested to disclose any actual or potential conflict of interest including any

financial, personal or other relationships with other people or organizations within three years of

beginning the submitted work that could inappropriately influence, or be perceived to influence,

their work. See also http://www.elsevier.com/conflictsofinterest. Further information and an

example of a Conflict of Interest form can be found at:

http://help.elsevier.com/app/answers/detail/a_id/286/p/7923.

Submission declaration

Submission of an article implies that the work described has not been published previously (except

in the form of an abstract or as part of a published lecture or academic thesis or as an electronic

preprint, see http://www.elsevier.com/sharingpolicy), that it is not under consideration for

publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the

responsible authorities where the work was carried out, and that, if accepted, it will not be published

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the written consent of the copyright-holder.

Changes to authorship

This policy concerns the addition, deletion, or rearrangement of author names in the authorship of

accepted manuscripts:

Before the accepted manuscript is published in an online issue: Requests to add or remove na

author, or to rearrange the author names, must be sent to the Journal Manager from the

corresponding author of the accepted manuscript and must include: (a) the reason the name should

be added or removed, or the author names rearranged and (b) written confirmation (e-mail, fax,

letter) from all authors that they agree with the addition, removal or rearrangement. In the case of

addition or removal of authors, this includes confirmation from the author being added or removed.

Requests that are not sent by the corresponding author will be forwarded by the Journal Manager to

the corresponding author, who must follow the procedure as described above. Note that: (1) Journal

Managers will inform the Journal Editors of any such requests and (2) publication of the accepted

manuscript in an online issue is suspended until authorship has been agreed.

100

After the accepted manuscript is published in an online issue: Any requests to add, delete, or

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noted above and result in a corrigendum.

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101

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Our online submission system guides you stepwise through the process of entering your article

details and uploading your files. The system converts your article files to a single PDF file used in

the peer-review process. Editable files (e.g., Word, LaTeX) are required to typeset your article for

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Please submit the names and institutional e-mail addresses of several potential referees. For more

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PREPARATION

Types of manuscript

Original papers

Articles should not exceed 12-15 typewritten pages or up to 5,000 words, including references,

tables and figures. Previously reported methods should be referenced only. The number of

references should not exceed 30 (except for review articles or reports on microarray data).

Short communications

Short communications should be condensed to 4-8 typewritten pages or not more than 2,500 words

including references and a maximum of two illustrations.

Review articles

Review articles will only be by invitation. Review articles can provide concise and critical updates

on a subject of current interest. Herbal drug-monographs are only acceptable if they contain the

newest pharmacological and toxicological issues and an outlook on future directions.

Prof. Hildebert Wagner Award

The "Prof. Hildebert Wagner Award" was created to honor the outstanding efforts of Prof. Wagner

for the journal Phytomedicine. This award will be granted to a graduate student or young post-

doctoral researcher who is the first author of a paper reviewed by the Editors of Phytomedicine to

be the best one in the Journal during the previous calendar year. The prize will be sponsored by

Elsevier with EUR 500 for the awardee and a certificate for every Co-Author. Additionally an

official notice will be published on the journal homepage of Phytomedicine

(http://www.elsevier.com/locate/phymed), on which the article will be available free of charge for

one year. The reviewing editors for the first contribution to be awarded in Phytomedicine will be

Prof. Hildebert Wagner himself, Prof. Alexander Panossian, and Prof. Susana Zacchino. To qualify,

nominees must be younger than 35 years and na outstanding contribution to the field must be

provided. Nominations can be made by first authors (resp. corresponding authors).

Nominations for the first "Prof. Hildebert Wagner Award" in 2016 can be done until June 30, 2016.

The announcement of the winner will be by end of October 2016. Please choose Award-Article

from the drop-down menu below, if you want your article to be considered for the Award.

Essential title page information

• Title. Concise and informative. Titles are often used in information-retrieval systems. Avoid

abbreviations and formulae where possible.

• Author names and affiliations. Please clearly indicate the given name(s) and family name(s) of

each author and check that all names are accurately spelled. Present the authors' affiliation

addresses (where the actual work was done) below the names. Indicate all affiliations with a

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lowercase superscript letter immediately after the author's name and in front of the appropriate

address.

Provide the full postal address of each affiliation, including the country name and, if available, the

e-mail address of each author.

