( PDF ) Desenvolvimento tecnológico e avaliação in vitro de matrizes hidrofílicas de norfloxacino contendo polioxietileno e hidroxipropilmetilcelulose

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UNIVERSIDADE FEDERAL DE SANTA CATARINA

CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-

GRADUAÇÃO EM FARMÁCIA

Desenvolvimento Tecnológico e Avaliação In Vitro de

Matrizes Hidrofílicas de

Norfloxacino Contendo

Polioxietile

no e Hidroxipropilmetilcelulose

Paulo Renato de Oliveira

Florianópolis

2010

UNIVERSIDADE FEDERAL DE SANTA CATARINA

CENTRO DE CIÊNCIAS DA SAÚDE

GRADUAÇÃO EM FARMÁCIA

Desenvolvimento Tecnológico e Avaliação In Vitro de

Norfloxacino Contendo

no e Hidroxipropilmetilcelulose

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Desenvolvimento Tecnológico e Avaliação In Vitro de

Matrizes Hidrofílicas de Norfloxacino Contendo

Polioxietileno e Hidroxipropilmetilcelulose

por

Paulo Renato de Oliveira

Tese apresentada ao Programa de Pós-Graduação em Farmácia da

Universidade Federal de Santa Catarina como requisito parcial à

obtenção do grau de Doutor em Farmácia.

Orientador: Prof. Dr. Marcos A. Segatto Silva

Florianópolis

2010

AGRADECIMENTOS

A Deus, pelos dons, caminhos, oportunidades e amigos que tive.

Ao Prof. Marcos Segatto por aceitar um “estranho no ninho”, pela

confiança no meu trabalho e pela orientação na formação acadêmica

(ciências farmacêuticas) e humana (ciência? filosofia? da vida).

Às Prof

. Simone Cardoso e Silvia Cuffini, sempre prontas a ajudar e

não deixar desanimar, tanto no laboratório como fora dele (histórias e

risadas!).

Aos Prof

. Eloir Schenkel, Flávio Reginatto e Celso Spada pela

amizade e ensinamentos em tantas reuniões.

Aos amigos e colegas de laboratório Lari, Silvinha, Rafa, Bruno, Gis,

Monika, Fabio, Hellen, Amarilis, Andrea TN, Andrea C$, Cinira, Chari,

Gabi, Geison, Cacá, Carlos, Solomon, pelo companheirismo, ajuda,

ensinamentos, desabafos, churrascos e tragos. Com certeza a amizade

não termina aqui!

À minha IC Cassiana Mendes, cuja ajuda, parceria, confiança e bom

humor foram de fundamental importância para a realização do trabalho.

Ringrazio il Prof. Paolo Colombo per l’opportunità di lavorare a Parma

e per aver guidato sapientemente la mia ricerca. Ringrazio tutti i miei

amici italiani, Fabio, Luca, Salvo, Camillo, Edo, Laura, Leti, La Robe,

(e tanti altri) con i quali oltre alle ore di laboratorio (ho imparato tanto!)

ho condiviso bevute di birra, vino, cene (si mangia bene in Italia) ed

sogni in cinque mesi indimenticabili della mia vita.

À Colorcon, Elofar, União Química, Farmacopéia Brasileira, Allcrom,

pela apoio e disponibilidade no fornecimento de materiais necessários à

realização deste trabalho.

Aos meus pais (Valmorim e Clara) e minha irmã Elenise, pelo apoio,

carinho e confiança.

À Larissa, amore mio, por estar na minha vida, fazendo tudo ter sentido.

Te amo!!

Ao CNPQ e CAPES pelo apoio financeiro.

À UFSC e PGFAR por possibilitarem a realização deste trabalho.

A todos que contribuíram de alguma forma para a realização deste

trabalho.

Se la vita ti sorride... ha una paresi!!

“Um homem precisa viajar. Por sua conta, não por meio de histórias,

imagens, livros ou TV. Precisa viajar por si, com seus olhos e pés, para

entender o que é seu. Para um dia plantar as suas próprias árvores e

dar-lhes valor. Conhecer o frio para desfrutar o calor. E o oposto.

Sentir a distância e o desabrigo para estar bem sob o próprio teto. Um

homem precisa viajar para lugares que não conhece para quebrar essa

arrogância que nos faz ver o mundo como o imaginamos, e não

simplesmente como é ou pode ser. Que nos faz professores e doutores

do que não vimos, quando deveríamos ser alunos, e simplesmente ir

ver”

Amyr Klink

RESUMO

O norfloxacino é um fármaco antimicrobiano amplamente utilizado para

o tratamento de infecções do trato urinário. A posologia indicada

normalmente é de 400 mg a cada 12 horas. Este trabalho teve como

objetivo desenvolver formulações de liberação prolongada contendo

norfloxacino para administração em dose única diária, com conseqüente

melhora na terapêutica. Durante a etapa de pré-formulação, estudos de

compatibilidade entre fármaco e excipientes não demonstraram

mudança significativa no perfil termoanalítico dos compostos, sugerindo

ausência de incompatibilidade. Metodologia analítica por cromatografia

líquida de alta eficiência foi desenvolvida e validada para o doseamento

e avaliação da estabilidade das formulações. Os comprimidos foram

obtidos através da compressão direta utilizando dois tipos de polímeros

hidrofílicos: hidroxipropilmetilcelulose (HPMC) ou polioxietileno

(POE), de diferentes massas moleculares, e em duas concentrações (20 e

30%), foram utilizados para obter as matrizes hidrofílicas. Através dos

estudos de estabilidade foi possível verificar que o teor não foi

influenciado pela temperatura e umidade, porém o revestimento ou

emblistagem das formulações em material adequado é necessário para

impedir a fotodegradação do fármaco. Os estudos de dissolução

demonstraram que conforme o aumento da massa molecular e da

concentração do polímero na formulação, o sistema hidrofílico apresenta

um mecanismo de liberação que muda de Super Caso-II (mais

dependente do relaxamento das cadeiras poliméricas e erosão da matriz)

para Transporte Anômalo (dependente da difusão do fármaco e

relaxamento/erosão matricial). As formulações que apresentaram melhor

desempenho in vitro foram HPMC K100 LV 30%, HPMC K4M 20%,

POE N60K 20% e POE N60K 30%. Os sistemas Dome Matrix

acoplados na configuração “void” demonstraram capacidade de

flutuação in vitro por aproximadamente 240 min, indicando possível

liberação prolongada e local-específica (estômago) in vivo. Este

resultado é de grande relevância terapêutica, uma vez que o fármaco

apresenta maior solubilidade em meio ácido. O trabalho desenvolvido

demonstra que os sistemas matriciais podem apresentar melhor

performance in vivo em comparação aos comprimidos convencionais de

liberação imediata disponíveis atualmente no mercado.

Palavras-chave: norfloxacino; liberação prolongada; comprimido

matricial; hidroxipropilmetilcelulose; polioxietileno; Dome Matrix

ABSTRACT

Norfloxacin is an antibacterial drug mainly used for the treatment of

urinary tract infections. The recommended dosage is 400 twice daily.

The aim of this research was to develop extended-release tablets for

once-a-day administration, with subsequent improvement of

therapeutics. Compatibility studies between the drug and excipients did

not evidenced difference in thermo-analytical profile of compounds, this

way suggesting the absence of incompatibility. A stability-indicating

liquid chromatographic method was developed and validated for the

assay of formulations. The matrix tablets were successfully obtained by

direct compression. Two polymers: hydroxypropylmethylcellulose and

poly(ethylene oxide), with different molecular weights, and in two

concentrations (20 and 30%) were used to obtain the formulations.

Stability studies showed that the assay was not influenced by

temperature and humidity, however tablet film-coating or opaque

blistering are necessary to ensure the photostability. Dissolution studies

showed that with the increase in molecular weight and concentration of

polymer in the formulation, the dissolution mechanism changed from

Super Case-II (a more polymer relaxation or erosion dependent) to

Anomalous Transport (drug-diffusion and polymer relaxation/erosion

dependent). The formulations containing HPMC K100 LV 30%, HPMC

K4M 20%, PEO N60K 20%, and PEO N60K 30% demonstrated a better

in vitro dissolution profile. Dome Matrix

systems assembled in “void”

configuration were able to float in vitro for up to 240 min, indicating a

possible in vivo extended and site-specific drug delivery system. This

result is of great therapeutic interest since norfloxacin is more soluble in

acid medium. The developed research demonstrated that matrix systems

could provide a better in vivo performance compared to the norfloxacin

conventional immediate-release tablets available in the market.

Keywords: norfloxacin; extended-release; matrix tablet;

hydroxypropylmethylcellulose; poly(ethylene oxide); Dome Matrix

SUMÁRIO

Lista de tabelas

............................................................................ xvii

Lista de figuras

............................................................................ xix

Lista de

abreviaturas

.................................................................. xxiii

Introdução

.................................................................................... 1

Objetivos

Objetivo geral................................................................................ 3

Objetivos específicos.................................................................... 3

Capítulo 1

Revisão bibliográfica.................................................................... 5

1. Norfloxacino............................................................................. 7

2. Formas farmacêuticas de liberação modificada........................ 10

3. Caracterização e estabilidade.................................................... 18

Capítulo 2

Caracterização térmica e estudos de compatibilidade

fármaco/excipiente........................................................................

Introdução..................................................................................... 25

Publicação científica:

Thermal characterization and

compatibility studies of norfloxacin for development of

extended release tablets.................................................................

Capítulo 3

Desenvolvimento e validação de metodologia analítica............... 41

Introdução..................................................................................... 43

Publicação científica:

Liquid chromatographic determination of

norfloxacin in extended release tablets........................................

Capítulo 4

Formulação, estabilidade e avaliação in vitro de comprimidos

de liberação prolongada contendo Norfloxacino.........................

Introdução.................................................................................... 69

Publicação científica:

Formulation, stability and in vitro

dissolution studies of norfloxacin extended-release matrix

tablets..

Capítulo 5

Desenvolvimento de sistemas Dome Matrix

de Norfloxacino... 99

Introdução..................................................................................... 101

Publicação científica:

Assembled modules technology for site-

specific prolonged delivery of norfloxacin...................................

103

Discussão geral

............................................................................ 127

Conclusõe

.................................................................................... 133

Referências

................................................................................... 137

xvii

LISTA DE TABELAS

Capítulo 1

Tabela 1. Tipos de polioxietileno disponíveis e respectivos massas

moleculares aproximadas.................................................................

Tabela 2. Tipos de hidroxipropilmetilcelulose disponíveis e

respectivas viscosidades...................................................................

Capítulo 3

Table 1. Inter-day and between-analysts precision data of the

method……………………………………………………………..

Table 2. Accuracy of the method…………………………………. 57

Table 3. Chromatographic conditions and range investigated

during robustness testing…………………………………………..

Capítulo 4

Table 1. Composition of tablets containing hydroxypropylmethyl

cellulose (HPMC) or poly(ethylene oxide) (PEO)………………...

Table 2. Pharmacopeial characteristcs of norfloxacin matrix

tablets………………………………………………………………

Table 3. Assay results of accelerated stability test………………... 81

Table 4. Assay results of photostability test………………………. 82

Table 5. Difference factor (ƒ1) and similarity factor (ƒ2)

calculated for uncoated and coated norfloxacin matrix tablets……

Table 6. Coefficients of determination (r

) obtained from

dissolution of norfloxacin uncoated formulations according to

different mathematical models…………………………………….

Table 7. Coefficients of determination (r

) obtained from

dissolution of norfloxacin coated formulations according to

different mathematical models…………………………………….

xviii

Capítulo 5

Table 1. Composition of the norfloxacin male and female modules 107

Table 2. Difference factor (ƒ1) and similarity factor (ƒ2)

calculated for the male and female modules………………………

113

Table 3. Mathematical modeling and drug release kinetics from

Dome Matrix

modules using Korsmeyer et. al and Peppas and

Sahlin equations...............................................................................

116

xix

LISTA DE FIGURAS

Capítulo 1

Figura 1. Estrutura química do norfloxacino.................................... 8

Figura 2. Diagrama simplificado do mecanismo de ação das

fluorquinolonas (RANG et al., 2008)...............................................

Figura 3. Diferentes protonações do norfloxacino dependentes do

pH: (a) neutra, (b) cation, (c) zwitterion e (d) anion (MUSA et al.,

2009).................................................................................................

Figura 4. Diagrama dos diferentes frontes existentes em um

sistema matricial hidratado (LOPES; LOBO; COSTA, 2005).........

Figura 5. Estrutura química do polioxietileno.................................. 13

Figura 6. Estrutura química da hidroxipropilmetilcelulose.............. 14

Figura 7. Representação de sistema flutuante (BARDONNET et

al., 2006)...........................................................................................

Figura 8. Módulos individuais da Dome Matrix e módulos

acoplados nas configurações “piled”, à esquerda e “void”, à

direita (LOSI et al., 2006)................................................................

Capítulo 2

Figure 1. Chemical structure of NFX (A), HPMC (B) and PEOs

(C)………………………………………………………………….

Figure 2. DSC and TG/DTG curves of norfloxacin (A), Polyox

WSR 301 (B), Polyox WSR N80 (C), and Methocel K100 LV CR

(D) in dynamic nitrogen atmosphere (50 mL min

-1

) and heating

rate of 10 ºC min

-1

………………………………………………...

Figure 3. SEM of NFX (A), Polyox WSR 301 (B), Polyox WSR

N80 (C), and Methocel K100 LC CR (D). The photomicrograph

A was taken at a magnification of 50x, and B, C and D of

100x………………………………………………………………..

Figure 4. X-ray diffraction spectra of NFX (A), Polyox WSR 301

(B), Polyox WSR N80 (C), and Methocel K100 LC CR (D)……...

Figure 5. DRIFT spectra of Norfloxacin (A), Polyox WSR 301

(B), Polyox WSR N80 (C), and Methocel K100 LC CR (D)……...

Figure 6. (A) TG curves obtained for the non-isothermic study of

NFX at 2.5, 5, 10, 15, and 20 ºC min

-1

. (B) Isothermal TG curves

of NFX obtained between 230 and 270 ºC, with a temperature

increment of 10 ºC…………………………………………………

Figure 7. DSC curves of NFX and excipients obtained in dynamic

nitrogen atmosphere (50 mL min

–1

) and heating of rate 10 °C

min

-1

………………………………………………………………..

Capítulo 3

Figure 1. Chemical structure of norfloxacin……………………… 45

Figure 2. LC chromatograms of norfloxacin (A) developed

formulation (1 µg/mL). (B) Decarboxylated degradant. (C) After

acidic condition. (D) After basic condition. (E) After photolytic

condition. (F) After oxidative condition. (G) After neutral

condition...........................................................................................

Figure 3. Mass spectra for (A) norfloxacin and (B) decarboxylated

degradant…………………………………………………………..

Figure 4. Normal plot of residuals and outlier T values for

responses (A) peak area RSD%, (B) assay, and (C) peak

symmetry…………………………………………………………..

Capítulo 4

Figure 1. Norfloxacin blistered matrix tablets: uncoated (A) and

coated (B)………………………………………………………….

Figure 2. Norfloxacin blistered matrix tablets after photostability

study: uncoated (A) and coated (B)………………………………..

Figure 3. Norfloxacin released vs. time of matrix tablets

containing HPMC K100 LV: 20% uncoated; 20% coated; 30%

uncoated; 30% coated………………...............................................

Figure 4. Norfloxacin released vs. time of matrix tablets

containing HPMC K4M: 20% uncoated; 20% coated; 30%

xxi

uncoated; 30% coated……………………………………............... 84

Figure 5. Norfloxacin released vs. time of matrix tablets

containing HPMC K100M: 20% uncoated; 20% coated; 30%

uncoated; 30% coated……………………………………………...

Figure 6. Norfloxacin released vs. time of matrix tablets

containing PEO N60K: 20% uncoated; 20% coated; 30%

uncoated; 30% coated……………………………………………...

Figure 7. Norfloxacin released vs. time of matrix tablets

containing PEO 301: 20% uncoated; 20% coated; 30% uncoated;

30% coated.......................................................................................

Figure 8. Norfloxacin released vs. time of matrix tablets

containing PEO 303: 20% uncoated; 20% coated; 30% uncoated;

30% coated.......................................................................................

Capítulo 5

Figure 1. Norfloxacin Dome Matrix

modules and assemblage: 1-

Male. 2- Female. 3- Void configuration assembled modules……...

108

Figure 2. Norfloxacin fraction released vs. time of Dome Matrix

modules containing 20% of HPMC: female module; male module;

void configuration............................................................................

111

Figure 3. Norfloxacin fraction released vs. time of Dome Matrix

modules containing 30% of HPMC: female module; male module;

void configuration............................................................................

112

Figure 4. Norfloxacin fraction released vs. time of Dome Matrix

modules containing 20% of PEO: female module; male module;

void configuration............................................................................

112

Figure 5. Norfloxacin fraction released vs. time of Dome Matrix

modules containing 30% of PEO: female module; male module;

void configuration............................................................................

113

Figure 6. Norfloxacin Fickian released fraction vs. time of Dome

Matrix

modules containing 20% of HPMC: female module; male

module; void configuration and 30% of HPMC: female module;

male module; void configuration.....................................................

117

Figure 7. Norfloxacin Fickian released fraction vs. time of Dome

xxii

Matrix

modules containing 30% of PEO: female module; male

module; void configuration..............................................................

119

Figure 8. Resultant weight vs. time of Dome Matrix

20% HPMC

K4M and 20% PEO N60K modules assembled in void

configuration....................................................................................

121

xxiii

LISTA DE ABREVIATURAS

µg Micrograma

ASC Área sob a curva da concentração plasmática versus

tempo

bioMEMS Sistemas bio-micro-eletro-mecânico

max

Concentração plasmática máxima

Da Daltons

DNA Ácido desoxirribonuclêico

DPR Desvio padrão relativo

DRIFT Infravermelho com transformada de Fourier de

reflexão difusa

DSC Calorimetria exploratória diferencial

h Hora

HPMC Hidroxipropilmetilcelulose

ITU Infecção do trato urinário

mg Miligrama

mL Mililitro

POE Polioxietileno

SEM Microscopia eletrônica de varredura

Tempo de meia-vida

TG Termogravimetria

max

Tempo para atingir a concentração plasmática máxima

XRPD Difração de raio-x de pó

INTRODUÇÃO

O norfloxacino pertence ao grupo das quinolonas, sendo um

agente antibacteriano de amplo espectro contra patógenos Gram-

positivos e Gram-negativos. É indicado terapeuticamente principalmente

para o tratamento de infecções do trato urinário. Seu mecanismo de ação

está baseado na inibição da enzima DNA girase e da topoisomerase IV,

inibindo a replicação do DNA bacteriano. A posologia recomendada

normalmente é de 400 mg a cada 12 horas e a duração do tratamento

varia de acordo com o diagnóstico (MANDELL, 1988; VAN

BAMBEKE et al., 2005; RANG et al., 2008; BOLON, 2009).

Recentemente a ANVISA, com intuído de diminuir o uso

indiscriminado de antibióticos e o surgimento de microorganismos

resistentes, determinou que a venda de medicamentos a base de

antimicrobianos somente poderá ser efetuada mediante receita de

controle especial, ficando uma via retida na farmácia (BRASIL, 2010).

O estudo de formulações contendo antibióticos que objetivam o

aumento da aderência do paciente ao tratamento, a manutenção da

eficácia terapêutica minimizando ou evitando concentrações plasmáticas

sub ou supraterapêuticas e a diminuição do surgimento de

microorganismos resistentes é de grande relevância, uma vez que

infecções bacterianas são bastante comuns e atingem milhões de pessoas

anualmente. Em termos mercadológicos, os comprimidos representam a

forma farmacêutica mais consumida, além disso, é a mais adequada para

produção em escala industrial possibilitando menor custo efetivo

(ALLEN; POPOVICH; ANSEL, 2007).

O estudo de propriedades no estado sólido e de compatibilidade

fármaco-excipientes são pré-requisitos no desenvolvimento racional de

medicamentos, pois diferentes formas cristalinas podem exibir distintas

propriedades físico-químicas, alterando a biodisponibilidade e

conseqüentemente a terapêutica (GIRON, 2002; AALTONEN et al.,

2009). O desenvolvimento e a validação da metodologia analítica

utilizada fornecem dados para assegurar a confiabilidade dos resultados

obtidos (SHABIR, 2003; BRASIL, 2003; ICH, 2005; STÖCKL, 2009).

Posteriormente estudos de perfil de dissolução e estabilidade são

realizados para avaliar a liberação do fármaco e a qualidade do produto

em desenvolvimento ou acabado.

As formas farmacêuticas de liberação prolongada objetivam o

aumento do tempo de ação farmacológica de substâncias ativas, a

diminuição de reações adversas, a redução da administração diária, a

manutenção da eficácia terapêutica, a otimização da aderência ao

tratamento e a não permissão ou minimização do aparecimento de

flutuação nas concentrações plasmáticas (AULTON, 2005; ALLEN;

POPOVICH; ANSEL, 2007; HOFFMAN, 2008). Possibilitam, também,

obter a mesma eficácia clínica com o decréscimo dos custos do

tratamento, sendo uma alternativa econômica quando comparada com

formas de dosagem de liberação imediata (SAKS; GARDNER, 1997).

O desenvolvimento de sistemas matriciais é ainda o modelo de

referência para inovações em liberação prolongada de fármacos na

industria farmacêutica devido ao fato de serem considerados confiáveis

em termos de liberação e facilidades de formulação e fabricação

(COLOMBO et al., 2009). Nestes sistemas, o mecanismo de liberação

do fármaco e sua cinética podem ser governados pelo intumescimento e

difusão do fármaco ou pelo intumescimento e erosão do polímero,

dependendo da porcentagem e da massa molecular do polímero utilizado

e das características fisico-químicas do fármaco (COLOMBO et al.,

2000; CONTI et al., 2007; OMIDIAN; PARK, 2008; PARK et al.,

2010). Dos agentes poliméricos disponíveis para a obtenção de

comprimidos matriciais, destacam-se o polioxietileno e a

hidroxipropilmetilcelulose, pois apresentam, entre outras características,

boa compressibilidade, boa capacidade de intumescência e atoxicidade.

Além da utilização de diferentes polímeros, o emprego de novas

tecnologias de fabricação representa alternativa para obtenção de formas

farmacêuticas diferenciadas, possibilitando melhor resultado

terapêutico. Um exemplo de nova tecnologia é a Dome Matrix

, onde

módulos individuais “male” e “female” são obtidos utilizando punções

especiais, e posteriormente pode-se acoplar os mesmos em diversas

combinações, formando inclusive sistemas flutuantes. Esta tecnologia

tem como característica possibilitar uma grande flexibilidade na

formulação e posterior liberação do fármaco (LOSI et al., 2006;

STRUSI et al., 2008; STRUSI et al., 2010).

Neste contexto, o presente estudo propõe o desenvolvimento e

avaliação in vitro de matrizes hidrofílicas e sistemas Dome Matrix

liberação modificada contendo norfloxacino.

OBJETIVOS

Objetivo geral

Desenvolvimento tecnológico de comprimidos matriciais de

liberação prolongada e sistemas Dome Matrix

flutuantes de

norfloxacino contendo os polímeros hidrofílicos polioxietileno (POE) e

hidroxipropilmetilcelulose (HPMC).

Objetivo específicos

 Verificar as características físico-químicas do norfloxacino e dos

polímeros POE e HPMC, obtendo resultados que servirão de

referência para avaliações da qualidade;

 Avaliar a compatibilidade entre o norfloxacino e diversos

excipientes farmacêuticos para o desenvolvimento racional das

matrizes;

 Desenvolver e validar metodologia indicativa da estabilidade por

cromatografia líquida em fase reversa;

 Produzir matrizes hidrofílicas de liberação prolongada de

norfloxacino através da compressão direta, utilizando polímeros de

diferentes massas moleculares e em duas concentrações;

 Realizar revestimento e emblistagem, para avaliar a influência

destes na estabilidade das matrizes;

 Obter módulos individuais Dome Matrix

“male” e “female” e

formar sistema flutuante;

 Avaliação das propriedades físico-químicas (farmacopéicas) dos

comprimidos;

 Caracterizar as formulações quanto aos seus perfis de dissolução;

 Comparar o modelo cinético de liberação do norfloxacino a partir

dos comprimidos matriciais através da aplicação de modelos

dependentes e independentes;

 Avaliar a relação entre os diferentes polímeros e concentrações

com o mecanismo de liberação do fármaco;

 Estudar a estabilidade acelerada em câmara climática e

fotoestabilidade das formulações desenvolvidas.

CAPÍTULO 1 – Revisão bibliográfica

1. NORFLOXACINO

A infecção do trato urinário (ITU) é afecção muito comum e

caracteriza-se pela presença de microorganismos, principalmente

bactérias, nas vias urinárias, seja na bexiga, próstata, sistema coletor ou

rins. A incidência é maior em mulheres, principalmente devido às

condições anatômicas como uretra mais curta e maior proximidade do

ânus com a uretra e o vestíbulo vaginal, o que possibilita a colonização

destes por enterobactérias (principalmente Escherichia coli)

(KRIEGER, 2002; WAGENLEHNER; NABER, 2006). As ITU

destacam-se não somente pela sua freqüência como também pela

possibilidade de complicações graves, como a insuficiência renal e a

septicemia. Aproximadamente 15% de todos os antibióticos prescritos

nos Estado Unidos são dispensados para tratamento de ITU, com um

custo estimado anual superior a 1 bilhão de dólares. Além disso, o custo

direta e indiretamente associado a esta enfermidade está estimado em

mais de 1,6 bilhões de dólares ao ano (WAGENLEHNER; NABER,

2006).

A classe de antibióticos das quinolonas, desde sua descoberta na

década de 60, tem sido extensivamente estudada e utilizada

clinicamente, pois apresenta uma série de vantagens, tais como

combinação de alta potência e amplo espectro de ação, boa

biodisponibilidade, formulações orais e intravenosas, altos níveis

plasmáticos, grande volume de distribuição e relativamente baixos

efeitos colaterais (ANDERSSON; MACGOWAN, 2003).

O norfloxacino (Figura 1), comercializado desde 1986,

pertencente ao grupo das quinolonas, é um agente bactericida de amplo

espectro contra patógenos Gram-positivos e Gram-negativos. Seu

mecanismo de ação está baseado na inibição da topoisomerase II (uma

DNA-girase bacteriana), enzima que produz um supernovelo negativo

no DNA, permitindo sua transcrição ou replicação (Figura 2) (RANG et

al., 2008). É indicado terapeuticamente principalmente para o

tratamento de infecções do trato urinário (cistite, pielite, cistopielite,

pielonefrite, prostatite crônica, epididimite). Também é indicado para o

tratamento de gastroenterites bacterianas, uretrite, faringite, proctite ou

cervicite gonocócicas, febre tifóide e na profilaxia da sepse em pacientes

com neutropenia intensa e da gastroenterite bacteriana. A posologia

recomendada normalmente é de 400 mg a cada 12 horas e a duração do

tratamento varia de acordo com o diagnóstico (MANDELL, 1988;

CHRISTIAN, 1996; EMMERSON; JONES, 2003).

Figura 1. Estrutura química do norfloxacino.

Figura 2.

Diagrama simplificado do mecanismo de ação das

fluorquinolonas (RANG et al., 2008).

Quimicamente, o norfloxacino é denominado como ácido 1

6-fluor-1,4-diidro-4-oxo-7-(1-piperazinil)-3-

quinolino carboxílico

(Figura 1)

. É um pó cristalino branco a amarelo claro, pouco solúvel em

água, metanol, etanol, acetato de etila e acetona,

facilmente solúvel em

ácido acético, ligeiramente solúvel em clorofórmio e insolúvel em éter

etílico. Possui massa molecular de 319,34 Daltons, fórmula molecular

(F. BRAS. IV, 2001).

A relação estrutura-atividade

das quinolonas é bastante

estud

ada, o átomo de flúor na posição 6 proporciona maior potência

contra organismos Gram-

negativos e o núcleo piperazínico na posição 7

é responsável pela atividade antipseudomonas

e aumento da ação contra

Diagrama simplificado do mecanismo de ação das

Quimicamente, o norfloxacino é denominado como ácido 1

-etil-

quinolino carboxílico

. É um pó cristalino branco a amarelo claro, pouco solúvel em

facilmente solúvel em

ácido acético, ligeiramente solúvel em clorofórmio e insolúvel em éter

etílico. Possui massa molecular de 319,34 Daltons, fórmula molecular

das quinolonas é bastante

ada, o átomo de flúor na posição 6 proporciona maior potência

negativos e o núcleo piperazínico na posição 7

e aumento da ação contra

Gram-positivos do norfloxacino (VAN BAMBEKE et al., 2005;

BOLON, 2009).

O NFX, composto anfótero, apresenta dois sítios de protonação,

o nitrogênio 4’ do anel piperazinil (básico) e o grupo carboxila

quinolona (ácido) (Figura 3), consequentemente apresenta duas

constantes de equilíbrio, com valores de pKa de aproximadamente 8,5 e

6,5, respectivamente. De acordo com o pH, este fármaco apresentará

diferentes formas. Em pH neutro será predominatemente um zwitterion

(com o grupo carboxílico deprotonado e o nitrogênio 4’ protonado). Em

pH 10, mais de 90% estará na forma aniônica. A forma catiônica é

obtida em pH igual ou inferior a 4,5 (YU; ZIPP; DAVIDSON, 1994;

MUSA et al., 2009). O norfloxacino exibe maior solubilidade em pH

inferior a 4,5 e superior a 8.

Figura 3. Diferentes protonações do norfloxacino dependentes

do pH: (a) neutra, (b) cation, (c) zwitterion e (d) anion (MUSA et al.,

2009).

Após administração oral de dose de 400 mg de norfloxacino, no

mínimo 30-40% é absorvida, a concentração plasmática máxima

atingida (C

max

) é de aproximadamente 1,5 µg/mL sendo obtida após 1

hora (t

max

). O tempo de meia-vida (t

) é de 3-4 horas com excreção

predominantemente renal através da filtração gromerular e secreção

tubular. A área sob a curva da concentração plasmática versus tempo

(ASC) é de 6,4 µg.h/mL. A administração concomitante com alimentos

e antiácidos pode diminuir a absorção (GADEBUSH; SHUNGU, 1991;

AL-RASHOOD et al., 2001).

O local preferencial de absorção do norfloxacino ou a proporção

de fármaco absorvido em cada parte do trato gastrointestinal não estão

descritos na literatura. Devido a sua alta solubilidade em meio ácido, a

solubilização no estômago e absorção neste local ou na parte proximal

do intestino delgado parecem ter grande influência para a

biodisponibilidade e efeito farmacológico dos comprimidos de liberação

convencional.

2. FORMAS FARMACÊUTICAS DE LIBERAÇÃO

MODIFICADA

As formas farmacêuticas de liberação prolongada objetivam o

aumento do tempo de ação farmacológica de substâncias ativas, a

diminuição de reações adversas, a redução da administração diária, a

manutenção da eficácia terapêutica, a otimização da aderência ao

tratamento e não permissão ou minimização do aparecimento de

flutuação nas concentrações plasmáticas (AULTON, 2005; ALLEN;

POPOVICH; ANSEL, 2007; HOFFMAN, 2008). Possibilitam, também,

obter a mesma a eficácia clínica com o decréscimo dos custos do

tratamento, sendo uma alternativa econômica quando comparada com

formas de dosagem de liberação imediata (SAKS; GARDNER, 1997).

Atualmente diversos sistemas de liberação modificadas de

fármacos são estudados, desde os baseados em polímeros hidrofílicos

biocompatíveis (mais relacionados aos processos “clássicos” de

produção de medicamentos), passando pelos micro e nano-estruturados

e chegando aos avançados sistemas bio-micro-eletro-mecânico

(bioMEMS), onde sistemas microeletrônicos são produzidos com

material biocompatível (por exemplo, polimetilmetacrilato) podendo ser

programados para liberar o fármaco, contido em reservatório, de acordo

com mudanças fisiológicas detectadas pelo sistema (COLOMBO et al.,

2009).

Uma alternativa na otimização do esquema posológico é o

desenvolvimento de formas de liberação modificadas, mais

especificamente, de um sistema matricial monolítico. Este sistema

tecnológico compreende a mistura comprimida de um fármaco e um

polímero hidrofílico. A formulação de fármacos em sistemas matriciais

inertes é um dos métodos de referência utilizados para o

desenvolvimento de liberação prolongada de fármacos, principalmente

devido a apresentar vantagens como: facilidade de manufatura e custo

reduzido de produção, uma vez que se utilizam equipamentos e

processos convencionais (tais como a compressão direta e granulação),

baixa influência das variáveis fisiológicas no processo de dissolução do

princípio ativo, facilidade no controle de liberação, versatilidade na

incorporação de substâncias ativas (inclusive em grandes quantidades)

(LOPES; LOBO; COSTA, 2005; COLOMBO et al., 2009).

Resumidamente, após a dissolução do fármaco na superfície do

comprimido na água ou no suco gástrico, o polímero hidrofílico hidrata-

se e intumesce rapidamente, formando uma camada externa de gel, com

propriedades ideais para controlar a liberação do fármaco. A camada de

gel torna-se uma barreira à entrada de mais água e à transferência de

fármaco, sendo que a liberação do fármaco ocorre na medida em que o

polímero passa do estado vítreo (matriz seca) para o gelatinoso

(COLOMBO et al., 1996; COLOMBO et al., 2000; SIEPMANN;

PEPPAS, 2001; LOPES; LOBO; COSTA, 2005; OMIDIAN; PARK,

2008). A liberação do fármaco, se solúvel, ocorre por difusão pela

camada de gel, e se for insolúvel, é liberado por erosão, seguida de

dissolução. Após a erosão, a nova superfície torna-se hidratada e forma

uma nova camada de gel (COLOMBO et al., 1996; BETTINI et al.,

2001; CONTI et al., 2007). Em uma observação macroscópica do

processo de intumescimento, podem ser verificadas três distintas áreas

(frontes). O primeiro fronte, o de intumescimento, pode ser observado

pela clara separação da região intumescida da região vítrea. O segundo

fronte, chamado de fronte de erosão, é a delimitação externa do sistema.

E o terceiro fronte, chamado de difusão, é caracterizado pela região

compreendida entre os frontes de intumescimento e erosão (Figura 4)

(COLOMBO et al., 1996; BETTINI et al., 2001; LOPES; LOBO;

COSTA, 2005). A formação do gel é importante para a resistência da

matriz e é controlada pela concentração, viscosidade e estrutura química

do polímero no estado gélico. A rápida formação dessa camada externa

é essencial para a estabilidade do sistema (SIEPMANN; PEPPAS, 2001;

LOPES; LOBO; COSTA, 2005; CONTI et al., 2007; PARK et al.,

2010).

Figura 4. Diagrama dos diferentes frontes existentes em um

sistema matricial hidratado (LOPES; LOBO; COSTA, 2005).

Dos agentes poliméricos disponíveis para a obtenção de

comprimidos matriciais, o POE (Figura 5) é um polímero bastante

utilizado devido à sua boa compressibilidade, não iônico, intumescível,

e de fácil manipulação. Para a produção de comprimidos, POE pode ser

usado como um excipiente até a concentração de 5-85% (HELLER et

al., 2002; PETROVIC et al., 2009; ROWE; SHESKEY; QUINN, 2009).

Devido a sua natureza não iônica, o pH do meio não exerce significativa

influência na solubilidade e, consequentemente, no perfil de dissolução

para o POE. Qualquer alteração pH dependente será devido ao fármaco

ou outros componentes da formulação (KIM, 1995, DOW, 2002). O

mecanismo de liberação pode ser governado pelo intumescimento e

difusão do fármaco ou pelo intumescimento e erosão do polímero,

dependendo da porcentagem e da massa molecular do POE usado. Os

tipos de POE disponíveis encontram-se descritos na tabela 1. No que

tange à sua concentração na formulação e sua massa molecular, os

dados de literatura demonstram haver uma relação direta entre estes

fatores e a liberação do fármaco a partir dos comprimidos (KIM, 1995;

MAGGI et al., 2002). Geralmente, para produtos de baixa massa

molecular o mecanismo que impera na liberação do fármaco é a razão de

erosão do polímero, uma vez que, para massa molecular alta, o

intumescimento do material polimérico é o passo dominante na cinética

de liberação controlada (APICELLA et al., 1993;

MAGGI et al., 2002;

JAMZAD; TUTUNJI; FASSIHI, 2005; JAMZAD;

FASSIHI, 2006).

Figura 5.

Estrutura química do polioxietileno

Tabela 1. Tipos de polioxietileno disponíveis e respectiv

moleculares aproximada

s (ROWE; SHESKEY; QUINN, 2009).

Polioxietileno Massa

molecular (Da)

WSR N-10 100 000

WSR N-80 200 000

WSR N-750 300 000

WSR N-3000 400 000

WSR 205 600 000

WSR 1105 900 000

WSR N-12K 1 000 000

WSR N-60K 2 000 000

WSR 301 4 000 000

WSR Coagulant 5 000 000

WSR 303 7 000 000

A HPMC, conhecida t

ambém como hipromelose (Figura 6

hoje o polímero mais comumente utilizado no desenvolvimento de

sistemas matriciais de liberação de fármacos. A HPMC K, a mais

amplamente utilizada como recurso para extensão da liberação de

fármacos, está disponível em diferentes massas moleculares e é

classificada de acordo com sua viscosidade (Tabela 2)

que o POE, a HPMC tem características não iônicas, não sofrendo

influência significativa do pH do meio na

solubilidade e

camada gélica formada (DOW, 2000).

MAGGI et al., 2002;

FASSIHI, 2006).

Estrutura química do polioxietileno

Tabela 1. Tipos de polioxietileno disponíveis e respectiv

as massas

s (ROWE; SHESKEY; QUINN, 2009).

molecular (Da)

ambém como hipromelose (Figura 6

) é

hoje o polímero mais comumente utilizado no desenvolvimento de

sistemas matriciais de liberação de fármacos. A HPMC K, a mais

amplamente utilizada como recurso para extensão da liberação de

fármacos, está disponível em diferentes massas moleculares e é

Da mesma forma

que o POE, a HPMC tem características não iônicas, não sofrendo

solubilidade e

viscosidade da

Onde R: H, CH

, ou CH

CH(OH)CH

Figura 6.

Estrutura química da hidroxipropilmetilcelulose

Tabela 2. Tipos de hidroxipropilmetilcelulose disponíveis e respectiva

viscosidades (2%, p/v, 20 ºC) (ROWE; SHESKEY; QUINN, 2009).

Hidroxipropilmetilcelulose Viscosidade (mPa s)

Methocel K3 Premium LV 3

Methocel K100 Premium LVEP 100

Methocel K4M Premium 4 000

Methocel K15M Premium 15 000

Methocel K100M Premium 100 000

Dois aspectos conferem uma adequada performance à HPMC

em relação à extensão da liberação de

fármacos, a rápida formação da

camada gélica durante e hidratação e a viscosidade relacionada ao tipo

de HPMC. Uma vez que a camada de gel é formada, a viscosidade dessa

regula a razão da liberação do fármaco. Essa regulação esta relacionada

principalmente

à propriedade de dissolução e difusão do fármaco na

camada hidratada do polímero, quando este tem características

hidrofílicas. Por outro lado, fármacos com características lipofílicas

terão sua liberação regulada preferencialmente pela erosão da matriz

polimérica. Entretanto, sabe-

se que ambos os mecanismos de liberação

atuam sinergicamente, salvo as proporcionalidades decorrentes das

características de solubilidade do fármaco em questão (

SIEPMANN

PEPPAS, 2001; LOPES; LOBO; COSTA, 2005;

OMIDIAN

2008).

De uma maneira geral,

o POE apresenta uma hidratação e

geleificação mais rápida que a HPMC, sendo o fármaco mais facilmente

dissolvido no meio e a matriz hidrofílica mais susceptível à erosão.

Consequentemente, há redução da eficiência do POE em relaç

controle da liberação do fármaco, o que pode ser corrigido com a

Estrutura química da hidroxipropilmetilcelulose

Tabela 2. Tipos de hidroxipropilmetilcelulose disponíveis e respectiva

viscosidades (2%, p/v, 20 ºC) (ROWE; SHESKEY; QUINN, 2009).

Dois aspectos conferem uma adequada performance à HPMC

fármacos, a rápida formação da

camada gélica durante e hidratação e a viscosidade relacionada ao tipo

de HPMC. Uma vez que a camada de gel é formada, a viscosidade dessa

regula a razão da liberação do fármaco. Essa regulação esta relacionada

à propriedade de dissolução e difusão do fármaco na

camada hidratada do polímero, quando este tem características

hidrofílicas. Por outro lado, fármacos com características lipofílicas

terão sua liberação regulada preferencialmente pela erosão da matriz

se que ambos os mecanismos de liberação

atuam sinergicamente, salvo as proporcionalidades decorrentes das

SIEPMANN

;

OMIDIAN

; PARK,

o POE apresenta uma hidratação e

geleificação mais rápida que a HPMC, sendo o fármaco mais facilmente

dissolvido no meio e a matriz hidrofílica mais susceptível à erosão.

Consequentemente, há redução da eficiência do POE em relaç

ão ao

controle da liberação do fármaco, o que pode ser corrigido com a

quantidade e a massa molecular do POE utilizado (MAGGI; BRUNI;

CONTE, 2000; JAMZAD; FASSIHI, 2006).

A tecnologia existente hoje permite modular a liberação de

fármacos no trato gastro-intestinal a partir de sistemas farmacêuticos ao

longo de períodos que podem chegar a 24 h. Porém, para alguns

fármacos, prolongamento do tempo de retenção gástrica em

determinadas circunstâncias pode ser útil para aumentar sua

biodisponibilidade e efeito terapêutico. No caso de fármacos absorvidos

principalmente no estômago ou na parte proximal do intestino delgado

ou ainda no caso de fármacos que sejam degradados em pH alcalino as

vantagens deste tipo de dispositivos tornam-se evidentes

(BARDONNET et al., 2006; BARROCAS et al., 2007). De acordo com

Bardonnet et al. (2006) estes sistemas podem ser classificados em:

sistemas flutuantes, sistemas expansíveis, sistemas muco-adesivos e

sistemas magnéticos.

Os sistemas flutuantes, onde a forma farmacêutica flutua no

suco gástrico (figura 7), são muito promissoras, uma vez que nesta

situação o sistema fica fisicamente afastado do piloro, dificultando seu

esvaziamento gástrico. Várias estratégias podem ser adotadas para esta

flutuação, por exemplo: sistemas hidrodinamicamente equilibrados,

sistemas de baixa densidade, sistemas geradores de gás (principalmente

) (BARDONNET et al., 2006) e mais recentemente, sistemas Dome

Matrix

Figura 7. Representação de sistema flutuante (BARDONNET et

al., 2006)

Em 2006 foi apresentada tecnologia inovadora para sistemas de

liberação modificada, caracterizada por grande flexibilidade na

produção, chamada Dome Matrix

(LOSI et al., 2006). Nesta tecnologia,

módulos individuais são acoplados e a liberação será influenciada pela

maneira com que estes módulos estão conectados entre si e pelas suas

composições individuais. Cada módulo tem a forma de um disco com as

bases curvas (uma côncava e outra convexa), conforme demonstrado na

Figura 8. Neste artigo é apresentada a Dome Matrix

obtida com

polímero hidrofílico HPMC. A forma não usual deste sistema foi

projetada para permitir o encaixe de uma base côncava em uma convexa

adjacente, formando desta maneira a configuração “piled”. Outra forma

possível de encaixe são dois módulos unidos pela base côncava, neste

caso haverá um espaço interno vazio, esta configuração é denominada

de “void”. Desta forma a dose medicamentosa pode ser facilmente

ajustada ou vários sistemas de liberação podem ser montados (STRUSI

et al., 2010).

Figura 8. Módulos individuais da Dome Matrix e módulos acoplados

nas configurações “piled”, à esquerda e “void”, à direita (LOSI et al.,

2006).