• Corresponding author. Clearly indicate who will handle correspondence at all stages of

refereeing and publication, also post-publication. Ensure that the e-mail address is given and

that contact details are kept up to date by the corresponding author. • Present/permanent address. If an author has moved since the work described in the article was

done, or was visiting at the time, a 'Present address' (or 'Permanent address') may be indicated as a

footnote to that author's name. The address at which the author actually did the work must be

retained as the main, affiliation address. Superscript Arabic numerals are used for such footnotes.

Example:

Anti-stress effects of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol in immobilized mice

Hyun A Oha, Dae-Eung Kimb, Hyuck Jai Choic, Nam Jae Kimc, and Dong-Hyun Kimac,* a

Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee

University, 26, Kyungheedae-ro, Dongdaemun-ku, Seoul 130-701, Republic of Korea b

Sempio Foods Company, 183, Osongsaengmyung-4ro, Cheongwongun, Chungcheongbukdo 363-

954, Republic of Korea c East-West Medical Research Institute, Kyung Hee University Medical Center, 23, Kyungheedae-

ro, Dongdaemun-ku, Seoul 130-872, Republic of Korea

* Corresponding author

Dong-Hyun Kim, Department of Life and Nanopharmaceutical Sciences, College of Pharmacy,

Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-ku, Seoul 130-701, Republic of Korea

Tel.: +82 2 961 0374; fax: +82 2 957 5030.

E-mail address: dhkim@khu.ac.kr (D.H. Kim).

**The phone, fax and email address of the corresponding author should be placed on the title page.

Abstract

A concise and factual abstract is required. Abstracts should summarize the contents of the article in

350 words or less. The abstract should be structured in the following format:

Background: In one or two sentences, summarize the scientific body of knowledge surrounding

your study and how this led to your investigation.

Hypothesis/Purpose: State the theory(ies) that you are attempting to prove or disprove by your

study or the purpose if no hypothesis exists.

Study Design: Identify the overall design of your study.

Methods: Succinctly summarize the overall methods you used in your investigation. For clinical

studies include the study population, type of intervention, method of data collection, and length of

he study.

Results: Report the most important results of your study. Only include positive results that are

statistically significant, or important negative results that are supported by adequate power. For

clinical studies report actual data, not just P values.

Conclusion: State the answer to your original question or hypothesis.Summarize the most important

conclusions that can be directly drawn from your study.

Graphical abstract

A Graphical abstract is mandatory for this journal. It should summarize the contents of the article in

a concise, pictorial form designed to capture the attention of a wide readership online. Authors must

provide images that clearly represent the work described in the article. Graphical abstracts should

be submitted as a separate file in the online submission system. Image size: please provide an image

with a minimum of 531 × 1328 pixels (h × w) or proportionally more. The image should be

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readable at a size of 5 × 13 cm using a regular screen resolution of 96 dpi. Preferred file types:

TIFF, EPS, PDF or MS Office files. See http://www.elsevier.com/graphicalabstracts for examples.

Authors can make use of Elsevier's Illustration and Enhancement service to ensure the best

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Keywords

Immediately after the abstract, provide a maximum of 6 keywords, using American spelling and

avoiding general and plural terms and multiple concepts (avoid, for example, 'and', 'of'). Be sparing

with abbreviations: only abbreviations firmly established in the field may be eligible. These

keywords will be used for indexing purposes.

Abbreviations

A section of abbreviations should precede the manuscript. Define abbreviations that are not

standard in this field in a footnote to be placed on the first page of the article. Abbreviations that are

unavoidable in the abstract must be defined at their first mention there, as well as in the footnote.

Ensure consistency of abbreviations throughout the article.

See "Uniform requirements for manuscripts submitted to biomedical journals" (1991) New England

Journal of Medicine 324:424–428.

Pagination and line numbers

Only manuscripts with page and line numbers will be reviewed.

Introduction

Provide an adequate background, avoiding a detailed literature survey or a summary of the results.

State the objectives of the work. No results of the study should be described in this section.

Material and methods

Provide sufficient detail to allow the work to be reproduced. Methods already published should be

indicated by a reference: only relevant modifications should be described.

This section should contain some subsections common for almost all studies: Plant names and parts

used (requirements see above) Study medication, herbal extracts (requirements see above) Chemical

compounds (requirements see above) Statistical analysis (requirements see above) Assays

(requirements see above) Animal studies (requirements see above) Study design (requirements see

above)

Results

Results should be clear and concise.

Discussion

This should explore the significance of the results of the work, not repeat them. A combined Results

and Discussion section is often appropriate. Avoid extensive citations and discussion of published

literature.