Quando a configuração “void” é obtida, devido à cavidade vazia

no interior do sistema matricial, é obtido um sistema flutuante. Nesta

configuração a velocidade de dissolução do fármaco é

significativamente menor em comparação com a liberação a partir de

módulos individuais (não-unidos). Isto indica que existem diferenças

biofarmacêuticas relevantes entres os módulos individuais e acoplados.

Além disso, a geometria do sistema tem influência no intumescimento

da matriz e consequentemente na liberação.

A presença de uma base côncava e uma convexa na matriz não

altera totalmente a cinética de liberação em comparação com

formulação de base lisa (convencional). A diferença na liberação

também está relacionada à maior área superficial inicial da Dome

Matrix

. Entretanto, quando as bases individuais são analisadas, a

velocidade de liberação e a cinética são diferentes. O comportamento de

intumescimento das bases curvas dá origem a diferentes velocidades e

mecanismos de liberação. Como diferentes módulos podem ser

combinados, incluindo aqueles com base lisa, o objetivo de aumentar a

flexibilidade na produção de comprimidos pode ser atingido. Além

disso, a velocidade de liberação pode ser modificada utilizando módulos

fabricados com polímeros de diferentes massas moleculares, que

resultam em matrizes com diferentes viscosidades.

O comportamento da configuração “void” na obtenção de uma

matriz flutuante foi estudado in vitro e in vivo (STRUSI et al., 2008). A

flutuação in vitro do sistema inicia imediatamente após imersão em água

e é mantida por mais de 5 horas. Estudos in vivo confirmaram os dados

in vitro, onde o sistema manteve-se no estomago de humanos por 214,5

± 54,2 minutos. A flutuação in vivo está correlacionada com os dados

obtidos in vitro, uma vez que o sistema somente é eliminado quanto

todo o conteúdo estomacal é eliminado, e não porque o sistema perde

sua capacidade de flutuação. De fato, a ingestão de alimentos e água

prolonga o tempo de residência no estômago.

Dessa maneira os módulos Dome Matrix

acoplados na

configuração “void” podem oferecer vantagens para fármacos que se

beneficiem de um tempo prolongado de residência gástrica. Este tempo

de permanência maior no estômago seria importante para uma

formulação de liberação prolongada contendo norfloxacino, uma vez

que ele é pouco solúvel em água, mas facilmente solúvel em soluções

ácidas (F. BRAS. IV, 2001), o que pode favorecer a absorção e

consequentemente a biodisponibilidade do medicamento, inclusive com

possibilidade de redução da dosagem em cada sistema matricial.

Em relação ao tratamento utilizando antibióticos, a pesquisa de

novas formas de liberação de fármacos é de grande importância, pois a

resistência dos microorganismos a estes fármacos, seus efeitos adversos

e a não aderência do paciente ao tratamento constituem problemas

terapêuticos importantes. Na maioria dos tratamentos as concentrações

plasmáticas sub e supraterapêuticas são responsáveis pela resistência

microbiana e efeitos adversos, respectivamente. Assim, para que estas

flutuações nas concentrações plasmáticas sejam minimizadas ou

eliminadas devem ser propostas alternativas terapêuticas, tais como,

modificação da forma farmacêutica vinculada ao fármaco, que é o

objeto de estudo deste projeto.

Algumas formulações farmacêuticas do norfloxacino em β-

ciclodextrinas, dispersões sólidas de PEG-6000, nanopartículas,

lipossomas, oligopeptídeos, sistema de liberação dérmica utilizando

quitosana e complexo com alumínio foram estudadas (GUYOT et al.,

1995; FAWAZ et al., 1996; MONTERO et al., 1996; COESSENS;

SCHACHT; DOMURADO, 1997; JEON et al., 2000; ROSEEUW et al.,

2003; DENKBAS et al., 2004; KAMAL et al, 2007; BREDA et al.,

2009). Entretanto, não existem relatos de desenvolvimento de

comprimidos matriciais de liberação prolongada contendo o antibiótico

norfloxacino.

3. CARACTERIZAÇÃO E ESTABILIDADE

Utilizando métodos de análise térmica como a calorimetria

exploratória diferencial (DSC) e termogravimetria (TG), é possível

inferir sobre as características térmicas da amostra, assim como sobre a

estabilidade, a identificação através da faixa de fusão e pureza pela

respectiva entalpia de fusão. Além disso, observações referentes ao

aparecimento, mudança ou desaparecimento de eventos endotérmicos ou

exotérmicos característicos em misturas binárias homogêneas

fármaco:excipiente (1:1, m/m) pode sugerir a interação entre os

compostos e uma possível incompatibilidade. Desta forma a DSC é uma

importante ferramenta para o estudo de pré-formulação de

medicamentos (BUCKTON; RUSSEL; BEEZER, 1991; GIRON, 2002;

LIZARRAGA; ZABALETA; PALOP, 2007). Os padrões de difração de

raios-X de pó e/ou monocristal de uma substância permitem identificá-

la, bem como fornecem informações sobre sua estrutura espacial, grau

de cristalinidade e polimorfismo (STEPHENSON; FORBES;

REUTZEL-EDENS, 2001;

NEWMAN; BYRN, 2003; RODRÍGUEZ-

SPONG et al., 2004; SHAH; KAKUMANU; BANSAL, 2006;

AALTONEN, 2009). Métodos de avaliação microscópica como a

microscopia eletrônica de varredura possibilitam a avaliação de

fármacos através da observação da homogeneidade da amostra e

determinação do tamanho e forma das partículas. O polimorfismo e a

estabilidade do norfloxacino, bem como a estrutura cristalina do

norfloxacino anidro, hidratos (dihidrato, sesquihidrato) e alguns co-

cristais estão descritos na literatura (ŠUŠTAR; BUKOVEC;

BUKOVEC, 1993; FLORENCE et al., 2000; BARBAS et al., 2006;

BASAVOJU; BOSTRÖM; VELAGA, 2006; ROY et al., 2008;

BARBAS; PROHENS; PUIGJANER, 2007; PUIGJANER et al., 2010).

A cromatografia líquida tem sido empregada para determinação

quantitativa de fármacos em matérias primas, formas farmacêuticas

acabadas, estudos de dissolução, estudos de estabilidade e em matriz

biológica, pois é uma técnica bastante conhecida e dominada, possuindo

características de resolução, precisão e exatidão significativas. O

desenvolvimento de métodos cromatográficos envolve a avaliação e

otimização de condições, incluindo etapas de preparação da amostra,

separação cromatográfica, detecção e quantificação. A validação é

necessária para demonstrar, através de estudos experimentais, que o

método atende às exigências das aplicações analíticas, assegurando a

confiabilidade e reprodutibilidade dos resultados obtidos (SHABIR,

2003; BRASIL, 2003; ICH, 2005; AHUJA, 2007; BRASIL, 2008;

STÖCKL; D’HONDT; THIENPONT, 2009; BOUABIDI et al., 2010).

Com relação ao norfloxacino, estão descritos na literatura

metodologias para sua quantificação, incluindo algumas para avaliação

da estabilidade, por exemplo: por cromatografia líquida

(PARASRAMPURIA; GUPTA, 1990; CHEN; LIU; WU, 1993;

HUSSAIN; CHUKWUMAEZE-OBIAJUNWA; MICETICH, 1995;

RAO; NAGARAJU, 2004; MOHAMMAD et al., 2007), fluorimetria

(STANKOV et al., 1993; DJURAJEVIC; JELIKIC-STANKOV;

STANKOV, 1995; EL WALILY; BELAL; BAKRY, 1996),

espectrofotometria (EL KHATEEB; RAZEK; AMER, 1998; EL

WALILY et al., 1999; RAHMAN; AHMAD; AZMI, 2004), eletroforese

capilar de zona e quimiluminescência (ALNAJJAR; ABUSEADA;

IDRIS, 2007a; ALNAJJAR; IDRIS; ABUSEADA, 2007b; LIU et al.,

2010), absorção atômica, condutimetria e colorimetria (RAGAB;

AMIN, 2004).

A absorção de fármacos após administração oral depende da sua

liberação da forma farmacêutica, da dissolução ou solubilização sob

condições fisiológicas e de permeabilidade através do trato

gastrintestinal. Os estudos de dissolução in vitro fornecem informações

úteis tanto para a pesquisa e desenvolvimento quanto para a produção e

controle de qualidade. A avaliação do perfil de dissolução permite a

comparação de produtos de diferentes fabricantes, otimização de

formulações e avaliação da influência de alterações realizadas na

formulação (MOORE; FLANNER, 1996; COSTA; LOBO, 2001;

DOKOUMETZIDIS; MACHERAS, 2006; AZARMI et al., 2007).

A estabilidade é o período de tempo em que uma forma

farmacêutica mantém suas propriedades dentro de limites pré-

estabelecidos. O produto deve preservar suas características

farmacotécnicas, organolépticas e microbiológicas, além de eficácia

terapêutica e ausência de toxicidade. Todos os fármacos estão sujeitos a

alguma forma de decomposição química ou física. Algumas classes

químicas são mais vulneráveis e tendem a se decompor mesmo em

condições brandas. A escolha dos excipientes também pode influenciar

na estabilidade física, química e biodisponibilidade. Desta forma, deve-

se respeitar a compatibilidade com os excipientes escolhidos, os quais

são componentes importantes contidos nas formulações que podem

significar melhorias das características, mas podem também reduzir a

eficácia de algumas preparações.

A segurança de um fármaco e da forma farmacêutica sólida na

qual está veiculado é afetada por vários fatores, como temperatura,

umidade, luz e ar, que aumentam a velocidade das reações intrínsecas. O

conhecimento do mecanismo de degradação é importante para definir

prazo de validade e condições de armazenagem específicas. Para

mimetizar o tempo de análise da degradação química e/ou física dos

comprimidos, submete-os a condições típicas de estresse, denominado

estudo acelerado de estabilidade. No Brasil, país classificado na região

climática IV (quente e úmido), as formulações são avaliadas após

permanecerem durante seis meses a 40 ± 2 ºC e 75 ± 5% de umidade

relativa. Um medicamento é considerado estável se suas características

físicas e químicas não sofrerem mudanças, restando acima de 90% da

concentração inicial (ICH, 1996; BRASIL, 2005; WHO, 2009).

Na exposição prolongada à luz, alguns grupos funcionais do

norfloxacino se decompõem (CÓRDOBA-BORREGO; CÓRDOBA-

DÍAZ; CÓRDOBA-DIAZ, 1999; MUSA; ERIKSSON, 2009). Além

disso, seu aquecimento prolongado em meio ácido produz um produto

de degradação descarboxilado, que está relacionado a precipitações em

formulações injetáveis e que também pode ser encontrado como

impureza em algumas matérias-primas (EL KHATEEB; RAZEK;

AMER, 1998; BORREGO; DIAZ; DIAZ, 1999; ALNAJJAR; IDRIS;

ABUSEADA, 2007b). A partir dos resultados dos estudos de

estabilidade, o formulador pode obter informações importantes sobre a

necessidade do revestimento da forma farmacêutica, tipo de embalagem

necessária para a comercialização do produto final, etc. Além disso, os

estudos de estabilidade podem comprovar uma incompatibilidade

fármaco/excipiente sugerida no estudo de pré-formulação.

Dessa maneira, a completa caracterização das matérias-primas de

fármacos e excipientes, o estudo minucioso do processo tecnológico

empregado na obtenção de um comprimido de liberação prolongada,

bem como a avaliação da qualidade do produto final, são etapas

fundamentais para que o produto tenha características tais que seja

viável sua produção em larga escala, com melhora na qualidade de vida

do paciente.

CAPÍTULO 2 - Caracterização térmica e estudos de

compatibilidade fármaco/excipiente.

INTRODUÇÃO

A caracterização no estado sólido de fármaco e excipientes é

etapa essencial para o desenvolvimento racional de um novo

medicamento. Os resultados obtidos por diversas técnicas servem como

parâmetros para a aquisição de matérias-primas com adequada

qualidade e para garantir a homogeneidade e reprodutibilidade na

produção industrial da formulação final, assegurando sua eficácia,

segurança e qualidade. Além disso, o polimorfismo e diferenças de

cristalinidade de fármacos devem ser investigados, uma vez que

diferentes formas cristalinas podem apresentar diferentes propriedades

físicas e físico-químicas que alteram a biodisponibilidade e terapêutica.

(BYRN et al., 2001; STEPHENSON; FORBES; REUTZEL-EDENS,

2001; GIRON et al., 2002).

No desenvolvimento de formulações é fundamental avaliar se

há interação química e/ou física entre o princípio-ativo e excipientes,

uma vez que estas interações podem afetar negativamente a estabilidade

e a biodisponibilidade da formulação final. Não existe protocolo padrão

determinando como realizar estudos de compatibilidade (KISS et al.,

2006; BRUNI et al., 2010). Normalmente métodos termoanalíticos

como DSC e TG são utilizados. Outras técnicas também podem ser

aplicadas para melhor compreensão e confirmação dos resultados

obtidos. De maneira geral, os estudos de compatibilidade são realizados

comparando-se o perfil termoanalítico do fármaco com o obtido de uma

mistura preparada na proporção 1:1 (p/p) fármaco:excipiente. O

surgimento, mudança ou desaparecimento de eventos térmicos são

considerados como interação, o que pode indicar uma possível

incompatibilidade (BRUNI et al., 2002; MURA; GRATTERI; FAUCCI,

2002; BERNARDI et al., 2009; BRUNI et al., 2010).

Neste capítulo são apresentados resultados da caracterização do

norfloxacino, POE e HPMC por DSC, TG, SEM, XRPD e DRIFT, bem

como o cálculo da energia de ativação do fármaco por método

isotérmico e não-isotérmico. Também são demonstrados estudos de

compatibilidade entre o norfloxacino e alguns excipientes que poderiam

compor a formulação matricial final.

Thermal Characterization and Compatibility Studies of Norfloxacin

for Development of Extended Release Tablets

P. R. Oliveira*, L. S. Bernardi, F. S. Murakami, C. Mendes and M. A. S.

Silva

Department of Pharmaceutical Sciences, Health Science Centre, Federal

University of Santa Catarina, 88040-900, Florianópolis-SC, Brazil

*Author for correspondence: prenato.oliveira@gmail.com

Abstract

Norfloxacin (NFX) is a synthetic antibacterial drug. The development of

extended release tablets improves the patients’ comfort and compliance,

resulting in lower discontinuation of the therapy; with consequently

decrease in bacterial resistance. In the present work, the thermal

behavior of NFX was investigated using TG and DSC techniques.

Isothermal and non-isothermal methods were employed to determine

kinetic data of decomposition process. Compatibility studies between

NFX and pharmaceutical excipients, including three hydrophilic

polymers were carried out in order to develop a new formulation of

NFX to obtain extended release tablets with an approved quality.

Keywords: Norfloxacin, Thermal characterization, Kinetic Studies,

Compatibility Studies

Introduction

Norfloxacin, chemically known as 1-ethyl-6-fluoro-1,4-

dihydro-4-oxo-7-(1-piperazinyl)-1-ethyl-fluoro-1.4-dihydro-4-oxo-7-(1-

piperazinyl)-3-quinoline-carboxylic acid (Fig. 1A) [1]. It is currently

used as a broad spectrum antibacterial drug, being the firstly selected

drug for the treatment of diseases caused by Campylobacter, E. coli,

Salmonella, Shigella and V. cholera [2-3]. The drug is also used for the

treatment of gonorrhoea as well as infection of eyes and urinary tract

[2]. Resistance in Gram-negative bacteria has become common, making

the therapeutic decisions more difficult. Increasing bacterial resistance

to currently available quinolones has reduced their effectiveness and

may compromise future use of this class of drugs [4-5].

The development of controlled-release formulations is a

successful area in the pharmaceutical industry because expenses of new

drug development are very high, and novel innovation is at an all-time

low. Hydrophilic matrices are one of the most used controlled delivery

systems in the world, due to the simple technology and low cost. Among

the various hydrophilic polymers employed, hydroxypropyl

methylcellulose (HPMC, Fig. 1B) is the most commonly used, due to its

versatility, compatibility with many drugs and safety [6] Nevertheless,

high molecular mass polyethylene oxides (PEOs, Fig. 1C) have been

proposed as an alternative to HPMC [7].

Figure 1. Chemical structure of NFX (A), HPMC (B) and PEOs (C).

Thermoanalytical techniques measure changes in physical

and/or chemical properties of the sample as a function of temperature.

There are many possible applications in pharmaceutical industry, for

example, identification, characterization of active and inactive

ingredients, routine analysis, quality control and stability study [8-9,].

Kinetic parameters (activation energy, frequency factor and reaction

order) can be measured by thermoanalytical methods according to

progress of reactions [10-12]. The successful formulation of a stable

and effective solid dosage form depends on the careful selection of the

excipients [8,13] and on the characterization of solid-state properties

using appropriate analytical methodologies [14-16]. The compatibility

studies using thermal analysis present advantageous to readily available

knowledge of any physical and chemical interactions between drugs and

excipients which might give rise to changes in chemical nature, stability,

solubility, absorption and therapeutic response of drugs [12]. In

particular differential scanning calorimetry has been proposed as a rapid

method for evaluating physicochemical interactions between

components of the formulation through comparison of thermal curves of

pure substances with curve obtained from a 1:1 mixture, and therefore

selecting excipients with suitable compatibility [10,15,17-21].

The aim of this study was to perform the physicochemical solid-

state characterization of norfloxacin and different polymers, to analyze

the kinetic parameters under isothermal and non-isothermal conditions,

and to carry out compatibility studies, to begin the development of a

new formulation of NFX extended release tablets.

Experimental

Materials

Norfloxacin (NFX) bulk material was kindly donated by União

Química Farmacêutica Nacional (Embu-Guaçu, SP, Brazil). The

polymers tested were: Polyox WSR N80 NF, Polyox WSR 301 NF, and

Methocel K100 Premium LV CR (all from Colorcon do Brasil, São

Paulo, Brazil). The pharmaceutical excipients tested were

microcrystalline cellulose, magnesium stearate, colloidal silicon dioxide,

lactose monohydrated and Opadry II White.

Methods

Differencial Scanning Calorimetry (DSC) and Thermogravimetric (TG)

Analysis

The DSC curves were obtained on Shimadzu DSC-60 cell

(Kyoto, Japan) using aluminum crucibles with about 1.5 mg of samples.

The temperature range was from 30 to 500 ºC at a heating rate of 10 ºC

min

-1

in dynamic N

atmosphere with the flow rate of 50 mL min

-1

. The

DSC equipment was preliminarily calibrated with standard reference of

indium (m.p. 156.6

C; ∆H

fus

=-28.54 J g

-1

) and zinc (m.p. 419.5

C).

The compatibility studies were performed with binary mixtures of NFX

and each excipient (1:1; m/m). TG experiments were measured on

Shimadzu thermobalance model TGA-50 (Kyoto, Japan) in the

temperature range of 30–800 °C, using platinum crucibles with

approximately 4 mg of samples, under dynamic N

atmosphere (50 mL

min

–1

) at a heating rate of 10 °C min

–1

. The equipment was preliminarily

calibrated with standard reference of calcium oxalate. Non-isothermal

kinetic investigation of NFX was performed from TG data by

application of Ozawa’s method [22]. The graph of mass loss versus

temperature of five TG curves was obtained at different heating rates

(2.5, 5.0, 10, 15, and 20 °C min

–1

), under N

atmosphere. For isothermal

method, the temperature was from 230 to 270 °C, with 10 °C

temperature increment, in N

atmosphere. A graphic of lnt vs 1/T (K

–1

)

was plotted and linear regression was applied.

X-ray powder diffraction (XRPD)

For characterization of crystallinity, X-ray diffraction patterns

were obtained on a Siemens diffractometer model D 5000, with tube of

CuKα, voltage of 40 kV and current of 40 mA, in the range of 3–40 (2θ)

with a pass time of 1 second.

Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFT)

The DRIFT spectra were measured in a Shimadzu

spectrophotometer (Prestige), in a scan range of 400 - 4000 cm

-1

with an

average of over 32 scans at a spectral resolution of 4 cm

-1

in KBr. A

background spectrum was obtained for each experimental condition.

Scanning Electron Microscopy (SEM)

The photomicrographs of NFX and the polymers were observed

by using a Phillips scanning electron microscope, model XL30. Samples

were mounted onto metal stubs using double-side adhesive tape,

vacuum-coated with gold (350 Å) in a Polaron E 5000 and directly

analyzed under SEM (N=50, 200, and 1000).

Results and discussion

DSC curve of norfloxacin (Fig. 2A) showed a sharp

endothermic event (T

peak

) at 219.4 °C (∆H

fusion

= -101.51 J g

-1

)

corresponding to melting point followed by an exothermal event. The

decomposition was defined in two major endothermic stages. This was

confirmed by TG/DTG curves that indicated thermal decomposition in

the following temperature range: 330–376 °C (∆m=47.4 %) and 421-

455 °C (∆m=27.8 %). The DSC curve of Polyox WSR 301 (Fig. 2B)

and Polyox WSR N80 (Fig. 2C) showed sharp endothermic peaks at

69.3 °C (∆H

fusion

= -162.70 J g

-1

) and 65.3 °C (∆H

fusion

= -180.36 J g

-1

respectively, corresponding to melting event. The TG/DTG curves

indicated one thermal decomposition step in the temperature range of

397–433 °C (∆m=95.9 %) for Polyox WSR 301 and 394–432 °C

(∆m=95.8 %) for Polyox WSR N80. The similarity of these thermal

decomposition profiles can be explained by the same polymer chemical

structure; the only difference is the molecular mass, 4,000,000 Da

(Polyox WSR 301) and 200,000 Da (Polyox WSR N80). Different

molecular masses usually are tested in the development of extended

release tablets because its increase leads to an increase in gel strength,

which tends to decrease the diffusion of the drug from the matrix. DSC

curve of Methocel K100 LV (Fig. 2D) showed a broad endothermic

event between 93-140 °C (∆H = -120.97 J g

-1

) and TG/DTG curves

indicated thermal decomposition in the temperature range of 360–394°C

(∆m=83.6 %).

Based on the photomicrographs obtained from scanning

electron microscopy, orthorhombic crystals were observed for NFX

(Fig. 3A). A particle size variation can be visualized for both Polyox

samples (Fig. 3B and 3C). An amorphous characteristic was observed

for Methocel K100 (Fig. 3D).

Figure 2. DSC and TG/DTG curves of norfloxacin (A), Polyox WSR

301 (B), Polyox WSR N80 (C), and Methocel K100 LV CR (D) in

dynamic nitrogen atmosphere (50 mL min

-1

) and heating rate of 10 ºC

min

-1

Figure 3. SEM of NFX (A), Polyox WSR 301 (B), Polyox WSR N80

(C), and Methocel K100 LC CR (D). The photomicrograph A was taken

at a magnification of 50x, and B, C and D of 100x.

X-ray powder diffraction studies were performed in order to

obtain more information about the crystalline characteristics. The 2θ

values of the diffraction peaks (Fig. 4) for NFX were 2θ = 7.87, 9.93,

10.63, 11.98, 13.38, 14.98, 16.08, 18.88, 20.78, and 25.08. For both

Polyox polymers, only two intensive peaks were observed: 2θ = 19.13

and 23.33. For Methocel K100, only two broad peaks, with low

intensity, between 2θ = 5.7-13.3 and 15.53–25.5, were observed,

indicating an amorphous state for this polymer.

Figure 4. X-ray diffraction spectra of NFX (A), Polyox WSR 301 (B),

Polyox WSR N80 (C), and Methocel K100 LC CR (D).

The IR spectra of quinolones are more representative in the

region 1800–1300 cm

−1

[23]. The IR spectrum (Fig. 5) of NFX exhibits

a stretching vibration band at about 1716 cm

−1

(–COOH stretching) and

1631 cm

−1

(pyridone keto). For Polyox WSR 301 and N80, it was

observed bands in 2915 and 1465 cm

-1

(streching –CH

-) and intense

bands in 1150 – 1085 cm

-1

, which is attributed to asymmetric axial

deformation C–O–C, characteristic of aliphatic ethers, confirming the

identification of the polymers. Methocel K100 IR spectra showed

absorption bands at 3440 cm

-1

(O-H stretching), 2904 cm

-1

(C-H

stretching), 1643 cm

-1

(C=O), and 1066 cm

-1

(C-O-C).

Figure 5. DRIFT spectra of Norfloxacin (A), Polyox WSR 301 (B),

Polyox WSR N80 (C), and Methocel K100 LC CR (D).

For non-isothermic study, the superposition of the TG curves of

NFX is shown in Fig. 6A. Ozawa’s method was applied in order to

determine the activation energy (Ea), Arrhenius frequency factor (A)

and order of reaction at the beginning of first thermal decomposition

step at around 300 to 350 °C. The Ea, calculated was 126 kJ mol

–1

, the

Arrhenius frequency factor was 4.029 x 10

min

-1

and order of reaction

followed a zero order reaction (n = 0).

Figure 6. (A) TG curves obtained for the non-isothermic study of NFX

at 2.5, 5, 10, 15, and 20 ºC min

-1

. (B) Isothermal TG curves of NFX

obtained between 230 and 270 ºC, with a temperature increment of 10

ºC.

The isothermal TG curves of NFX are illustrated in Fig. 6B.

These curves were used to obtain a graphic of lnt vs. the reciprocal of

temperature 1/T (K

–1

). From this linear regression method, the equation

obtained was y= –16.098x + 26.382 (R=0.9982). The activation energy

calculated from the product of 16.098 with the molar gas constant

(R=8.314) and was Ea=134 kJ mol

–1

. This result is in agreement with

the value obtained from the dynamic method. The selection of adequate

excipients for a new formulation is based on the characteristics of the

drug and its compatibility with other components. Moreover, excipients

can influence the dissolution profile affecting the bioavailability of the

drug. The results from the compatibility studies between NFX and

excipients are shown in Fig. 7, where the DSC curves can be considered

as superposition of the curves of pure compounds indicating that there is

no interaction, and therefore no physical-chemical incompatibility.

Figure 7. DSC curves of NFX and excipients obtained in dynamic

nitrogen atmosphere (50 mL min

–1

) and heating of rate 10 °C min

-1

Conclusion

The thermal behaviour and the solid-state characterization of

NFX and the polymers were carried out by means of TG, DSC, DRIFT,

SEM, and XRPD. The obtained isothermal and non-isothermal kinetic

parameters can be used as reference values for the routine quality

control of NFX. The results demonstrated the applicability of DSC as a

fast screening tool for selection of adequate excipients at the early stages

of pre-formulation studies. No interaction was observed for NFX and

the tested excipients, making feasible the development of a high quality

formulation of NFX extended release tablets.

Acknowledgments

The authors wish to thank CNPq and CAPES for the support.

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[23] Shaikh AR, Giridhar R, Yadav MR. Bismuth-norfloxacin complex:

synthesis, physicochemical and antimicrobial evaluation. Int J Pharm

2007;332:24-30.

CAPÍTULO 3 – Desenvolvimento e validação de metodologia

analítica.

INTRODUÇÃO

A análise do fármaco é necessária nas diversas fases do

desenvolvimento farmacêutico, como estudos de formulação e controle

de qualidade da forma farmacêutica. Após o desenvolvimento analítico

a validação da metodologia é necessária para atestar, de forma

documentada, que o procedimento fornece resultados reprodutíveis,

precisos, exatos, específicos e confiáveis, adequados aos fins

pretendidos (SHABIR, 2003; BRASIL, 2003; ICH, 2005; STÖCKL,

2009).

Para atingir o objetivo de se avaliar a estabilidade de

formulações farmacêuticas, o método analítico deve demonstrar

adequada capacidade de separar e, se possível, identificar os produtos de

degradação formados. Para tanto, submete-se o fármaco a diversas

condições de stress, tais como: hidrólise ácida, hidrólise básica, fotólise,

oxidação, temperatura (ICH, 2005; BRASIL, 2005; ALSANTE et al.,

2007; BRASIL 2008). Desta forma, procura-se demonstrar que se

alguma substância for formada, o método será capaz de separar e até

quantificar.

O presente capítulo tem como objetivo demonstrar os resultados

do desenvolvimento e validação de metodologia analítica para

quantificação de norfloxacino em sistemas matriciais de liberação

prolongada. Com o objetivo de avaliar se o método é adequado para os

ensaios de estabilidade (em câmara climática, fotoestabilidade) foi

realizado estudo de especificidade em condições de stress e a produção

de um produto de degradação (norfloxacino descarboxilado) de acordo

com procedimento descrito na literatura (EL KHATEEB; RAZEK;

AMER, 1998).

Liquid Chromatographic Determination of Norfloxacin in Extended

Release Tablets

Paulo R. Oliveira*

, Larissa S. Bernardi

, Cassiana Mendes

, Simone G.

Cardoso

, Maximiliano S. Sangoi

, and Marcos A. S. Silva

a Department of Pharmaceutical Sciences, Health Science Centre,

Federal University of Santa Catarina, 88040-900, Florianópolis-SC,

Brazil.

b Faculty of Pharmacy, Federal University of Rio Grande do Sul,

90610-000, Porto Alegre-RS, Brazil.

* Corresponding author: prenato.oliveir[email protected]

Abstract

A stability indicating reversed-phase liquid chromatography

method is developed and validated for the determination of norfloxacin

in a new formulation of extended release tablets. The LC method is

carried out on a Luna C

column (150 x 4.6 mm), maintained at 40 ºC.

The mobile phase is composed of phosphate buffer 0.04 M, pH 3.0

/acetonitrile (84:16, v/v), run at a flow rate of 1.0 mL/min, and detection

at 272 nm. The chromatographic separation was obtained within 10 min

and it is linear in the concentration range of 0.05-5 µg/mL. Validation

parameters such as the specificity, linearity, precision, accuracy, and

robustness were evaluated, giving results within the acceptable range.

Moreover, the proposed method was successfully applied for the assay

of norfloxacin in the developed formulations.

Keywords: Norfloxacin, Extended release, Liquid chromatography,

Method validation.

Introduction

Norfloxacin, chemically known as 1-ethyl-6-fluoro-1,4-

dihydro-4-oxo-7-(1-piperazinyl)-1-ethyl-fluoro-1.4-dihydro-4-oxo-7-(1-

piperazinyl)-3-quinoline-carboxylic acid (Figure 1) (1), is currently used

as a broad spectrum antibacterial drug, being the first choice drug for the

treatment of diseases caused by Campylobacter, E. coli, Salmonella,

Shigella and V. cholera (2,3). The drug is also used for the treatment of

gonorrhea as well as infection of eyes and urinary tract (2). Resistance

in Gram-negative bacteria has become common, making the therapeutic

decisions more difficult. Increasing bacterial resistance to currently

available quinolones has reduced their effectiveness and may

compromise future use of this class of drugs (4,5).

Figure 1. Chemical structure of norfloxacin.

The development of controlled-release formulations is a

successful area in the pharmaceutical industry because expenses of new

drug development are very high, and true innovation is at an all-time

low. Hydrophilic matrices are one of the most used controlled delivery

systems in the world, due to the simple technology and low cost. Among

the various hydrophilic polymers employed, hydroxypropyl

methylcellulose (HPMC) is the most commonly used, due to its

versatility, compatibility with many drugs and safety (6). Nevertheless,

high molecular weight polyethylene oxides (PEOs) have been proposed

as an alternative to HPMC (7). The correct choice of the hydrophilic

polymer, molecular weight and quantity in the matrix formulation can

provide an appropriate combination of swelling, dissolution or erosion

mechanisms to control drug release kinetics (8,9).

Moreover, drug release at a constant rate is often desirable, to

maintain the plasmatic levels of the drug in the therapeutic range,

thereby avoiding the peak and valley profile characteristics of

conventional dosage forms in a multidose regime (10), which, for

antibiotics, is very important, mainly in combination with a better

patient compliance to the therapy, to avoid the discontinuation of the

treatment and the development of resistant microorganisms.

Analytical methods have been published for the determination

of NFX in pharmaceuticals by means of spectrophotometry, liquid

chromatography (LC) and capillary electrophoresis, and some of them

are listed in the references (11-16).

As the excipients (including polymeric ones) and the

technological process can affect the stability of the active

pharmaceutical ingredient and the evaluation of the stability-indicating

capability of LC methods becoming mandatory by the surveillance

agencies (17,18), the aim of the present work was to develop and

validate a stability-indicating method for the quality assessment of the

new formulation of norfloxacin extended release tablets.

Experimental

Chemical and reagents

Norfloxacin reference standard was kindly provided by

Brazilian Pharmacopeia and norfloxacin raw material was from União

Química Farmacêutica Nacional (Embu-Guaçu, SP, Brazil). The

polymers used in this study, Polyox

WSR N80, Polyox

WSR 301,

Polyox

WSR 303, Polyox

N60K, Methocel

K100 LV, Methocel

K100M, and Methocel

K4M were kindly provided by Colorcon do

Brazil Ltda (São Paulo, SP, Brazil). Others excipients used were:

microcrystalline cellulose (Microcel

102, Blanver, Itapevi, SP, Brazil),

magnesium stearate (M. Cassab, São Paulo, SP, Brazil), and colloidal

silicon dioxide (Aerosil

, Labsynth, Diadema, SP, Brazil). HPLC-grade

acetonitrile was purchased from Tedia (Fairfield, OH, USA). For all the

analyses, ultrapure water was purified using a Milli-Q Gradient System

(Millipore, Bedford, USA).

Samples

The composition of each NFX tablet formulation was: NFX

(700 mg), polymer (20 or 30%), magnesium stearate (1%), colloidal

silicon dioxide (0.5%), and microcrystalline cellulose (qs 1,07 g). For

the production of the extended release tablets, NFX and excipients were

mixed for 10 min and then compressed by direct compression (Fellc

compressing model F-10/8, São Paulo, SP, Brazil).

Methods

Liquid chromatography (LC)

A Shimadzu LC system (Shimadzu, Kyoto, Japan) was used

equipped with a SCL-10A

system controller, LC-10 AD pump, DGU-

14A degasser, CTO-10AS

column oven, SPD-10A

UV detector, and

a SPD-M10A

photodiode array detector. The detector was set at 272

nm and peak areas were integrated automatically by computer using a

Shimadzu Class VP



V 6.12 software program. The experiments were

carried out on a reversed-phase Phenomenex (Torrance, USA) Luna C

column (150 mm x 4.6 mm I.D., with a particle size of 5 µm and pore

size of 100 Å). A security guard holder (4.0 mm x 3.0 mm I.D.) was

used to protect the analytical column. The LC system was operated

isocratically at 40 ºC using a mobile phase of phosphoric acid 0.04 M,

pH 3.0/acetonitrile (84:16, v/v). This was filtered through a 0.45 µm

membrane filter and run at a flow rate of 1.0 mL/min. The injection

volume was 20 µL for both standard and samples.

Diffuse Reflectance Infrared Fourier Transform Spectroscopy

(DRIFT)

The DRIFT spectra were measured in a Prestige

spectrophotometer (Shimadzu, Kyoto, Japan), in a scan range of 400 -

4000 cm

-1

with an average of over 32 scans at a spectral resolution of 4

-1

in KBr. A background spectrum was obtained for each

experimental condition.

Mass spectrometry (MS)

The MS experiments were performed on a triple quadrupole

mass spectrometer (Micromass, Manchester, UK), model Quattro LC,

equipped with an electrospray ionization (ESI) source in positive mode,

set up in scan mode, using a Masslynx (v 3.5) software program. The

samples were introduced into the mass spectrometer by direct infusion

at 10 µL/min, diluted in mobile phase. The best response for NFX was

obtained with electrospray capillary potential of 3 kV, cone voltage of

30 V, RF lens voltage of 0.3 V, source temperature of 120 ºC, and ESI

probe temperature of 400 ºC. The mass spectrometry data were acquired

in the m/z range between 100 and 550 amu.

Procedure

Preparation of reference solutions

The stock solutions of norfloxacin were prepared by weighing

50 mg, transferred to individual 50 mL volumetric flasks, dissolved with

0.2 mL of acetic acid glacial, and diluted to volume with mobile phase,

obtaining a concentration of 1 mg/mL. The stock solutions were stored

at 2-8 °C protected from light. Working standard solutions were

prepared daily by diluting the stock solutions to an appropriate

concentration in mobile phase.

Preparation of decarboxylated norfloxacin (DCN)

The hydrolysis of NFX during prolonged heating of its acid

solution yields a decarboxylated degradant (DCN), which was prepared

based on a described procedure (11). 250 mg of NFX was refluxed with

70 ml of hydrochloric acid 2 M at 150 °C for 48 h, protected from light.

Then, the solution was cooled and adjusted to pH 7.5 with sodium

hydroxide 2 M. After that, the solution was evaporated under vacuum to

dryness. The residue was extracted with ethanol and filtered. To verify

the identity of the obtained product, a sample was analyzed by means of

DRIFT, LC-MS, and the proposed LC method.

Preparation of sample solutions

To prepare the sample stock solution, the obtained extended

release tablets were crushed to a fine powder. An appropriated amount

was transferred into an individual 50 mL volumetric flask, dissolved

with 0.2 mL of acetic acid glacial, and diluted to volume with mobile

phase, obtaining a concentration of 1 mg/mL of the active

pharmaceutical ingredient. This solution was stored at 2-8 °C protected

from light. Working sample solutions were prepared daily by diluting

the sample stock solutions to an appropriate concentration in mobile

phase.

Validation of the method

Analytical method development and validation play a major

role in the discovery, development, and manufacture of pharmaceuticals

(19). The International Conference on Harmonization (ICH) (20)

requires the stress testing to be carried out to elucidate the inherent

stability characteristics of the active substance. A stability-indicating

method is the one that quantifies the drug and also resolves its

degradation products (17,18,21). The method was validated to quantify

NFX in a new formulation of extended release tablets by the

determination of the following parameters: specificity, linearity,

accuracy, precision, robustness, and quantitation and detection limits.

Specificity

In order to determine the specificity of the method, a placebo

solution was analyzed to evaluate the absence of interference from the

formulation excipients (including the polymers) on the NFX peak.

Moreover, the specificity was determined by subjecting a sample

solution (1 mg/mL) to accelerated degradation by acidic, basic, neutral,

oxidative, and photolytic conditions. After the procedures, the samples

were diluted in mobile phase to a final concentration of 1 µg/mL. A

sample solution in 5 M hydrochloric acid and 5 M sodium hydroxide,

both refluxed at 100 ºC for 24 h, were used for the acidic and basic

hydrolysis, respectively. The oxidative degradation was induced by

storing the sample solution in 30% hydrogen peroxide, at ambient

temperature for 24 h, protected from light. Photodegradation was

induced by exposing the samples to 200 watt hours/square meter of near

ultraviolet light. Then, the specificity of the method was established by

determining the peak purity of norfloxacin in degradated samples using

a PDA detector.

Linearity and range

Linearity was determined by constructing three independent

calibration curves. For the construction of each calibration curve seven

standard concentrations of NFX in the range of 0.05–5 µg/mL were

prepared in mobile phase. Three replicates of 20 µL injections were

made for the standard solution to verify the repeatability of the detector

response at each concentration. The peak areas of the chromatograms

were plotted against the concentrations of NFX to obtain the calibration

curve. The seven concentrations of the standard solutions were

subjected to regression analysis by the least squares method to calculate

calibration equation and correlation coefficient.

Precision and accuracy

The precision of the method was determined by repeatability

and intermediate precision. Repeatability was examined by six

evaluations of the same concentration sample, on the same day, under

the same experimental conditions. The intermediate precision was

assessed by carrying out the analysis on three different days (inter-days)

and also by other analysts performing the analysis in the same

laboratory (between-analysts). The accuracy was evaluated by the

recovery of known amounts (0.3, 0.5 and 0.7 µg/mL) of the reference

substance added to a sample solution (containing 0.50 µg/mL of NFX

and tablet excipients) to obtain solutions with final concentrations of

0.80, 1.0, and 1.2 µg/mL, corresponding to 80, 100, and 120% of the

nominal analytical concentration, respectively. The accuracy was

calculated as the percentage of the drug recovered from the formulation

matrix.

Limits of quantitation (LQ) and detection (LD)

The LQ was taken as the lowest concentration of analyte in a

sample that can be determined with acceptable precision and accuracy,

and the LD was taken as the lowest absolute concentration of analyte in

a sample that can be detected but not necessarily quantified. The LD and

LQ were calculated from the slope and the standard deviation of the

intercept of the mean of three calibration curves, determined by a linear

regression model, as defined by ICH.

Robustness

Two approaches are possible to evaluate robustness, either an

one-variable-at-a-time (OVAT) procedure or an experimental design

procedure. The OVAT procedure varies the levels of one factor while

keeping the other factors at nominal levels, to evaluate the effect of this

former factor on the method response(s). When applying an

experimental design, the effect of a given factor is calculated at several

level combinations of the other factors. Thus, in an experimental design,

a reported factor effect is an average value for the whole domain, and it

represents more globally what is happening around the nominal situation

(22-24).

The robustness was determined by analyzing the same samples

under a variety of conditions of the method parameters, such as: flow

rate, column temperature, changing the mobile phase composition and

pH. The response surface method (RSM) design was applied to evaluate

the relationships between one or more measured responses. Moreover,

the D-optimal criteria was used to select design points to minimize the

variance associated to the estimates of specified model coefficients, with

a low number of experiments.

System suitability

The system suitability was carried out to evaluate the resolution

and reproducibility of the system for the analysis to be performed, using

six replicate analyses of the drug at a concentration of 1 µg/mL. The

parameters evaluated were peak area, retention time, theoretical plates,

and asymmetry.

Analysis of the extended release tablets

For the quantitation of NFX in the extended release tablets,

twenty tablets of each batch were separated, accurately weighed and

crushed to a fine powder. An appropriate amount of each tablet was

transferred into an individual 50 mL volumetric flask, dissolved with 0.2

mL of acetic acid glacial, sonicated for 15 min, diluted with mobile

phase (sonicated again for 15 min), and diluted to volume, obtaining the

NFX final concentration of 1 mg/mL (stock solutions). For the analysis,

the stock solutions were diluted to appropriate concentrations with

mobile phase. An aliquot of 20 µL was injected for the analysis and the

amount of each drug per tablet calculated against the respective

reference standard.

Results and discussion

To obtain the best chromatographic conditions, the mobile

phase was optimized to provide adequate peak symmetry and

sensitivity. Potassium phosphate, sodium phosphate, sodium acetate,

formic acid, and phosphoric acid buffers were tested. Methanol was

tested as the organic solvent; however a broad and non-symmetric peak

was obtained. The use of phosphoric acid 0.04 M (pH 3.0) in

combination with acetonitrile, which was optimized to 16%, at 40 ºC,

resulted in a relatively short retention time of 6.6 min, better peak

symmetry (1.11), and a simple mobile phase (without salt buffer

addition). For the selection of the best wavelength detection a PDA

detector was used. The optimized conditions of the LC method were

validated for the analysis of NFX in the developed tablets and a typical

chromatogram obtained by the proposed LC method is shown in Figure

2A.

A stability-indicating method is defined as an analytical method

that accurately quantitates the active ingredients without interference

from degradation products, process impurities, excipients, or other

potential impurities. Forced degradation studies should be the first step

in method development. The presence of degradants and impurities in

pharmaceutical formulations can result in changes in their chemical,

pharmacological and toxicological properties affecting their efficacy and

safety. Therefore, the adoption of stability-indicating methods is always

required to control the quality of pharmaceuticals during and after the

production. This greatly contributes to the possibility of improving drug

safety (17,18, 25,26).

Figure 2. LC chromatograms of norfloxacin (A) developed formulation

(1 µg/mL). (B) Decarboxylated degradant. (C) After acidic condition.

(D) After basic condition. (E) After photolytic condition. (F) After

oxidative condition. (G) After neutral condition. Chromatographic

conditions: Luna C

column (150 x 4.6 mm, 5 µm), 40 ºC; mobile

phase: phosphate buffer 0.04 M, pH 3.0 /acetonitrile (84:16, v/v); flow

rate: 1.0 mL/min; detection: 272 nm.