Conclusions

The main conclusions of the study may be presented in a short Conclusions section, which may

stand alone or form a subsection of a Discussion or Results and Discussion section.

Acknowledgements

Collate acknowledgements in a separate section at the end of the article before the references and do

not, therefore, include them on the title page, as a footnote to the title or otherwise. List here those

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individuals who provided help during the research (e.g., providing language help, writing assistance

or proof reading the article, etc.).

Units

Follow internationally accepted rules and conventions: use the international system of units (SI). If

other units are mentioned, please give their equivalent in SI.

Math formulae

Please submit math equations as editable text and not as images. Present simple formulae in line

with normal text where possible and use the solidus (/) instead of a horizontal line for small

fractional terms, e.g., X/Y. In principle, variables are to be presented in italics. Powers of e are often

more conveniently denoted by exp. Number consecutively any equations that have to be displayed

separately from the text (if referred to explicitly in the text).

Footnotes

Footnotes should be used sparingly. Number them consecutively throughout the article. Many word

processors can build footnotes into the text, and this feature may be used. Otherwise, please indicate

the position of footnotes in the text and list the footnotes themselves separately at the end of the

article. Do not include footnotes in the Reference list.

References

Citation in text

Please ensure that every reference cited in the text is also present in the reference list (and vice

versa). Any references cited in the abstract must be given in full. Unpublished results and personal

communications are not recommended in the reference list, but may be mentioned in the text. If

these references are included in the reference list they should follow the standard reference style of

the journal and should include a substitution of the publication date with either 'Unpublished results'

or 'Personal communication'. Citation of a reference as 'in press' implies that the item has been

accepted for publication.

Reference links

Increased discoverability of research and high quality peer review are ensured by online links to the

sources cited. In order to allow us to create links to abstracting and indexing services, such as

Scopus, CrossRef and PubMed, please ensure that data provided in the references are correct.

Please note that incorrect surnames, journal/book titles, publication year and pagination may

prevent link creation. When copying references, please be careful as they may already contain

errors. Use of the DOI is encouraged.

Web references

As a minimum, the full URL should be given and the date when the reference was last accessed.

Any further information, if known (DOI, author names, dates, reference to a source publication,

etc.), should also be given. Web references can be listed separately (e.g., after the reference list)

under a different heading if desired, or can be included in the reference list.

References in a special issue

Please ensure that the words 'this issue' are added to any references in the list (and any citations in

the text) to other articles in the same Special Issue.

Most Elsevier journals have a standard template available in key reference management packages.

This covers packages using the Citation Style Language, such as Mendeley

(http://www.mendeley.com/features/reference-manager) and also others like EndNote (http://

www.endnote.com/support/enstyles.asp) and Reference Manager (http://refman.com/support/

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rmstyles.asp). Using plug-ins to word processing packages which are available from the above sites,

authors only need to select the appropriate journal template when preparing their article and the list

of references and citations to these will be formatted according to the journal style as described in

this Guide. The process of including templates in these packages is constantly ongoing. If the jornal

you are looking for does not have a template available yet, please see the list of sample references

and citations provided in this Guide to help you format these according to the journal style.

If you manage your research with Mendeley Desktop, you can easily install the reference style for

this journal by clicking the link below: http://open.mendeley.com/use-citation-style/phytomedicine

When preparing your manuscript, you will then be able to select this style using the Mendeley plug-

ins for Microsoft Word or LibreOffice. For more information about the Citation Style Language,

visit http://citationstyles.org.

Reference style

Text: All citations in the text should refer to:

1. Single author: the author's name (without initials, unless there is ambiguity) and the year of

publication;

2. Two authors: both authors' names and the year of publication;

3. Three or more authors: first author's name followed by 'et al.' and the year of publication.

Citations may be made directly (or parenthetically). Groups of references should be listed first

alphabetically, then chronologically.

Examples: 'as demonstrated (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). Kramer et al.

(2010) have recently shown ....'

List: References should be arranged first alphabetically and then further sorted chronologically if

necessary. More than one reference from the same author(s) in the same year must be identified by

the letters 'a', 'b', 'c', etc., placed after the year of publication.

Examples:

Reference to a journal publication:

Wagner., H., Ulrich-Merzenich, G., 2009. Synergy research: Approaching a new generation of

phytopharmaceuticals. Phytomedicine 16, 97–110.