For NFX, its decarboxylated degradant has a particular

significance since the pharmacological activity of the drug depends on

the carboxylic group (27). On the other hand, this degradant was

recorded as impurity in the bulk form and precipitate in the injection

formulation (11). The prepared degradant (DCN) and the intact drug

were submitted to FTIR, MS and LC analysis. The spectrum scan of

NFX standard exhibited a strong stretching vibration band at 1715 cm

−1

together with a broad band around 2500–3500 cm

−1

characterizing

carbonyl and hydroxyl moieties of the carboxylic group, respectively.

These two bands, in the spectrum of DCN, corresponded in position to

that obtained for NFX standard, but the relative intensity decreased,

indicating decarboxylation. The mass spectra obtained is shown in

Figure 3A, with characteristic signal on m/z 320 amu for NFX[H

] and

m/z 361 amu for NFX[CH

CN-H

], which is an adduct produced with

NFX and acetonitrile (41 Da) from the mobile phase. Adduct is defined

as an ion formed through the interaction between two species, usually an

ion and a molecule, containing all the atoms of one specie plus one or

several atoms of the other (28). In Figure 3B is shown the signal on m/z

276 amu for DCN[H

], supporting the identity of the degradation

product. Moreover, the degraded sample was analyzed by the proposed

LC method (Figure 2B) and its retention time was used to assume the

identity of this product in the samples subjected to stress studies.

Figure 3. Mass spectra for (A) norfloxacin and (B) decarboxylated

degradant.

The specificity of the analytical method for NFX was indicated

in Figure 2, where the excipients did not interfere on NFX peak. Under

acidic condition, one additional peak was observed with the same

retention time of DCN, thus confirming its identity. The basic and

photolytic conditions generated one additional peak. Under oxidative

and neutral conditions there was no change in the area and no additional

peak was detected. The studies with the PDA detector showed that the

norfloxacin peak was free from any coeluting peak, with values of peak

purity index higher than 0.9999, thus demonstrating that the proposed

method is specific.

The calibration curves constructed for norfloxacin were found

to be linear in the 0.05–5 µg/mL

range. The value of the determination

coefficient calculated (r

=0.9999, y=186846 ± 1960x – 951.8 ± 890.6,

where, x is concentration and y is the peak absolute area) indicated the

linearity of the calibration curve for the method. The validity of the

assay was verified by means of ANOVA, which demonstrated

significant linear regression and non-significant linearity deviation (P <

0.01).

The precision evaluated as the repeatability of the method was

studied by calculating the relative standard deviation (RSD) for six

determinations of the concentration of 1 µg/mL performed on the same

day and under the same experimental conditions. The RSD value

obtained was 0.98%.

The intermediate precision was assessed by analyzing two

samples of the pharmaceutical formulation on three different days (inter-

day); the RSD values obtained were 0.59% and 0.39%. Between-

analysts precision was determined by calculating the RSD for the

analysis of two samples of the pharmaceutical formulation by three

analysts; the values were found to be 0.05% and 0.28% (Table 1).

Table 1. Inter-day and between-analysts precision data of the method.

Sample

Inter-day

Between-analysts

Day

Recovery

(%)

RSD

(%)

Analysts

Recovery

(%)

RSD

(%)

1 99.99

0.59

A 100.00

0.05

2 101.07

B 99.91

3 100.96

C 100.00

1 99.06

0.39

A 101.72

0.28

2 99.63

B 101.15

3 98.88

C 101.46

Mean of three replicates

RSD = Relative standard deviation

The accuracy was assessed from three replicate determinations of three

different added standard solutions containing 0.3, 0.5 and 0.7 µg/mL of

NFX. The results are shown in table 2, with a mean value of 99.90% and

RSD of 0.97%, demonstrating that the method is accurate within the

desired range.

Table 2. Accuracy of the method.

Added

Concentration

(µg/mL)

Mean concentration

found

(µg/mL)

RSD

(%)

Accuracy

(%)

0.30 0.30 1.09 100.48

0.50 0.50 1.16 100.23

0.70 0.69 0.40 98.70

Mean of three replicates

RSD = Relative standard deviation

For the calculation of the LD and LQ, the calibration equation

for NFX was generated by using the mean values of the three

independent calibration curves. The mean of the slope and the standard

deviation of the intercept of the independent curves were 186846 and

951.8 respectively. The values calculated for the LD and LQ were 0.01

and 0.05 µg/mL, respectively. The LQ evaluated experimentally for

NFX was also 0.05 µg/mL and was included in the calibration curve of

the method.

To evaluate the robustness of an analytical method usually the

OVAT approach is applied, however it is not recommended. The most

important reason is that when the factors are examined in given

intervals, the effects are estimated for a smaller domain around the

nominal levels with the OVAT than with the experimental design

approach. Moreover, the OVAT approach requires more (too many)

experiments, especially when the number of examined factors becomes

larger, and secondly, the importance of factor interactions cannot be

taken into account (22,23). The experimental ranges of the selected

variables evaluated are given in Table 3. The analysis of variance

ANOVA was performed and the model terms (variables) were not

significant (P > 0.05). The normal plot of residuals and outlier T for the

responses evaluated are shown in Figure 4, and the results demonstrated

that the method was robust. Moreover, the stability of the analytical

solution was analyzed and it was found to be stable up to 48 h (99.65%,

assay).

Table 3. Chromatographic conditions and range investigated during

robustness testing.

Experi-

mental

Factors Responses

ACN

(%)

Flow

(ml/min)

pH Temp

(

RSD

(%)

Assay

(%)

Peak

Symmetry

1 18.00 1.00 2.7 45.00 0.43 99.78 1.09

2 16.00 1.20 3.3 35.00 0.31 100.48 1.10

3 16.00 1.20 2.7 45.00 0.35 99.84 1.09

4 14.00 1.00 3.3 45.00 0.35 100.23 1.10

5 16.00 1.20 3.3 45.00 0.49 99.99 1.09

6 14.00 1.00 2.7 35.00 0.54 100.17 1.11

7 14.00 0.80 3.0 35.00 0.41 100.05 1.11

8 14.00 0.80 2.7 40.00 0.28 100.56 1.10

9 14.00 1.20 3.0 35.00 0.89 100.02 1.10

10 18.00 0.80 3.0 35.00 0.52 100.41 1.10

11 18.00 0.80 3.3 40.00 0.17 99.61 1.09

12 16.00 1.20 2.7 35.00 0.44 100.41 1.10

13 14.00 1.00 3.3 45.00 0.22 99.76 1.08

14 18.00 1.00 2.7 45.00 0.31 100.12 1.09

15 14.00 0.80 3.0 35.00 0.70 100.34 1.10

16 16.00 0.80 2.7 35.00 0.63 99.90 1.09

17 18.00 1.00 2.7 35.00 0.38 100.37 1.09

Mean of three replicates

Figure 4. Normal plot of residuals and outlier T values for responses

(A) peak area RSD%, (B) assay, and (C) peak symmetry.

The system suitability results showed that the parameters were

within the suitable range. The RDS values calculated for peak area and

retention time were 0.98 and 0.15%, respectively. The mean asymmetry

and theoretical plates ± RSD were 1.11 ± 0.34% and 8226 ±1.3%,

respectively.

The LC method validated in this paper was applied for the

determination of norfloxacin in the new extended release formulations,

without prior separation of the excipients. The values obtained ranged

from 99.43 to 102.35%.

Conclusion

The results of the validation studies showed that the LC method

is specific, accurate and possesses significant linearity and precision

characteristics without any interference from the formulation excipients

and degradation products. Moreover, the proposed method was

successfully applied for the quantitative analysis of norfloxacin in the

developed extended release dosage forms.

Acknowledgments

The authors wish to thank CAPES and CNPq for the financial

support, and Allcrom for kindly providing the Phenomenex analytical

column.

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CAPÍTULO 4 – Formulação, estabilidade e avaliação in vitro de

comprimidos de liberação prolongada contendo Norfloxacino.

Publicação científica: Oliveira, P.R.; Bernardi, L.S.; Mendes, C.; Silva,

M.A.S. Formulation, stability and in vitro dissolution studies of

norfloxacin extended-release matrix tablets. Manuscrito em preparação.

INTRODUÇÃO

A tecnologia de produção de sistemas matriciais de liberação

prolongada é relativamente simples, possibilitando inclusive sua

obtenção por compressão direta (COLOMBO et al., 2009). O estudo do

tipo de polímero utilizado, da sua massa molecular e concentração na

formulação é etapa fundamental para o desenvolvimento destes

sistemas, objetivando uma liberação adequada in vivo (LOPES; LOBO;

COSTA, 2005; JAMZAD; FASSIHI, 2006).

A estabilidade de produtos farmacêuticos depende de fatores

ambientais como temperatura, umidade, luz e de outros relacionados ao

próprio produto como propriedades físicas e químicas do fármaco, além

do processo de fabricação. A estabilidade acelerada é realizada para

estudar a degradação química e/ou mudanças físicas de um produto

farmacêutico, utilizando condições forçadas de armazenamento. Como o

Brasil está classificado em zona IV (clima quente e úmido), este estudo

é realizado em câmara climática em temperatura de 40 ± 2 ºC e umidade

relativa de 75 ± 5% (BRASIL, 2005; WHO, 2009). A estabilidade da

formulação frente à luz UV também deve ser avaliada (ICH, 1996;

BRASIL, 2008). Os resultados obtidos são utilizados para estabelecer

prazo de validade e recomendar condições de armazenamento. Neste

capítulo, além dos comprimidos, avaliou-se a influência do revestimento

e da embalagem na estabilidade.

O ensaio de perfil de dissolução é de fundamental importância

no desenvolvimento de comprimidos de liberação prolongada

(JORGENSEN; BHAGWAT, 1998; CHEN et al., 2010; MOURÃO et

al., 2010), pois através dele pode-se avaliar o resultado das alterações

realizadas na formulação e planejar a etapa seguinte. A partir da analise

dos perfis é possível saber a cinética e o mecanismo de liberação do

fármaco a partir da matriz, bem como a relação destes com a

composição da formulação.

As equações utilizadas nesta etapa para avaliação da cinética

foram: zero-ordem (COSTA; LOBO, 2001), primeira-ordem (COSTA;

LOBO, 2001), Higuchi (HIGUCHI, 1963; COSTA; LOBO, 2001) e

Korsmeyer-Peppas (COSTA; LOBO, 2001; KORSMEYER et al.,

1983). Além disso, através da equação de Korsmeyer-Peppas pode-se

calcular o mecanismo de liberação predominante: difusão fickiana,

transporte Caso-II (relaxamento e erosão das cadeias poliméricas),

transporte anômalo (combinação dos dois mecanismos citados) ou

transporte super Caso-II (aumento da plasticidade das cadeias

poliméricas, relaxamento e erosão poliméricas).

Formulation, stability and in vitro dissolution studies of norfloxacin

extended-release matrix tablets

Paulo Renato Oliveira*, Larissa Sakis Bernardi, Cassiana Mendes,

Marcos A. Segatto Silva.

Department of Pharmaceutical Sciences (J/K - 207), Health Science

Centre, Federal University of Santa Catarina, 88040-900, Florianópolis-

SC, Brazil.

* Corresponding author: prenato.oliveir[email protected]

Abstract

The aim of this research was to develop a new hydrophilic matrix

system containing norfloxacin (NFX) and to carry out stability and in

vitro release studies. Extended-release tablets are usually intended for

once-a-day administration with benefits to the patient and lower

discontinuation of the therapy, which for antibacterial drugs is very

important since it can result in a decrease of bacterial resistance.

Formulations were developed with hydroxypropylmethylcellulose

(HPMC) and poly(ethylene oxide) (PEO) as hydrophilic polymers, with

different molecular weights (MWs) and concentrations (20 and 30%).

The tablets were found to be stable (6 months at 40 ± 2 ºC and 75 ± 5%

relative humidity) and the film-coating process is recommended to avoid

NFX photodegradation. The dissolution profiles demonstrated an

extended-release of NFX for all developed formulations, however for

those containing high MW polymers, at the end of the analysis, the drug

was not completely released. Dissolution curves analyzed using the

Korsmeyer exponential equation showed that drug release was

controlled by both drug diffusion and polymer relaxation or erosion

mechanisms. A more erosion controlled system was obtained for the

formulations containing lower MW and amount of polymer. With the

increase in MW and amount of polymer in the formulation, the gel layer

became stronger and the dissolution was more drug-diffusion dependent

(with decreasing in exponent n values). Formulations containing

intermediate MW polymers or high concentration (30%) of low MW

polymers demonstrated a combination of extended and complete in vitro

drug release. This way, these formulations could provide an increased

bioavailability in vivo.

Keywords: Norfloxacin; hydroxypropylmethylcellulose; poly(ethylene

oxide); hydrophilic polymers; extended-release; dissolution studies.

1. Introduction

Hydrophilic matrix tablets are among the most popular orally

administered controlled release systems. Despite having been around

since four decades, matrices are still the reference starting point for

innovations in drug delivery. It can be due to the fact that they are

considered quite reliable in terms of drug delivery, simple technology

and low-cost of manufacture. Moreover, matrices can be continuously

innovated as new materials for formulation become commercially

available [1-5].

The matrix tablets are usually composed of active

pharmaceutical ingredients (APIs) and hydrophilic swellable polymers.

When the system is exposed to the aqueous medium, water will be

absorbed and a gel layer will be formed. This viscous gel layer may

hinder water penetration and become the rate-controlling step during gel

formation. The gel strength is important in the matrix performance and

is dependent on the chemical structure, concentration and viscosity of

the polymer used. Depending on the mechanical properties of the gel

layer, drug release is controlled by different mechanisms and kinetics.

Polymer swelling, drug dissolution, drug diffusion, and matrix erosion

are the basic phenomena leading to the drug release from swellable

matrices [6-12]. Additionally, drug load and solubility can influence the

release mechanism and kinetics.

Hydroxypropylmethylcellulose (HPMC) is a propylene glycol

ether of methylcellulose and is widely used as a matrix former in oral

controlled release tablet formulations [1]. One of its most important

characteristics is the high swellability, which has a significant effect on

the release kinetics of an incorporated drug. Furthermore, HPMC is

compatible with numerous drugs, accommodates high levels of drug

loading and can be easily incorporated to form matrix tablets by direct

compression or granulation [9,13-16]. The availability of a wide range

of viscosity grades also allows the formulator to modify the release of

drugs from HPMC matrix tablets according to therapeutic need.

High molecular weight poly(ethylene oxide) (PEOs) have been

proposed as an alternative to HPMC in controlled release dosage forms

[17]. They are important polymers for the pharmaceutical industries

mainly because of their non-toxicity, high water-solubility and

swellability, insensitivity to the pH of the biological medium and ease of

production. PEOs swell and form a compact gel layer on the surface of

the tablet which is responsible for the controlled drug release [17-22].

They are also available in a wide range of molecular weights, thus

allowing the formulator to control the mechanism of drug release to

achieve the therapeutic goal.

Norfloxacin (NFX) is a synthetic broad spectrum antibacterial

drug being the firstly selected drug for the treatment of diseases caused

by Campylobacter, E. coli, Salmonella, Shigella and V. cholera [23,24].

The drug is also used for the treatment of urinary tract infections as well

as gonorrhoea and infection of eyes [23]. The recommended dosage is

usually 400 mg twice daily. The half-life of NFX in serum and plasma is

3-4 hours and only approximately 30-40% of an oral dose is absorbed

[25,26]. Increasing bacterial resistance to currently available antibiotics,

including the quinolone class, has reduced their effectiveness, making

the therapeutic decisions more difficult and may compromise future use

of this class of drugs [27-31].

The development of an extended release formulation that could

improve the bioavailability of NFX and reduce the administration

schedule may improve the patients’ comfort and compliance, resulting

in lower discontinuation of the therapy; with consequently decrease in

bacterial resistance. The correct choice of the hydrophilic polymer,

molecular weight and quantity in the matrix formulation can provide an

appropriate combination of polymer swelling, erosion or drug diffusion

mechanisms to control drug release. Thus, the aim of this work was to

develop and carry out stability and in vitro dissolution studies of a new

formulation of norfloxacin extended-release tablets.

2. Materials and Methods

2.1 Materials

Norfloxacin (NFX) was purchased from Zhejiang Neo-Dankong

pharmaceutical (Zhejiang, China). Hydroxypropylmethylcellulose

(HPMC) K100 LV (apparent viscosity: 100 mPa s, 2% in water at 20

ºC), HPMC K4M (4 000 mPa s), HPMC K100M (100 000 mPa s), and

poly(ethylene oxide) (PEO) N60K (2 000 kDa), PEO 301 (4 000 kDa)

and PEO 303 (7 000 kDa) were kindly donated by Colorcon (São Paulo,

Brazil). The pharmaceutical excipients used were: microcrystalline

cellulose (Microcel 102, Blanver, Itapevi, Brazil), magnesium stearate

(M. Cassab, São Paulo, Brazil), and colloidal silicon dioxide (Aerosil®,

Labsynth, Diadema, Brazil).

2.2 Methods

2.2.1 Preparation of matrix tablets

A powder blend containing NFX, polymer and microcrystalline

cellulose was prepared and mixed for 15 min, followed by addition of

magnesium stearate and colloidal silicon dioxide with a further 5 min

mixing. The modules having the composition reported in Table 1 were

prepared by direct compression using a 19 x 8 mm punch set. (Fellc

compressing model F-10/8, São Paulo, Brazil).

Table 1. Composition of tablets containing hydroxypropylmethyl

cellulose (HPMC) or poly(ethylene oxide) (PEO).

Composition For one tablet For one tablet

Norfloxacin 700 mg 700 mg

Polymer

HPMC K100 LV

HPMC K4M

HPMC K100M

PEO N60K

PEO WSR 301

20 % 30 %

PEO WSR 303

Magnesium stearate 1 % 1 %

Colloidal silicon dioxide 0.5 % 0.5 %

Microcrystalline cellulose q.s. q.s.

Total weight 1.07 g 1.07 g

2.2.2 Characterization of tablet formulation

Tablets were characterized by weight, hardness, friability,

dimension, and loss on drying according to pharmacopeial limits

[33,34]. The average weight was obtained for at least 20 units. Hardness

was determined for at least 10 tablets using a Hardness Tester (298-AT,

Nova Ética, Vargem Grande Paulista, Brazil), and adopting a minimum

hardness of 3 kgf as the acceptance criterion. For each formula, friability

was evaluated for a sample of 20 tablets, using the acceptance criterion

of a maximum loss of 1.5% of the initial weight. Dimension was

evaluated measuring 10 tablets with a paquimeter. Loss on drying was

carried out with 2 g of sample, in vacuum, at 105 ºC for 2 h.

2.2.3 Tablet coating and blistering

A tablet coating solution was formed by adding 30 g of Opadry

II White (Colorcon, São Paulo, Brazil) to 120 g of purified water and

stirring for 2 min. An amount corresponding to 50% of each formulation

batch was placed in a Rama Cota RD conventional coating machine.

Tablets were preheated until the bed temperature reached 45 °C. Pan

rotation was set to 40 rpm and tablets were coated using a Binks Model

460 spray gun operating at 2 Bar. The coating solution was pumped at a

rate of 5.9-9.6 g/min using a peristalic pump. Tablet bed temperature

was maintained between 42-45 °C during the spray coating process.

After coating, an amount of coated and uncoated tables were blistered in

transparent PVC blister and sealed with an aluminium foil.

2.2.4 NFX tablets assay

NFX quantification assay was carried out according to a

previously validated method [35]. Briefly, the LC system was operated

isocratically at 40 ºC using a mobile phase composed by phosphoric

acid 0.04 M, pH 3.0/acetonitrile (84:16; v/v), eluted at a flow rate of 1.0

mL/min. A reversed-phase Phenomenex (Torrance, USA) Luna C

column (150 mm x 4.6 mm I.D., with a particle size of 5 µm and pore

size of 100 Å) was used and the detector was set at 272 nm. The

injection volume was 20 µL.

To prepare the sample stock solution, the manufactured

extended-release tablets were crushed to a fine powder. An appropriated

amount was transferred into an individual 50 mL volumetric flask,

dissolved with 0.2 mL of glacial acetic acid, and diluted to volume with

mobile phase, obtaining a concentration of 1 mg/mL of the API. The

NFX standard stock solutions were prepared by weighing 50 mg,

transferred to 50 mL volumetric flasks, dissolved with 0.2 mL of acetic

acid glacial, and diluted to volume with mobile phase, obtaining a

concentration of 1 mg/mL. Both sample and standard stock solutions

were stored at 2-8 °C protected from light. Working solutions were

prepared daily by diluting the stock solutions to an appropriate

concentration in mobile phase.

2.2.5 Stability tests

The manufactured tablets were submitted to accelerated

stability test. Samples of each batch (non-coated, coated, with and

without blister) were maintained for 6 months in a accelerated stability

chamber (420 CLD, Nova Ética, Vargem Grande Paulista, Brazil) at 40

± 2 ºC and 75 ± 5% relative humidity [36,37]. For photostability tests,

samples were exposed to an overall illumination of not less than 1.2

million lux [38]. The illumination was measured with a Digital Lux

Meter (MLM-1011, Minipa, São Paulo, Brazil). Protected samples

(wrapped in aluminium foil) were used as dark controls to evaluate the

contribution of thermally induced change to the total observed change.

2.2.6 Drug release study

Drug release studies were performed based on pharmacopeial

methods using USP apparatus II Vankel 7000 dissolution tester (Varian

Technology Group, Cary, USA), with paddle rotation of 75 rpm, in 900

ml of buffer pH 4.0 at 37.0 ± 0.5 °C [33,34]. At specified time intervals,

5 mL samples were withdrawn, filtered and quantified in a UV

spectrophotometer (Varian Cary 50 bio, Cary, USA) at the wavelength

278 nm.

2.2.7 Analysis of drug release

The analysis of the values obtained in dissolution tests is easier

when mathematical formulas that express the dissolution results as a

function of some of the dosage forms characteristics are used. NFX

release kinetic was evaluated according to the following models: zero

order, first order, Higuchi, and Korsmeyer-Peppas.

Additionally, the

difference factor (ƒ1) and similarity factor (ƒ2) were used to compare

the dissolution profiles.

2.2.7.1 Zero-order model

Drug dissolution from pharmaceutical dosage forms that do not

disaggregate and release the drug slowly (assuming that area does not

change and no equilibrium conditions are obtained) following a 'steady-

state release' can be represented by the following equation (eq. 1) [39]:

(eq. 1)

Where Qt is the fraction of drug released at time t, Q

is the initial

amount of drug in the solution (most times Q

= 0); k

is the zero-order

release constant. The pharmaceutical dosage forms following this profile

release the same amount of drug by unit of time and it is the ideal

method of drug release in order to achieve a pharmacological prolonged

action.

t o o

Q Q k t

= +

2.2.7.2 First-order model

The drug dissolution is assumed to decline exponentially and

the release rate is proportional to the residual amount of drug in the

dosage form (eq. 2) [39]:

(eq. 2)

Where Qt is the fraction of drug released at time t, Q

is the initial

amount of drug in the solution; k

is the first-order release constant. The

pharmaceutical dosage forms following this dissolution profile release

the drug by unit of time in a way that is proportional to the amount of

drug remaining in its interior.

2.2.7.3 Higuchi model

The most widely used model to describe drug release from

matrices, derived from Higuchi for a planar matrix. It describes the drug

release mechanism as a diffusion process based on Fick’s law,

dependent on the square root of time (eq. 3) [39,40].

(eq. 3)

Where Qt is the fraction of drug released at time t and K

is the Higuchi

dissolution constant.

2.2.7.4 Korsmeyer-Peppas model

This model is generally used to analyze the release of

pharmaceutical polymeric dosage forms when the release mechanism is

not well known or when more than one type of release phenomena could

be involved (eq. 4) [39,41].

(eq. 4)

where Mt/M∞ is the fraction of drug released, k is the kinetic constants

characteristic of the drug/polymer, n is the diffusional exponent for drug

release. Dissolution values in the range of 5 – 60% were used to fit

release data.

log log

2.303

k t

Q Q= +

t H

Q K t

∞

2.2.7.5 Difference factor (ƒ1) and similarity factor (ƒ2)

The relevance of the difference between the release curves were

assessed using difference factor ƒ1 and similarity factor ƒ2, calculated

by eq. (5) and (6), respectively [42,43]:

(eq. 5)

(eq. 6)

where Rt and Tt are the percentages released at each time point.

An ƒ1 value up to 15 (0-15) and ƒ2 value between 50 and 100 implies

similarity between two release profiles. Only one more point after the

85% of drug has released was used for the equation.

3. Results and Discussion

Norfloxacin matrix tables were successfully obtained by direct

compression (Fig. 1). Different polymers and molecular weights did not

interfere in the technological process. The pharmacopeial characteristics

of the manufactured tablets are summarized in Table 2. These results

demonstrated that the tablets were reliable on hardness and friability,

which are important characteristics for the further step of coating.

Consistent hardness of the tablet surface enables the coating to

“lock” into the surface. If the surface is too soft, the impingement of the

solution can erode the tablet. Too hard a surface will not allow the

solution to impinge and adhere, and the coating will peel away. Both of

these coating defects can also occur by over- or under-applying the

coating solution or by applying the coating with too much or too little

force [44-47]. The film-coating (Opadry II) applied on the NFX tablets

surface is non-functional, however it can improve the final quality by

protecting the hygroscopic polymer from absorbing humidity and

preventing photodegradation of the drug. NFX coated tablets showed an

uniform, smooth and shiny surface, without coating defects (Fig. 1).

From Table 2, it can be observed that the weight and hardness increased

about 3% and 9%, respectively, demonstrating the influence of the

coating process. The loss on drying analysis (Table 2) showed that the

1 1

1 ( ) / .100

n n

i i

f Rt Tt Rt

= =

 

= −

 

 

∑ ∑

0.5

2 50.log 1 ( ) .100

f Rt Tt

−

 

 

 

 

= + −

 

 

 

 

 

 

 

∑

coated tablets have a lower amount of volatile matter, probably due to

the loss of water absorbed during the coating process at 42-45 °C.

Figure 1. Norfloxacin blistered matrix tablets: uncoated (A) and coated

(B).

Table 2. Pharmacopeial characteristcs of norfloxacin matrix tablets.

Formulation Weight

(g)

Hardness

(KgF)

Water Loss

(%)

Friability

(%)

Uncoat.

Coated

Uncoat.

Coated

Uncoat

Coated

Uncoat.

HPMC K100LV 20%

1.1154

1.1472

16.3 17.0 7.75 7.29 0.021

HPMC K100LV 30%

1.1025

1.1321

14.8 16.4 7.93 7.60 0.014

HPMC K4M 20% 1.1033

1.1314

13.1 15.1 7.08 6.60 0.034

HPMC K4M 30% 1.0795

1.1119

13.0 14.5 7.29 7.11 0.018

HPMC K100M 20% 1.1048

1.1351

17.3 17.9 6.68 5.85 0.022

HPMC K100M 30% 1.0967

1.1277

14.3 16.2 7.02 7.21 0.024

PEO N60K 20% 1.0858

1.1193

14.2 15.7 7.02 5.08 0.017

PEO N60K 30% 1.0854

1.1131

16.9 17.7 6.77 5.64 0.021

PEO 301 20% 1.0831

1.1162

16.6 18.8 7.16 6.91 0.018

PEO 301 30% 1.1014

1.1306

16.0 17.5 7.09 7.01 0.023

PEO 303 20% 1.0748

1.1090

14.4 15.6 6.13 5.55 0.024

PEO 303 30% 1.0734

1.1077

14.6 15.8 5.51 4.22 0.019

mean of twenty determinations;

mean of ten determinations

The assay determination of NFX demonstrated that all

formulations were in the range from 99.43 to 102.35% (Table 3).

Therefore, the coating process did not influence on the assay of the

drug.

Table 3. Assay results of accelerated stability test.

Formulation Time zero After 6 months

Blister Without blister

Uncoat

(%)

Coated

(%)

Uncoat

(%)

Coated

(%)

Uncoat

(%)

Coated

(%)

HPMC K100LV 20%

101.98

101.09 101.16

101.51

102.03

101.18

HPMC K100LV 30%

101.47

101.81 101.87

103.01

100.87

101.74

HPMC K4M 20% 101.45

100.42 100.44

102.98

101.92

101.08

HPMC K4M 30% 100.88

99.31 99.12 99.45 99.91 98.09

HPMC K100M 20% 102.35

101.74 99.14 101.25

103.22

102.80

HPMC K100M 30% 102.25

101.58 99.66 99.31 99.02 101.80

PEO N60K 20% 102.06

101.72 102.89

98.47 101.44

100.26

PEO N60K 30% 99.43 99.92 98.49 99.05 99.44 99.03

PEO 301 20% 99.48 99.35 101.24

97.51 101.75

100.31

PEO 301 30% 99.71 100.50 99.03 100.49

100.61

100.36

PEO 303 20% 99.98 99.50 99.79 97.11 100.62

97.36

PEO 303 30% 99.95 99.36 99.16 97.78 99.10 99.35

Accelerated stability testing was carried out to provide evidence

of how the quality of the manufactured tablets may change with time

under the influence of environmental factors such as temperature and

humidity. Brazil, being considered with hot and humid climate is

classified in the region IV [36]. According to this classification, the

accelerated stability study was carried out for 6 months in a climatic

chamber at 40 ± 2 ºC and 75 ± 5% relative humidity. The obtained

results are shown in Table 3. All formulations were considered stable

since after 6 months a change from the initial assay of 5% or more was

not observed [36]. The presence of coating and/or blister did not

influence in the stability of the developed tablets. Additionally, the

chromatographic profiles did not show any additional degradation peak.

Light testing should be an integral part of stress testing and

recommends evaluation of the photostability of a formulation to

demonstrate that light exposure does not result in unacceptable changes

[36,38]. For this study, the following formulations were selected:

HPMC K100 LV (20 and 30%) and PEO N60K (20 and 30%). At the

end of the exposure period (about five days), equivalent of not less than

1.2 million lux, samples were examined for changes in appearance and

for assay. It was observed a color change from pale-yellow to dark-

yellow in NFX raw material and uncoated tablets. The transparent

blister (primary packing) did not have any protecting influence in the

formulations (Fig. 2). Prolonged exposure of NFX bulk drug, tablets and

specially in solution under direct sunlight or fluorescent light results in

the formation of ethylenediamine degradation product [48,49]. Since the

chromatograms did not show additional peaks and a significant decrease

of drug content was not observed (Table 4), it seems that the

ethylenediamine degradant requires an exposure time and/or intensity

higher than the used in this research to be significantly formed.

Nonetheless, to prevent drug exposure to light and degradation, it would

be recommended the coating process or light-protective blister for the

formulations.

Table 4. Assay results (%) of photostability test.

Formulation Blister Without blister

Uncoated

(%)

Coated

(%)

Uncoated

(%)

Coated

(%)

HPMC K100LV 20% 100.80 99.22 100.30 99.71

HPMC K100LV 30% 100.25 100.36 99.96 100.60

PEO N60K 20% 98.78 100.35 99.54 99.26

PEO N60K 30% 98.17 100.79 99.36 99.06

Figure 2. Norfloxacin blistered matrix tablets after photostability study:

uncoated (A) and coated (B).

Two concentrations (20 and 30%) of different MWs HPMC or

PEO polymers were used to manufacture the NFX matrix tablets used in

this study (Table 1). The dissolution test was carried out under sink

conditions, defined as the volume of medium being at least three times

higher than that necessary to obtain a saturated solution of the drug [33].

Samples were withdrawn from the dissolution medium at the following

times: 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 24 h. The first time

point at 0.5 h was included to study if the product presents a burst effect

(with an excessive early drug release), while the final time point shows

whether or not the intended dose is fully delivered.

NFX release profiles are shown in Fig. 3 – Fig. 8. The polymers

used have different average MWs and therefore they differ in

controlling drug release from matrix tablets. An extended-release of

NFX was obtained for all formulations manufactured, demonstrating

that the mechanical strength of the viscous-gel layer was strong enough

to maintain its integrity and drug release. Faster dissolution was

obtained for formulations containing lower MW polymer and

concentration (20%) (Fig. 3, Fig. 6). The tablets containing HPMC

K100 LV showed the fast dissolution profile, with complete drug release

at about 6 – 8 h (Fig. 3).

Figure 3. Norfloxacin released vs. time of matrix tablets containing

HPMC K100 LV: 20% uncoated(); 20% coated (); 30% uncoated

(); 30% coated ().

Figure 4. Norfloxacin released vs. time of matrix tablets containing

HPMC K4M: 20% uncoated(); 20% coated (); 30% uncoated ();

30% coated ().

100

0 2 4 6 8 10

Norfloxacin Released (%)

Time (h)

100

0 5 10 15 20 25

Norfloxacin Released (%)

Time (h)

Figure 5. Norfloxacin released vs. time of matrix tablets containing

HPMC K100M: 20% uncoated(); 20% coated (); 30% uncoated

(); 30% coated ().

Figure 6. Norfloxacin released vs. time of matrix tablets containing PEO

N60K: 20% uncoated(); 20% coated (); 30% uncoated (); 30%

coated ().

100

0 5 10 15 20 25

Norfloxacin Released (%)

Time (h)

100

0 5 10 15 20 25

Norfloxacin Released (%)

Time (h)

Figure 7. Norfloxacin released vs. time of matrix tablets containing PEO

301: 20% uncoated(); 20% coated (); 30% uncoated (); 30%

coated ().

Figure 8. Norfloxacin released vs. time of matrix tablets containing PEO

303: 20% uncoated(); 20% coated (); 30% uncoated (); 30%

coated ().

100

0 5 10 15 20 25

Norfloxacin Released (%)

Time (h)

100

0 5 10 15 20 25

Norfloxacin Released (%)

Time (h)

For the formulations containing HPMC K100M (20 and 30%,

Fig. 5) and PEO 303 30% (Fig. 8), the NFX release was not complete at

24 h. Due to the high MW and/or concentration of polymer in the

formulations, the swelling was too slow and the gel strength was very

high, resulting the central part of the tablet not being fully wetted or

hydrated (a “dry core”), with incomplete drug release. Probably, these

formulations would exhibit an inadequate performance in vivo, since an

incomplete polymer gelification and drug release would be obtained

while passing through stomach and small intestine.

It seems that the coating process somehow influenced the NFX

dissolution profile (Fig. 3 – Fig. 8), and a relation with the polymer MW

could be suggested. In general, coated formulations exhibited faster drug

release than uncoated ones. The faster NFX release may be due to the

coating process temperature that resulted in lower residual humidity

tablets (Table 2) and consequently a faster water uptake and polymer

swelling in the dissolution medium.

For HPMC K100 LV formulations, due to the lower MW, water

uptake, polymer hydration and gelification is faster than dissolution of

the coating film. In this case, the coating may have worked as a

“barrier”, and drug release was delayed. For PEO 301 and PEO 303 the

dissolution profiles were overlapped, demonstrating no influence of the

coating. It can be explained since high MW polymers forms a stronger

gel layer, with lower water uptake rate and drug release, hence

influencing drug diffusion and dynamics of matrix erosion. However,

the influence of the coating process was not relevant based on the

difference (ƒ1) and similarity (ƒ2) parameters calculated (Table 5).

Table 5. Difference factor (ƒ1) and similarity factor (ƒ2) calculated for

uncoated and coated norfloxacin matrix tablets.

Formulation ƒ1 ƒ2

HPMC K100LV 20% 5.47 71.72

HPMC K100LV 30% 8.50 62.32

HPMC K4M 20% 9.22 70.03

HPMC K4M 30% 14.23 66.36

HPMC K100M 20% 9.22 73.89

HPMC K100M 30% 9.27 77.02

PEO N60K 20% 13.77 56.35

PEO N60K 30% 14.66 57.81

PEO 301 20% 2.00 91.85

PEO 301 30% 3.12 88.95

PEO 303 20% 4.90 83.40

PEO 303 30% 2.76 93.30

Dissolution profiles were analyzed for zero-order, first-order

and Higuchi models with the equations up to 12 h of drug release,

except for HPMC K100 LV 20 and 30% formulations where the

equations were analyzed for up to 6 and 8 h, respectively. The analysis

according to Korsmeyer-Peppas was carried out with the diffusional

exponential equation up to 60% of drug released [41]. Calculation of the

exponent n identifies the prevalent mechanism of release. For

cylindrical systems, n = 0.45 indicates diffusion-controlled (Fickian)

drug release and n = 0.89 indicates swelling/erosion-controlled drug

release (Case-II transport). Values of n between 0.45 and 0.89 can be

regarded as an indicator for the superposition of both phenomena,

indicating that the drug delivery was not controlled only by diffusion,

but also by significant polymer relaxation or erosion mechanisms

(anomalous transport). The n > 0.89 values reveals a super case-II

transport. This mechanism could result from an increased plasticization

at the relaxing boundary (gel layer) and is also related to polymer

relaxation and erosion mechanisms [41,50].

In general, data of all matrices provided better fit to Korsmeyer-

Peppas model (Table 6 and Table 7). None formulation fitted to Higuchi

equation, this way demonstrating that NFX release mechanism was not

only a diffusion process dependent on the square root of time.

Table 6. Coefficients of determination (r

) obtained from dissolution of

norfloxacin uncoated formulations according to different mathematical

models.

Formulation Zero order

First order

Higuchi

Korsmeyer-Peppas

HPMC K100LV 20%

0.9794 0.9247 0.9685 0.9952 0.9623

HPMC K100LV 30%

0.9794 0.8538 0.9681 0.9985 0.9761

HPMC K4M 20% 0.9943 0.9916 0.9604 0.9963 0.7115

HPMC K4M 30% 0.9834 0.9954 0.9801 0.9986 0.6593

HPMC K100M 20%

0.9866 0.9962 0.9744 0.9978 0.6838

HPMC K100M 30%

0.9759 0.9909 0.9878 0.9995 0.6422

PEO N60K 20% 0.9976 0.9302 0.9372 0.9994 0.9485

PEO N60K 30% 0.9978 0.9513 0.9127 0.9990 1.0027

PEO 301 20% 0.9978 0.9591 0.9180 0.9978 0.8900

PEO 301 30% 0.9978 0.9795 0.9308 0.9971 0.8771

PEO 303 20% 0.9935 0.9934 0.9598 0.9982 0.7863

PEO 303 30% 0.9932 0.9931 0.9589 0.9979 0.7866

Table 7. Coefficients of determination (r

) obtained from dissolution of

norfloxacin coated formulations according to different mathematical

models.

Formulation Zero order

First order

Higuchi

Korsmeyer-Peppas

HPMC K100LV 20%

0.9827 0.9719 0.9649 0.9990 0.9283

HPMC K100LV 30%

0.9898 0.9571 0.9588 0.9999 0.8867

HPMC K4M 20% 0.9833 0.9981 0.9807 0.9998 0.7270

HPMC K4M 30% 0.9784 0.9921 0.9821 0.9984 0.7041

HPMC K100M 20%

0.9829 0.9973 0.9794 0.9994 0.7404

HPMC K100M 30%

0.9755 0.9917 0.9825 0.9980 0.7406

PEO N60K 20% 0.9759 0.9716 0.9685 0.9975 0.9892

PEO N60K 30% 0.9955 0.9766 0.9468 0.9992 1.0019

PEO 301 20% 0.9970 0.9593 0.9277 0.9979 0.8522

PEO 301 30% 0.9911 0.9586 0.9216 0.9917 0.8550

PEO 303 20% 0.9934 0.9889 0.9553 0.9968 0.7803

PEO 303 30% 0.9924 0.9898 0.9538 0.9975 0.7757

The formulations containing PEO demonstrated also a good fit

to zero-order kinetics. The exponent n calculated (Table 6, Table 7) for

Korsmeyer-Peppas equation confirmed this to PEO N60K (n between

0.94 - 1.0) and to PEO 301 (n about 0.87), indicating super case-II and

case-II transport mechanism, respectively, as also evidenced by quasi-

linear release profiles (Fig. 6 and Fig. 7). It can be due to the lower MW

of these polymers in comparison to PEO 303. In the case of low

viscosity gelling agents, erosion of the swollen polymer is the major

release factor, generally leading to zero-order release kinetics [13]. For

the tablets containing PEO 303, the exponent n obtained (about 0.78)

indicated that drug delivery was controlled by diffusion and polymer

relaxation/erosion mechanisms. Moreover, the dissolution kinetic

equation fit was very similar to zero and first-order release.

HPMC K100LV formulations demonstrated a similar release

profile to PEO N60K, where a super case-II transport mechanism was

obtained due to the dissolution of polymeric matrix and relaxation of the

polymer chain, with zero-order release. Most of matrix tablets

containing HPMC K4M and HPMC K100M fitted better to first-order

than to zero-order kinetic (Table 6 and Table 7). Moreover, the n

exponent calculated (between 0.64 – 0.74) indicated the significant

influence of both drug diffusion and polymer relaxation/erosion to drug

release. It can be explained base on the fact that high viscosity polymers

can form a mechanically stable gel and polymer

dissolution/disintegration will be lower [13]. Therefore, the diffusion-

controlled mechanism will have more influence on drug release from the

swollen matrix.

Based on the dissolution profiles, HPMC K100 LV 30%,

HPMC K4M 20%, PEO N60K 20%, and PEO N60K 30% matrices

presented a combination of polymer type, MW, concentration, and

complete drug release that could result in a formulation able to resist to

the destructive forces within the gastro-intestinal tract, providing a

superior in vivo performance. In fact, the results obtained confirm that

gels showing lower strength and texture, usually derived from low MW

polymers, have lower resistance to the fluid erosion action and the

release of the active molecule is mainly due to polymer relaxation and

chains disentanglement, leading to drug delivery kinetic towards an

erosion/relaxation mechanism, with exponent n ≥ 0.89. On the other

hand, when the MW or polymer concentration is increasing, the gel

layer formed will be concomitantly characterized by higher strength and

consistence, being less susceptible to erosion and chains

disentanglement, with drug release mechanism tending to diffusion

(with decreasing exponent n values).

4. Conclusions

In this study the development of a stable extended-release

dosage form containing norfloxacin was demonstrated. The film-coating

of tablets was necessary to avoid a photo-induced color changing of the

active pharmaceutical ingredient. The dissolution studies showed that

according to the increase in polymer molecular weight and

concentration, the matrix changed from a more erodible system (with

zero-order release) to a system with dissolution controlled by drug

diffusion and polymer relaxation/erosion mechanisms. The formulations

containing intermediate molecular weight HPMC or PEO or high

concentration (30%) of low molecular weight polymers (HPMC K100

LV 30%, HPMC K4M 20%, PEO N60K 20%, and PEO N60K 30%) are

more promising, since a combination between gel structure and

complete in vitro drug release was obtained. This prolonged and

complete in vitro release profile is expected to lead to an increased

bioavailability, however in vivo studies are necessary to confirm this

possibility. Based on an improved bioavailability combined with a

reduced frequency of administration, an improved patient compliance

and decreased bacterial resistance could be achieved.

Acknowledgements

The authors would like to thank CAPES for the financial

support and Colorcon do Brasil for kindly donating the polymers and

providing the equipments necessary for coating and blistering processes.

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CAPÍTULO 5 – Desenvolvimento de sistemas Dome Matrix

Norfloxacino.

100

101

INTRODUÇÃO

Dentre os sistemas gastro-retentivos de fármacos, os sistemas

flutuantes apresentam a vantagem de manter a forma farmacêutica

fisicamente afastada do piloro dificultando seu esvaziamento gástrico

(BARDONNET et al., 2006). Um sistema com esta característica foi

desenvolvido pelo grupo do Prof. Paolo Colombo, na Università degli

Studi di Parma – Italia. Este sistema, denominado Dome Matrix

basicamente consiste em dois comprimidos com uma base côncava e

uma convexa ligados entre si de forma que exista uma câmara com ar no

seu interior, sendo esta responsável pela flutuação (LOSI et al., 2006).