Reference to conference proceedings:

Argyropoulos D, Kudadam J, Müller J, 2009. Color degradation of lemon balm (Melissa officinalis

L.) as affected by the drying process. In: 5th International Technical Symposium on Food

Processing, Monitoring Technology in Bioprocesses and Food Quality Management, Potsdam,

Germany, August 31– September 2, pp. 730–736.

Willcox, M.L., Graz, B., Falquet, J., Diakite, C., Giani, S., Diallo, D., 2011. A "reverse

pharmacology" approach for developing an anti-malarial phytomedicine. Malaria J. 10 (Suppl. 1),

S8.

Reference to a book:

Cramer, J.A., Spilker, B., 1998. Quality of Life and Pharmacoeconomics. An Introduction.

Lippincott- Raven, Philadelphia.

Reference to a chapter in an edited book:

Cragg, G.M., Boyd, M., 1996. Drug discovery and development at the National Cancer Institute: the

role of natural products of plant origin. In: Balick, M.J., Elisabetsky, E., Laird, S.A. (Eds.),

Medicinal Plan Resources of the Tropical Forest. Columbia University Press, New York, pp. 101–

136.

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Journal abbreviations source

Journal names should be abbreviated according to the List of Title Word Abbreviations:

http://www.issn.org/services/online-services/access-to-the-ltwa/.

Appendices

If there is more than one appendix, they should be identified as A, B, etc. Formulae and equations in

appendices should be given separate numbering: Eq. (A.1), Eq. (A.2), etc.; in a subsequent

appendix,

Eq. (B.1) and so on. Similarly for tables and figures: Table A.1; Fig. A.1, etc.

Video data

Elsevier accepts video material and animation sequences to support and enhance your scientific

research. Authors who have video or animation files that they wish to submit with their article are

strongly encouraged to include links to these within the body of the article. This can be done in the

same way as a figure or table by referring to the video or animation content and noting in the body

text where it should be placed. All submitted files should be properly labeled so that they directly

relate to the video file's content. In order to ensure that your video or animation material is directly

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size of 150 MB. Video and animation files supplied will be published online in the electronic

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and the print version for the portions of the article that refer to this content.

AudioSlides

The journal encourages authors to create an AudioSlides presentation with their published article.

AudioSlides are brief, webinar-style presentations that are shown next to the online article on

ScienceDirect. This gives authors the opportunity to summarize their research in their own words

and to help readers understand what the paper is about. More information and examples are

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an invitation e-mail to create an AudioSlides presentation after acceptance of their paper.

Supplementary material

Elsevier accepts electronic supplementary material to support and enhance your scientific research.

Supplementary files offer the author additional possibilities to publish supporting applications,

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descriptive caption for each file. For more detailed instructions please visit our artwork instruction

pages at http://www.elsevier.com/artworkinstructions.

Data deposit and linking

Elsevier encourages and supports authors to share raw data sets underpinning their research

publication where appropriate and enables interlinking of articles and data. Please visit

http://www.elsevier.com/about/research-data for more information on depositing, sharing and using

research data.

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Ensure that the following items are present:

1. One author has been designated as the corresponding author with contact details: Full postal

address E-mail address Tel / fax number

2. All necessary files have been uploaded separately Author Agreement Cover letter Manuscript

Tables Figures Graphical Abstract (mandatory) Supplementary material (if needed)

3. Correct order within the manuscript: Title Page (Heading, Author names (the superscripts behind

the names which indicates the Institutes/affiliation of the authors have to be a,b,c,.... and * for the

corresponding author in addition), Institutes/affiliation, Corresponding Author with full address,

Word count) Abstract: has to be structured into Background, Hypothesis/Purpose, Study Design,

Methods, Results, Conclusion Keywords not more than 6 Abbreviations Introduction Materials and

methods Results and discussion Conclusion Acknowledgments Conflict of interest (mandatory)

References Table legends Figure legends Page and line numbers throughout the manuscript

4. References about 30 (have to be numbered) References are in the correct format for this jornal

References in alphabetical order All references mentioned in the Reference list are cited in the text,

and vice versa Citation according to our journal style

5. Choose the correct section for your article

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has been obtained for use of copyrighted material from other sources (including the Internet)

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visit our customer support site at http://support.elsevier.com.

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ANEXO C: Comprovante de submissão do artigo “α-Terpineol, a monoterpene alcohol,

complexed with β-cyclodextrin exerts antihyperalgesic effect in animal model for fibromyalgia

aided with docking study” na Phytomedicine - Journal of Phytotherapy and Phytopharmacology.