O norfloxacino apresenta uma maior solubilidade em meio

ácido (F. BRAS. IV, 2001) consequentemente, uma maior permanência

no estômago pode resultar em maior biodisponibilidade para o fármaco.

Esta representa uma outra estratégia para melhorar a resposta in vivo

alterando a formulação farmacêutica, sem alterar quimicamente o

fármaco. Com este sistema, uma maior biodisponibilidade para o

norfloxacino pode ser obtida a partir de uma permanência do sistema no

estômago (maior solubilidade do fármaco no meio ácido), combinada a

uma liberação prolongada, obtida através da utilização de polímeros

hidrofílicos (HPMC e POE) na formulação.

Neste estudo avaliou-se a dissolução de módulos individuais e

na configuração flutuante “void” em fluido gástrico simulado, bem

como estudos de flutuação in vitro, já prevendo o comportamento in

vivo destas formulações.

102

103

Assembled Modules Technology for Site-specific Prolonged Delivery of

Norfloxacin

Paulo Renato Oliveira

, Larissa Sakis Bernardi

, Orazio Luca Strusi

Salvatore Mercuri

, Marcos A. Segatto Silva

, Paolo Colombo

, Fabio

Sonvico

Department of Pharmaceutical Sciences, Federal University of Santa

Catarina, Quality Control Laboratory, J/K 207, 88040-900,

Florianópolis-SC, Brazil.

Department of Pharmacy, University of Parma, Viale G.P. Usberti

27/A, Parma, Italy

* Corresponding author. Dipartimento Farmaceutico. Università degli

Studi di Parma, Viale G.P. Usberti 27/A, 43125, Parma, Italy. Tel.: +39

0521905086; fax: +39 0521905006.

E-mail address: Fabio.sonv[email protected]

104

Abstract

The aim of this research was to design and study norfloxacin (NFX)

release in floating coditions from compressed hydrophilic matrices of

hydroxypropylmethylcellulose (HPMC) or poly(ethylene oxide) (PEO).

Module assembling technology for drug delivery system manufacturing

was used. Two differently cylindrical base curved matrix/modules,

identified as female and male, were assembled in void configuration by

friction interlocking their concave bases obtaining a floating release

system. Drug release and flotation behavior of this assembly was

investigated. Due to the higher surface area exposed to the release

medium, faster release was observed for individual modules compared

to their assembled configuration, independently on the polymer used

and concentration. The release curves analyzed using the Korsmeyer

exponential equation and Peppas & Sahlin binomial equation showed

that the drug release was controlled both by drug diffusion and polymer

relaxation or erosion mechanisms. However, convective transport was

predominant with PEO and at low content of polymers. NFX release

from PEO polymeric matrix was more erosion dependent than HPMC.

The assembled systems were able to float in vitro for up to 240 min,

indicating that this drug delivery system of norfloxacin could provide

gastro-retentive site-specific release for increasing norfloxacin

bioavailability.

Keywords

Norfloxacin; Dome Matrix

; Release Modules; Floating dosage form

Graphical Abstract

105

1. Introduction

Modified-release formulations are valuable developments in

pharmaceutical industry since, if compared to the new drug application

expenses, product innovation remains affordable. Among the different

approaches adopted for oral prolonged-release dosage forms,

hydrophilic matrices are the most used delivery systems, due to the

simple technology and manufacturing [1-4].

An innovative drug delivery platform based on hydrophilic

matrices, named module-assembling technology (Dome Matrix

), has

been presented [5]. In this technology, release modules made as

swellable polymeric matrices are fixed together in a firm structure

forming the drug delivery system. The individual module in its typical

shape is a cylindrical tablet having one concave and one convex base,

designed for allowing their assembling by inserting the convex into

concave base. In dependence on modules assemblage, different system

configurations can be made. Piled configurations are obtained by

stacking two or more modules convex base into concave base. A

peculiar assembly obtained by sticking the concave base of one module

to the concave base of another module made feasible the construction of

floating systems intended to keep the drug release into the stomach. This

configuration, named “void”, is characterized by an inner empty space

that makes buoyant the assembly [6]. Referring to the module

assembling for delivery system manufacturing, two differently shaped

matrix/modules, identified as female and male, have been constructed.

The friction interlocking of the complementary concave bases of these

modules drives their assemblage in void configuration.

Using this technology, the individual dose administered can be

easily adjusted or, if the composition of modules is different, multiple

release kinetics can be achieved. In addition, module assemblage can

allow the delivery of two drugs in a single unit at a specific time and at a

proper rate and duration, characterizing the flexibility of the Dome

Matrix

technology [7].

Norfloxacin (NFX) is a synthetic broad-spectrum antibacterial

drug firstly selected for the treatment of diseases caused by

Campylobacter, E. coli, Salmonella, Shigella and V. cholera [8, 9]. The

drug is mainly used for the treatment of urinary tract infections. [8].

Development of bacterial resistance to currently available antibiotics,

due to the lack of patient compliance, suggests an appropriate dosing of

quinolonic drugs [10-14]. NFX is very slightly soluble in water;

106

however, its solubility increases sharply at pH below 4.0 and above

10.0, due to the amphoteric nature of the drug [15, 16]. The

recommended dosage is usually 400 mg twice daily. The half-life of

NFX in serum and plasma is 3-4 hours; only approximately 30-40% of

an oral dose is absorbed and the fecal recovery accounts for 30% of the

administered dose [15].

Since NFX is more soluble in acidic media and better absorbed

from the upper part of the gastrointestinal tract, a prolonged gastric

residence of dose form is expected to lead to an increased dissolution

rate. Dome Matrix

modules assembled in void configuration floated for

up to 4 hrs on gastric content (6). Based on the gastro-retention

evidence, the development of a gastro-retentive site-specific drug

delivery system of NFX could improve the drug bioavailability and

simplify the administration schedule. This would favor the patient

convenience and compliance and result in less erratic absorption,

hampering bacterial resistance development.

Thus, the aim of this work was to design and study a floating

norfloxacin prolonged delivery system manufactured with Dome

Matrix

modules. In this paper, the formulations and the performances

of the NFX non-assembled modules made with two different polymers

(HPMC and PEO) at two concentrations (20 and 30 % w/w) and the

corresponding void configurations have been examined. In vitro studies

on floatation behavior and the release profile of the system have been

carried out.

2. Materials and Methods

2.1 Materials

Norfloxacin was purchased from Zhejiang Neo-Dankong

Pharmaceutical (Zhejiang, China). Hydroxypropylmethylcellulose

(Methocel K4M, viscosity 2% (p/v) solution 4000 mPa s) and poly-

(ethylene oxide) (Polyox N60K, MW 2 x 10

Da, viscosity 2% (p/v)

solution 3060 mPa s) were kindly donated by Colorcon (Gallarate,

Italy). Magnesium stearate (Eigemann & Veronelli S.p.A., Milan, Italy),

colloidal silicon dioxide (Aerosil

, Evonik Degussa S.p.A., Ravenna,

Italy), and talc (A.C.E.F., Fiorenzuola D’Arda, Italy) were

pharmacopoeia grade. The compatibility of NFX with excipients was

described elsewhere [17]. NaCl anhydrous (A.C.E.F., Fiorenzuola

107

D’Arda, Italy), HCl 37% and NaOH anhydrous (Carlo Erba S.p.A.,

Milan, Italy) were used to prepare the simulated gastric fluid.

2.2 Methods

2.2.1 Matrix modules preparation

NFX, hydrophilic polymer and talc were blended in a Turbula

mixer (WAB, Basel, Switzerland) for 15 min, followed by the addition

of magnesium stearate and colloidal silicon dioxide with a further 5

minutes mixing. The modules composition is reported in Table 1. Male

and female modules were prepared by direct compression in a single

punch tableting machine (EKO Korsch, Berlin, Germany) equipped with

special sets of cylindrical punches of 7.4 mm diameter, having the tip

surface concave or convex. In the assembling procedure, one male and

one female module were manually interlocked concave-to-concave

bases to give rise to a void configuration system (Figure 1).

Table 1. Composition of the norfloxacin male and female modules

Composition

Polymer 20%

Polymer 30%

Norfloxacin 100 100

HPMC K4M or PEO N60K 20 30

Talc 4.8 5.2

Magnesium stearate 1.2 1.3

Colloidal silicon dioxide 0.6 0.7

Total weight 126.6 137.2

108

Fig. 1 Norfloxacin Dome Matrix

modules and assemblage: 1- Male. 2-

Female. 3- Void configuration assembled modules.

2.2.2 In vitro drug release

Drug release studies were performed using USP apparatus II

(Erweka DT6R, Heusenstamm, Germany) with paddle rotation of 50

rpm, in 900 ml of simulated gastric fluid without pepsin (USP 29) at

37.0±0.5 °C. At specified time intervals, 5 mL samples were withdrawn,

filtered and quantified by a validated UV spectrophotometric method

(Jasco V530, Tokyo, Japan) at the wavelength 278 nm.

Drug release data were analyzed according to Korsmeyer Eq.

(1) [18] and Peppas and Sahlin Eq. (2) [19]:

(1)

(2)

∞

m m

d r

k t k t

∞

= +

109

where M

∞

is the fraction of drug released, k is the kinetic constants

characteristic of the drug/polymer combination, n is the diffusional

exponent for drug release, k

and k

are diffusion and relaxation rate

constants, respectively, and m is the purely Fickian diffusion exponent

for a device of any geometrical shape that exhibits controlled release.

The value of 0.425 was used in this analysis according to the aspect

ratio of the matrix. These mathematical models are capable of

describing the solute release kinetics and mechanism from polymeric

hydrophilic matrices and were used to fit release fractions in the range

of 5–60%. The Peppas and Sahlin equation allows the calculation of the

fraction of drug released due to Fickian mechanism, F, as in Eq. (3):

(3)

Release curves were compared using difference factor ƒ1 and similarity

factor ƒ2, calculated by Eq. (4) and (5), respectively [20, 21]:

(4)

(5)

where Rt and Tt are the percentages released at each time point. ƒ1

value up to 15 (0-15) and ƒ2 value between 50 and 100 implies

similarity between two release profiles.

2.2.3 Floatation behavior

The flotation characteristics of the void assembled modules

were assessed using an apparatus constructed according to Timmermans

and Moes [22]. A digital camera pictured the swollen system during the

1 1

1 ( ) / .100

n n

i i

f Rt Tt Rt

= =

 

= −

 

 

∑ ∑

0.5

2 50.log 1 ( ) .100

f Rt Tt

−

 

 

 

 

= + −

 

 

 

 

 

 

 

∑

110

immersion in the medium for determining the volume of the floating

object. The resultant-force due to the sinking of the system in simulated

gastric fluid without enzyme (USP 29) at 37 ± 0.5 °C was measured

during time by the weighing part of the apparatus (precision 0.1 mg).

The resultant-force is the difference between the buoyancy and gravity

forces both acting on the system submerged in water, according to the

following equation Eq. (6):

(6)

where F

result

is the resultant force, d

is the density of the medium in

which the object is sunk, d

is the apparent density of the solid, g is the

gravity acceleration and V the volume of the object. This force was

measured as weight by the balance inserted in the apparatus.

3. Results and discussion

3.1 In vitro drug release

The modules have been designed to allow their assemblage by

interlocking the curved bases of disc. Two differently shaped Dome

Matrix

modules were manufactured in this study to obtain NFX

prolonged release floating drug delivery systems i.e., a “female” module

and “male” module (Figure 1). In particular, as the picture shows, the

protrusion on the concave base rim of male module , was designed in

order to fit the concavity on the concave base of the module without

protrusion on the rim, i.e., female. Thus, facing the concave base of

male module to the concave base of the female one and exerting a light

pressure, the two modules interlock by clicking giving rise to one-piece

assembled system characterized by the presence of an empty internal

space.

The norfloxacin release profiles of the Dome Matrix

modules

and their assemblies are shown in Figures 2-5. The release of NFX

observed for the female modules, independently on the polymer used

and its concentration, was faster compared to the male modules.

However, the curves were found pretty similar based on the difference

(ƒ1) and similarity (ƒ2) parameters calculated (Table 2). This difference

between the two shapes of modules was already observed with other

( ) .

result f s

F d d g V

= −

drugs having different solubility and formulated as hydrophilic matrices

with HPMC [7

, 23]. Moreover, even when inert polymers or compounds

were used to obtain the modules (such as Tapioca starch derivatives

[23]), the female module showed a faster release profile than the male.

Apart the different initial surface area, the release differe

also assigned to the swelling kinetics of the two modules. The male and

female modules have different concavity size due to the protrusion on

the rim of male concave base. We observed that the swelling determined

the fill-up with jellified po

lymer of the concavity of the male module

but not of the female module that has a larger concavity. As a

consequence, the female module erodes/dissolves more quickly than the

male one. Finally, the individual modules exhibited no floatation.

Fig. 2

Norfloxacin fraction released vs. time of Dome Matrix

containing 20% of HPMC: female module (Ο); male module (

configuration (

) (mean values ± standard deviation, n=6)

111

drugs having different solubility and formulated as hydrophilic matrices

, 23]. Moreover, even when inert polymers or compounds

were used to obtain the modules (such as Tapioca starch derivatives

[23]), the female module showed a faster release profile than the male.

Apart the different initial surface area, the release differe

nce has to be

also assigned to the swelling kinetics of the two modules. The male and

female modules have different concavity size due to the protrusion on

the rim of male concave base. We observed that the swelling determined

lymer of the concavity of the male module

but not of the female module that has a larger concavity. As a

consequence, the female module erodes/dissolves more quickly than the

male one. Finally, the individual modules exhibited no floatation.

Norfloxacin fraction released vs. time of Dome Matrix

modules

); male module (

); void

) (mean values ± standard deviation, n=6)

112

Fig. 3

Norfloxacin fraction released vs. time of Dome Matrix

containing 30% of HPMC: female module (Ο); male module (

configuration () (mean values ± standard deviation, n=6)

Fig. 4

Norfloxacin fraction released vs. time of Dome Matrix

containing 20% of PEO: female module (Ο); male module (

configuration () (mean values ± standard deviation, n=6)

Norfloxacin fraction released vs. time of Dome Matrix

modules

); male module (

); void

Norfloxacin fraction released vs. time of Dome Matrix

modules

); male module (

); void

Fig. 5

Norfloxacin fraction released vs. time of Dome Matrix

containing 30% of PEO: female module (Ο); male module (

configuration () (mean values ± standard deviation,

n=6)

Table 2. Difference factor (ƒ1) and similarity factor (ƒ2) calculated

for the male and female modules

Formulation ƒ1 ƒ2

HPMC K4M 20% 4.84

71.41

HPMC K4M 30% 6.37

64.96

PEO N60K 20% 13.86

50.18

PEO N60K 30% 7.54

58.54

The release studies of the void configuration demonstrated that

NFX release rate was significantly slowed down in comparison to the

release of individual modules, independently on the polymer and

concentration. The void assembled modules containing 20% HPM

released about 80% of drug in 270 min maintaining the floatation up to

240 min (Figure 2). In correspondence of this last time, assembled

modules disintegrated completely with the consequent impairment of

floatation capacity. This was evidently reflecte

d by the release profile

that in between 200-

250 minutes had a sudden increase of fraction

113

Norfloxacin fraction released vs. time of Dome Matrix

modules

); male module (

); void

n=6)

Table 2. Difference factor (ƒ1) and similarity factor (ƒ2) calculated

71.41

64.96

50.18

58.54

The release studies of the void configuration demonstrated that

NFX release rate was significantly slowed down in comparison to the

release of individual modules, independently on the polymer and

concentration. The void assembled modules containing 20% HPM

released about 80% of drug in 270 min maintaining the floatation up to

240 min (Figure 2). In correspondence of this last time, assembled

modules disintegrated completely with the consequent impairment of

d by the release profile

250 minutes had a sudden increase of fraction

114

release rate. The significant increase in the NFX release values standard

deviations after 200 minutes indicates the variability of the void

configuration disintegration time. The floating capability of the Dome

Matrix

in void configuration was already studied in humans, showing

that the system remained in the stomach after a light standard meal for

214.5 ± 54.2 min [6]. The prolonged gastric residence could be

beneficial for NFX absorption that due to the favored dissolution in acid

can be quickly absorbed in stomach or in the first intestinal tract [24,

25].

The assembled modules containing 30% HPMC had a different

performance in terms of release and floatation. The void configuration

system remained floating until 480 minutes, the end of the release

experiment (Figure 3). At 210 min, which corresponded to the average

time of void configuration gastro-residence, about 55% of NFX was

released. The 80% release was achieved at about 400 min; at this time,

the system could already be eliminated from the stomach.

High molecular weight poly-(ethylene oxide) have been

proposed as an alternative to HPMC. The correct choice of this

hydrophilic polymer molecular weight and quantity in the matrix

formulation can provide an appropriate combination of swelling,

dissolution or erosion mechanisms to control drug release kinetics [26-

29]. For the formulations containing PEO, the drug release rate of

individual modules was faster than the correspondent modules made

with HPMC. The same was also observed with the void configurations

in comparison to the release of the HPMC assembled modules (Figures

4 and 5).

However, similarly to HPMC system, the void assembled

modules made with 20% PEO maintained the floating characteristic up

to 240 min. After this time, assembled systems completely disintegrated

with impairment of floating capacity. Despite the different polymer, also

with this polymer the disintegration of the assembled system was

reflected by an evident slope increase in the profile fraction released

versus time after 200 min (Figure 4). Similarly, the increase in standard

deviation of NFX release values due to the variability of disintegration

time can be observed. The floatation time corresponded to about 80%

NFX released.

Compared to HPMC composition, the Dome Matrix

modules

containing 30% PEO (Figure 5) showed faster release rate for both the

modules than for the assembly. The floating time was prolonged until

115

330 min by the higher polymer concentration. At 210 min, about 60% of

NFX was released and the 80% release was achieved at about 300 min.

3.2 Analysis of drug release

The release profiles were analyzed with the Korsmeyer

diffusional exponential equation up to 60% of drug released [18].

Release data analysis was carried out also with the Peppas and Sahlin

equation in order to calculate the Fickian fraction released of each

profile [19]. Parameter values are listed in Table 3. Calculation of the

exponent n identifies the prevalent mechanism of release. For planar

systems, n = 0.5 indicates diffusion-controlled (Fickian) drug release

and n = 1.0 indicates swelling/erosion-controlled drug release (Case-II

transport). Values of n between 0.5 and 1.0 can be regarded as a

superposition of both phenomena, indicating that the drug delivery was

not controlled only by diffusion, but also significantly by polymer

relaxation or erosion mechanisms (anomalous transport). In general, the

data of all release systems studied provided good fit to the different

models (Table 3), supporting the adaptability of both models to fit the

release data with the systems and polymers studied.

116

Table 3. Mathematical modeling and drug release kinetics from

Dome Matrix

modules using Korsmeyer et. al and Peppas and

Sahlin equations (mean values ± SD; n=6).

Modules Korsmeyer et. al equation

Peppas and Sahlin equation

n ± 95% CI r

x 10

HPMC K4M 20%

Male 0.686 ± 0.044 0.9994 29 ± 7.6 9.1 ± 1.2

0.9992

Female 0.758 ± 0.021 0.9996 17 ± 3.6 13 ± 0.8 0.9995

Void 0.688 ± 0.011 0.9986 14 ± 3.4 4.5 ± 0.5

0.9996

HPMC K4M 30%

Male 0.767 ± 0.040 0.9983 16 ± 6.9 9.4 ± 1.3

0.9999

Female 0.732 ± 0.049 0.9998 15 ± 5.6 12 ± 1.2 0.9981

Void 0.718 ± 0.014 0.9998 14 ± 1.8 4.2 ± 0.3

0.9995

POE N60K 20%

Male 0.841 ± 0.025 0.9977 -4.8 ± 3.4 16 ± 0.7 0.9979

Female 0.921 ± 0.029 0.9995 -17 ± 5.2 22 ± 1.0 0.9993

Void 0.816 ± 0.014 0.9993 -0.4 ± 2.0 7.4 ± 0.3

0.9997

POE N60K 30%

Male 0.679 ± 0.015 0.9911 18 ± 3.4 11 ± 0.7 0.9940

Female 0.742 ± 0.024 0.9975 15 ± 6.1 15 ± 1.3 0.9983

Void 0.670 ± 0.013 0.9982 15 ± 1.4 4.9 ± 0.2

0.9996

In the case of HPMC modules, the n values from Korsmeyer

equation were in the range 0.686 - 0.767 indicating anomalous (non-

Fickian) transport as evidenced by the quasi-linear release profiles [33].

The k

and k

values for male and female modules revealed the

relevance of drug diffusion or polymer relaxation and erosion.

Calculations showed that the fraction of drug released due to Fickian

mechanism was the lowest for the female module compared to the male

module and void assembly (Figure 6). Fickian fraction released was

very similar for formulations containing 20 and 30% of HPMC

assembled in void configuration. This result fits well with the similar n

exponents obtained with Korsmeyer equation (Table 3). The

contribution of Fickian diffusion to NFX release was around 50% for

the assembled system at the beginning of the release experiment and

tended to decrease with time. The percentages of drug released by

Fickian mechanism for individual modules, were lower than 40% with

the exception of the male module containing 20% of HPMC that

behav

ed similarly to the assembled system. Considered that the

difference between the assembled system and the individual modules is

the non-

accessibility of the concave bases to the release medium after

the assemblage, it seems straightforward to consider that

penetration, chain disentanglement, build-up of gel-

layer thickness and

erosion of the matrix occurred differently affecting the balance between

Fickian and relaxation release mechanism. These results corresponded

to a relevant anomalous Fickian r

elease typical of swellable matrices

due to an important contribution of the polymer swelling and erosion

mechanisms to drug delivery.

Fig. 6

Norfloxacin Fickian released fraction vs. time of Dome Matrix

modules containing 20% of HPMC: female module (



(); void configuration (

) and 30% of HPMC: female module (

male module (); void configuration (

) (mean values ± standard

deviation, n=6)

In the case of PEO polymer, n diffusional exponents for the

PEO concentration of 20% were in the range 0.816 -

0.921, indicating a

release mechanism very close to Case II transport. The negative values

of k

and the high values of k

in Peppas and Sahlin equation indicate

117

Fickian mechanism for individual modules, were lower than 40% with

the exception of the male module containing 20% of HPMC that

ed similarly to the assembled system. Considered that the

difference between the assembled system and the individual modules is

accessibility of the concave bases to the release medium after

the assemblage, it seems straightforward to consider that

solvent

layer thickness and

erosion of the matrix occurred differently affecting the balance between

Fickian and relaxation release mechanism. These results corresponded

elease typical of swellable matrices

due to an important contribution of the polymer swelling and erosion

Norfloxacin Fickian released fraction vs. time of Dome Matrix



); male module

) and 30% of HPMC: female module (

);

) (mean values ± standard

In the case of PEO polymer, n diffusional exponents for the

0.921, indicating a

release mechanism very close to Case II transport. The negative values

in Peppas and Sahlin equation indicate

118

that the drug release is predominantly controlled by polymer relaxation

or erosion [30]. Due to the negative values of k

obtained, the Fickian

release fraction was not calculated in this case. Likely, the PEO gel

layer was weaker in comparison to HPMC and could be more rapidly

removed by the dissolution medium; therefore, NFX/PEO matrix system

is more susceptible to the erosion process. In general, PEO polymer is

considered more soluble than HPMC, but, its molecular weight and

amount in the formulation allow tailoring the drug release kinetics [26,

27, 29, 31].

For the 30% PEO concentration, the diffusional exponents

between 0.670 - 0.742 indicated anomalous Fickian transport profiles

[32, 33]. The similarity between k

and k

values revealed that the drug

release was controlled by drug diffusion and polymer relaxation and

erosion mechanisms. Figure 7 showed that also in this case the fraction

of drug released due to Fickian mechanism was the lowest for the

female module. Fickian fraction release values for void configuration

were very similar to those obtained for 20 and 30% of HPMC

assembled in the same configuration, confirming also the n diffusional

exponents obtained (Table 3). As observed for HPMC, the NFX release

due to Fickian contribution initially was about 50% and decreased with

time (Figure 7). It was impressive to see the linearity of the release

profiles obtained with this polymer considered that a quasi-linear

dissolution profile (Fig. 4 and Fig. 5) could be observed up to 80% of

NFX released.

Fig. 7

Norfloxacin Fickian released fraction vs. time of Dome Matrix

modules containing 30% of PEO: female module (Ο); male module (

void configuration (

) (mean values ± standard deviation, n=6)

3.3 In vitro floatation behavior of assembled modules

Originally, the dome-

shaped modules were designed for

facilitating th

eir assembly by insertion of the convex base into the

concave in the aim to build up a pile. The possibility to proceed to a

different module assembly was discovered later when glue or

ultrasounds were used to firmly attach the modules by creating links

tween the flat surfaces of the concave base rims held in contact. In

this study, two new types of dome modules described elsewhere [6],

having the rim of concavity modified in order to favor a firm concave

to-concave assembly by simple interlocking were us

ed (see Figure 1).

The floatation behavior of the void assembled NFX system, made of

two swellable modules, was studied in vitro. In particular, it was

determined the force required to hold the system submerged in water

and its variation over time, accordi

ng to [6, 22]. The individual male and

female modules never floated.

Based on the floating time observed in the dissolution profiles,

two formulations (20% HPMC and 20% PEO) were selected for this

study. The floatation behavior, expressed as resultant

versus time, is shown in Figure 8. The profiles show that the systems did

119

Norfloxacin Fickian released fraction vs. time of Dome Matrix

Ο); male module (

);

) (mean values ± standard deviation, n=6)

shaped modules were designed for

eir assembly by insertion of the convex base into the

concave in the aim to build up a pile. The possibility to proceed to a

different module assembly was discovered later when glue or

ultrasounds were used to firmly attach the modules by creating links

tween the flat surfaces of the concave base rims held in contact. In

this study, two new types of dome modules described elsewhere [6],

having the rim of concavity modified in order to favor a firm concave

ed (see Figure 1).

The floatation behavior of the void assembled NFX system, made of

two swellable modules, was studied in vitro. In particular, it was

determined the force required to hold the system submerged in water

ng to [6, 22]. The individual male and

Based on the floating time observed in the dissolution profiles,

two formulations (20% HPMC and 20% PEO) were selected for this

weight variation

versus time, is shown in Figure 8. The profiles show that the systems did

120

not float immediately but it started to float between 5-10 min after

submersion in medium; at 10 min a positive resultant-weight of

approximately 4 and 2 mg was measured for PEO and HPMC,

respectively. Then, the resultant-weight values increased attaining peak

values of approximately 42 mg for HPMC and 45 mg for PEO, both at

125 min. After this peak, the resultant-weight had a continuous slow

decrease for HPMC, while an abrupt decrease was observed for PEO.

This PEO profile could anticipate the beginning of disintegration system

manifested by a partial exit of air bubbles from the inside of the void

module. The buoyancy variation during time can be explained as the

volume and weight change of the immersed system due to water uptake

and swelling. Therefore, the augmentation of resultant-weight over time

was due to the swelling of the system, which reinforced the capability of

the system to float. The gastric residence time demonstrated in vivo

(214.5 ± 54.2 min) was derived from an experiment after a standard

meal without any additional food [6]. In case where a prolonged gastric

residence time is requested and the food intake after drug administration

is allowed, a more durable Dome Matrix

system could be constructed.

In this case, formulations containing higher amount of polymers (HPMC

or PEO) should be studied.

Fig. 8 Resultant weight vs. time of Dome Matrix

20% HPMC K4M

() and 20% PEO N60K () modules assembled

in void configuration

(mean values ± standard deviation, n=3)

4. Conclusion

This study revealed a prolonged linear release of norfloxacin in

simulated gastric fluid when the drug is formulated in Dome Matrix

modules with hydroxypropylmethylcellulose but

, in particular, with

poly(ethylene oxide). Two modules, “male and female” have been

manufactured and were interlocked to form the “void” configuration.

This assembly exhibited in vitro floatation up to 240 min, while the

individual male and female modules

never floated. The mechanism of

NFX release from Dome Matrix

modules and void assemblies was

mainly governed by the swelling and erosion of the polymer matrix. The

diffusive contribution to drug release was lower than 50% and decreased

during release time. In conclusion, a site-

specific prolonged drug

delivery system of norfloxacin was achieved when the modules were

assembled in the void configuration; the release kinetics was strongly

linear, independently on floating conditions. However, in vivo studies

are necessary to confirm the possibility for this linear release gastro

retentive system to increase norfloxacin bioavailability and reduce dose

administration.

121

20% HPMC K4M

in void configuration

This study revealed a prolonged linear release of norfloxacin in

simulated gastric fluid when the drug is formulated in Dome Matrix

, in particular, with

poly(ethylene oxide). Two modules, “male and female” have been

manufactured and were interlocked to form the “void” configuration.

This assembly exhibited in vitro floatation up to 240 min, while the

never floated. The mechanism of

modules and void assemblies was

mainly governed by the swelling and erosion of the polymer matrix. The

diffusive contribution to drug release was lower than 50% and decreased

specific prolonged drug

delivery system of norfloxacin was achieved when the modules were

assembled in the void configuration; the release kinetics was strongly

linear, independently on floating conditions. However, in vivo studies

are necessary to confirm the possibility for this linear release gastro

retentive system to increase norfloxacin bioavailability and reduce dose

122

Acknowledgments

The authors wish to thank the Brazilian agency CNPq (National

Counsel of Technological and Scientific Development) for the financial

support.

123

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127

DISCUSSÃO GERAL

128

129

O norfloxacino é um agente antibacteriano amplamente

utilizado para o tratamento de infecções no trato urinário. Normalmente

a posologia indicada é de 400 mg duas vezes ao dia (MANDELL, 1988;

CHRISTIAN, 1996; EMMERSON; JONES, 2003) e não se encontra

disponível comercialmente formulação de liberação prolongada, para

administração em dose única diária. Para o desenvolvimento racional de

medicamentos, as etapas de caracterização das matérias-primas e

estudos de pré-formulação são de fundamental importância, pois através

da investigação da compatibilidade entre fármaco-excipiente é possível

selecionar excipientes adequados para proceder com o desenvolvimento

farmacotécnico (BRUNI et al., 2002; GIRON et al., 2002; BRUNI et al.,

2010).

A caracterização do norfloxacino e excipientes, bem como os

estudos de compatibilidade, foram apresentados na forma de publicação

científica no capítulo 2. Através de DSC pode-se avaliar que o fármaco

apresenta ponto de fusão de aproximadamente 219 ºC, seguido de

evento exotérmico, sem perda de massa (observado por TG). Como não

há evento endotérmico antes da fusão, pode-se inferir que a matéria-

prima é a forma anidra e não dihidrato ou sesquihidrato. A degradação

do fármaco ocorre em duas etapas sucessivas em temperaturas entre

330–376 ºC e 421–455 ºC. Através das imagens obtidas por SEM e dos

padrões de XRPD demonstrou-se alto grau de cristalinidade para a

amostra, o que é importante para o desenvolvimento de comprimidos,

umas vez que substâncias amorfas tendem a ser menos estáveis em

relação às cristalinas. O polímero hidrofílico POE também demonstrou

características cristalinas, com reflexões bem definidas, e a HPMC

mostrou-se amorfa.

O norfloxacino apresenta três formas anidras (A, B e C) e suas

temperaturas de fusão são 219 ºC (A), 212 ºC (B) e 207 ºC (C)

(BARBAS; PROHENS; PUIGJANER, 2007). A estrutura cristalina da

forma A foi determinada por difração de raio-x de monocristal, sendo

triclínica, grupo espacial P-1 (BASAVOJU, S.; BOSTRÖM, D.;

VELAGA, 2006; PUIGJANER et al., 2010). Baseando-se

principalmente no ponto de fusão (219 ºC) e no perfil termoanalítico

pode-se inferir que a forma utilizada nesta tese é a forma A.

Para o estudo de compatibilidade, uma vez que não se

encontram relatos de interação/incompatibilidade do norfloxacino,

selecionaram-se os excipientes farmacêuticos que possivelmente

comporiam a formulação final. Utilizou-se mistura física fármaco-

excipiente na proporção 1:1 (p/p) e analisou-se através de DSC. As

130

curvas obtidas foram consideradas como superposição das curvas

individuais dos compostos da mistura, não sendo observados

deslocamentos ou desaparecimento do ponto de fusão do norfloxacino,

demonstrando ausência de incompatibilidade.

A cromatografia líquida possibilita a separação e quantificação

de diferentes componentes de uma formulação farmacêutica através da

escolha adequada dos parâmetros do sistema como colunas, fase móvel

e método de detecção. Apesar de existir método farmacopeico para

comprimidos de norfloxacino de liberação imediata (F. BRAS. IV,

2001), este requer um tempo de estabilização da coluna analítica por 8

horas e não pode ser utilizado para estudos de estabilidade sem prévia

avaliação. Além disso, como se trata de nova formulação, é necessário

método analítico adequado. Desta maneira, desenvolveu-se e validou-se

procedimento para a determinação de norfloxacino nos comprimidos

matriciais conforme demonstrado no capítulo 3. A metodologia

consistiu na utilização de coluna C

mantida a 40 ºC, detecção no UV a

272 nm, fase móvel composta por tampão fosfato 0,04 M (pH 3,0) e

acetonitrila na proporção 84:16 (v/v), com vazão de 1,0 mL/min.

Na avaliação da especificidade foi possível a identificação de

um composto de degradação, o norfloxacino descarboxilado. Além

disso, nos cromatogramas obtidos, os picos dos produtos de degradação

encontraram-se resolvidos em relação ao pico do norfloxacino,

demonstrando a especificidade e que o procedimento também pode ser

usado como indicativo da estabilidade. O método apresentou-se linear

na faixa de 0,5-5 µg/mL (r

= 0,9999). Os dados obtidos para a

repetibilidade (DRP ≤ 0,98), precisão intermediária (DRP ≤ 0,59) e

exatidão (99,90% ± 0,97%) estão dentro dos valores preconizados. A

avaliação da robustez através da superfície de resposta demonstrou que

mudanças pequenas e deliberadas em diversos fatores não influenciaram

significativamente a metodologia. Desta forma, demonstrou-se que o

método proposto cumpre os requisitos preconizados pela literatura,

podendo ser empregado para a análise pretendida.

O desenvolvimento, estudos de estabilidade e avaliação in vitro

de formulações com tecnologia de liberação prolongada contendo

norfloxacino foi apresentado como publicação científica no capítulo 4.

A partir do estudo de pré-formulação, além dos polímeros hidrofílicos

POE e HPMC, foram selecionados como excipientes a celulose micro-

cristalina (diluente), o estearato de magnésio (lubrificante) e o dióxido

de silício coloidal (deslizante, absorvente).

131

Para o desenvolvimento das formulações avaliou-se o processo

de granulação via seco e via úmida, porém não se observou diferença

entre estes e a compressão direta na obtenção dos comprimidos. Dessa

forma, optou-se pelo processo de compressão direta, devido a maior

simplicidade e rapidez. Devido ao fato da liberação prolongada ser

obtida através dos polímeros hidrofílicos, foram avaliados dois tipos

(POE e HPMC) de diversas massas moleculares e duas concentrações

(20 e 30%) nas formulações. Os valores de concentração foram

selecionados pois estão na faixa normalmente utilizada na indústria

farmacêutica para a fabricação de comprimidos de liberação prolongada.

Dessa forma a obtenção dos comprimidos por compressão direta e a

quantia de polímero na formulação levaram em conta uma possível

transposição para escala industrial.

O equivalente a 50% do total de comprimidos produzidos foi

submetido ao processo de revestimento. Para estudar a influencia da

presença de blister, realizou-se a emblistagem de comprimidos

revestidos e não revestidos. A avaliação da qualidade foi determinada

segundo as características farmacopéicas de variação de peso, dureza,

friabilidade e perda por dessecação e os resultados obtidos estão de

acordo com os valores preconizados.

O estudo de estabilidade acelerada, realizado em condições

recomendadas para zona climática IV (40 ± 2 ºC e 75 ± 5% umidade

relativa) por 6 meses (BRASIL 2005, WHO, 2009) demonstrou que não

houve alteração na aparência ou teor de principio ativo. Porém, na

avaliação da fotoestabilidade, foi evidenciada a necessidade do

revestimento dos comprimidos ou a presença de blister opaco, uma vez

que houve alteração na coloração dos comprimidos.

Os perfis de dissolução foram realizados utilizando método

baseado nas farmacopéias brasileira e americana, utilizando tampão pH

4 (F. BRAS. IV, 2001; USP 30, 2007). Os resultados in vitro

demonstraram que conforme aumentava-se a massa molecular e a

concentração do polímero hidrofílico, a liberação do fármaco tornava-se

mais lenta, inclusive com algumas formulações apresentando

incompleta dissolução após 24 horas.

Através da análise dos perfis pela equação de Korsmeyer-

Peppas (KORSMEYER et al., 1983) pode-se concluir que a liberação do

norfloxacino foi controlada pela difusão do fármaco e

intumescimentos/erosão do sistema matricial. Quando o comprimido

continha menor concentração e/ou menor massa molecular do polímero

o mecanismo de liberação predominante, indicado pelo valor do

132

expoente n ≥ 0,89, era o Caso-II ou Super Caso-II (intumescimento do

polímero, relaxamento da matriz ou liberação mediante erosão). Quando

aumentava-se a massa molecular e/ou a concentração de polímero na

matriz o mecanismo de liberação mudava para transporte anômalo (0,45

< n > 0,89), mostrando crescente influência da difusão do fármaco na

dissolução.

As formulações contendo POE ou HPMC de massas

moleculares intermediárias (HPMC K4M 20%, POE N60K 20% e POE

N60K 30%) ou alta concentração de baixa massa molecular (HPMC

K100 LV 30%) foram consideradas mais promissoras para futuros testes

in vivo, pois resultaram em combinação entre dissolução total do

norfloxacino e estrutura da camada gelatinosa formada. Baseado na

liberação prolongada e total do fármaco, uma melhor biodisponibilidade

é esperada. Para melhor compreensão e correlação com o que poderá

ocorrer in vivo, estudos em diferentes pHs ou em fluidos gástrico e

intestinal simulados estão sendo planejados como continuação desta

tese. Dependendo dos resultados obtidos as formulações poderão ser

otimizadas, inclusive com a possibilidade da utilização de polímeros de

diferentes massas moleculares na mesma formulação para obtenção do

perfil de liberação adequado.

Além da fabricação de sistemas matriciais utilizando

compressoras com punções convencionais, a inovação tecnológica

permite a renovação e a otimização destas técnicas clássicas. Um

exemplo disso foi o desenvolvimento dos sistemas de acoplamento

Dome Matrix

. Baseado nesta tecnologia, foi estudada a possibilidade

de obter sistema flutuante para liberação prolongada e local específica

de norfloxacino, conforme descrito no capítulo 5. Utilizou-se duas

concentrações, 20 e 30% de HPMC e, pela primeira vez, POE. Módulos

individuais “male” e “female” foram obtidos por compressão direta

utilizando punções especiais (LOSI et al., 2006). O acoplamento destes

permitiu a obtenção da configuração flutuante “void”, devido a presença

de ar no interior do sistema.

Além das formulações demonstradas neste capítulo (contendo

HPMC K4M e POE N60K, ambos a 20 e 30%) foram produzidas outras

(contendo 20 e 30%) de: HPMC K100 LV, HPMC K15M, POE N12K e

POE 301. Para os polímeros de baixa massa molecular (HPMC K100

LV e POE N12K) obteve-se dissolução in vitro muito rápida e pouco

tempo de flutuação. Ao contrário, com os polímeros de massa molecular

mais alta (HPMC K15M e POE 301) a velocidade de dissolução era

demasiada lenta, tanto para os módulos individuais como para o sistema

133

flutuante. A dosagem de 200 mg para a configuração “void” é inferior à

recomendada normalmente para adultos de 400 mg. Porém, devido a

esta tecnologia estar ainda em desenvolvimento, outros punções com

capacidade superior (e inferior) de carga estavam sendo desenvolvidos

quando da realização desta etapa. Diferentes processos tecnológicos

como granulação a seco e via úmida não resultaram em melhora em

comparação à compressão direta.

Nos estudos de dissolução observou-se maior velocidade de

liberação do norfloxacino nos módulos individuais em relação à

configuração flutuante, o que pode ser explicado devido à maior área

superficial em contato com o meio. Esta característica já havia sido

observada com outros fármacos de diferentes solubilidades e formulados

como matrizes hidrofílicas (CASAS et al., 2010; STRUSI et al., 2010)

Os perfis de dissolução analisados de acordo com as equações de

Korsmeyer-Peppas e Peppas-Sahlin demonstraram que a liberação era

controlada tanto pela difusão do fármaco como pelo intumescimento e

erosão da matriz. Da mesma forma que para os comprimidos descritos

no capítulo 4, os sistemas Dome Matrix

fabricados com POE e menor

concentração de polímero apresentam mecanismo de liberação mais

dependente da erosão, o que pode ser evidenciado pelos perfis de

dissolução quase lineares e nos maiores valores do expoente n obtidos.

A HPMC K4M apresenta uma viscosidade média de 4000 mPa s (2%;

p/v) que pode ser considerada semelhante a do POE N60K (3060 mPa s,

2%, p/v). Dessa forma fica evidente o caráter mais erodível do POE em

relação à HPMC, o que está de acordo com o descrito por Jamzad e

Fassihi (2006). A contribuição da difusão de fick para a liberação do

norfloxacino era inicialmente menor que 50% e diminuía de acordo com

o tempo.

Os módulos na configuração “void” iniciaram a flutuar em

menos de 10 minutos e mantiveram esta capacidade por pelo menos 240

minutos, o que está de acordo com o tempo médio de permanência

estudado in vivo de 214, 5 ± 54,2 minutos (STRUSI et al., 2008). Com o

aumento da concentração de polímero na formulação, aumenta-se a

capacidade flutuante do sistema. A flutuação in vitro obtida sugere um

sistema gastro-retentivo in vivo, o que poderia aumentar a

biodisponibilidade do norfloxacino, uma vez que este fármaco é mais

solúvel em meios ácidos, com conseqüente redução da posologia.

134

135

CONCLUSÕES

136

137

 As matérias-primas de norfloxacino e polímeros (HPMC e POE)

foram caracterizadas e os resultados obtidos podem ser utilizados

como parâmetros de controle de qualidade para futuros estudos;

 Estudos de pré-formulação foram realizados e não observou-se

incompatibilidade entre fármaco e excipientes;

 O método desenvolvido e validado, por cromatografia líquida em

fase reversa, foi utilizado para análise de norfloxacino nas matrizes

desenvolvidas e estudos de estabilidade;

 Comprimidos matriciais foram obtidos por compressão direta e

cumpriram com as características farmacopéicas preconizadas;

 O processo de revestimento tem influencia sobre o perfil de

dissolução dos comprimidos, porém esta não foi considerada

biofarmaceuticamente relevante;

 Os estudos de estabilidade sugerem que as formulações são

estáveis frente à temperatura e umidade, porém o revestimento e/ou

presença de blister opaco deve ser recomendado para prevenir a

fotodegradação do princípio-ativo;

 As formulações contendo POE ou HPMC de massas moleculares

intermediárias (HPMC K4M 20%, POE N60K 20% e POE N60K

30%) ou alta concentração de baixa massa molecular (HPMC K100

LV 30%) apresentaram melhor perfil de liberação in vitro;

 O mecanismo de liberação do norfloxacino depende da

concentração e massa molecular do polímero utilizado;

 Sistemas Dome Matrix

acoplados na configuração “void”

permitiram flutuação in vitro, sugerindo liberação local específica

(gastro-retentiva) in vivo para o fármaco;

 Estudos in vivo são necessários para confirmar a possibilidade de

aumento na biodisponibilidade e redução do regime posológico do

norfloxacino através do uso de sistemas matriciais e Dome

Matrix

138

139

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