In vitro – In vivo Correlation Study of Colon-targeted Metronidazole

Microparticle in Corncob Hemicellulose Capsule

Gabena Indrayani Dalimunthe

and Ida Fauziah

Department of Pharmaceutical Technology, Faculty of Pharmacy,

Universitas Muslim Nusantara Al Washliyah, Medan, Indonesia

Department of Biology, Faculty of Science and Technology, Universitas Medan Area, Medan, Indonesia

Keywords: Colon-targeted, Metronidazole, In vitro, In vivo

Abstract: Colon-targeted therapy requires a strategy to keep the medicine pass the stomach and release in the colon,

which is hard to reach by conventional dosage form of metronidazole in gelatin capsule. In this study,

microparticles dosage form of metronidazole which was placed inside corncob-hemicellulose capsule shell

need to be compared with conventionally gelatin capsule of metronidazole preparation to examine whether

it meets medications standards. The aim of this study was to find out the in vitro and in vivo assay

correlation of metronidazole microparticle which covered by corncob hemicellulose capsules. In vitro test

was carried out to observe the profile of differences in the percent release of metronidazole from various

formulations with various media and times. It was performed using a dissolution tester, in an artificial

stomach medium of pH 1.2 for 6 hours in artificial intestinal medium of pH 7.4 for 10 hours, and in artificial

colonic medium of pH 8 for 10 hours. The in vivo test design was conducted using six rabbits. The drug

released in plasma was measured by HPLC using 1% glacial acetic acid solvent in aqua bidest and

methanol-water with a ratio of 80: 20. The test was performed using the cross over design method.

Metronidazole microparticle capsules were administered orally according to the test design (metronidazole

in microparticles and metronidazole in conventional forms). Based on the plotted graph (data retrieval was

started from the drug released in the colon because the drug began to be absorbed at that time), a correlation

value was obtained (R

= 0.8785). It can be stated that there is a correlation between the formulations tested

in vitro and in vivo because the correlation value was greater than 0.8, it is assumed to have a correlation.

1 INTRODUCTION

Gelatin capsule has been used for decades in various

therapy as well as for gastrointestinal diseases. The

emerging problems are including the enzymatic

reactions in the upper gastrointestinal tract which

induce premature release of medicine before it

reaches the colon (Nicholas et al. 2011, Lee et al.

2020). Colon Drug Delivery Systems (CDDS) has

become a focus in in drug research and development

resulting in modifications of capsules by using

animal and plant origins material in order to improve

drug bioavailability in the colon (Amidon et al.

2015, Oladzadabbasabadi et al. 2017, Yang et al.

2020). Plant products has become more preferable in

recent years in contrast with products that are made

from animal origin material which could be due to

numerous reasons including consumers views and

beliefs (Cliceri et al. 2018).

The phenomenon has no exception for

medications. Therefore, rapid drug development is

required to enhance efficacy and fit consumers and

patient’s preference. In vitro and in vivo

investigations are paramount in drug development to

confirm the effectiveness which were carried out by

examining the relationship between dissolution and

bioavailability, resulting in the concept of in vitro in

vivo correlation. In the last few years, the concept

and application of in-vitro-in vivo correlation for

pharmaceutical dosage forms has become a major

focus of attention of the pharmaceutical industry,

academics and the regulatory sector (Maity et al.

2016).

From a biopharmaceutical perspective, in vitro-in

vivo (IV-IVC) correlation is a mathematical

prediction that describes the relationship between

the in vitro nature of the dosage form (drug release

rate) and the relevant in vivo response (plasma drug

Dalimunthe, G. and Fauziah, I.

In vitro - In vivo Correlation Study of Colon-targeted Metronidazole Micropar ticle in Corncob Hemicellulose Capsule.

DOI: 10.5220/0010613400002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 561-565

ISBN: 978-989-758-556-2

561

concentration, urine, amount of drug absorbed).

IVIVC is a tool for the development of drug dosage

forms, since IVIVC can assist in the selection of

drug formulations with suitable and acceptable

dissolution criteria, these predictions can be used as

estimates or substitutes for further bioequivalence

studies (Emami, 2006; Qiu and Duan, 2017)

The in vitro test was carried out to find out the

profile of differences in the percent release of

metronidazole from various formulas with various

mediums and times with a stirring speed of 100 rpm,

medium volume of 900 ml at 370.5°. Meanwhile, in

vivo testing is a test that is carried out using

experimental animals to determine the metabolism

of a compound in the body. Animals used in in vivo

experiments must be from mammals, because the

results can be applied to humans (Chow et al. 2003).

2 MATERIALS AND METHOD

2.1 Materials

Metronidazol (E Merck), NaOH 0.1 N, (E Merck),

CaCl

(E Merk), KH

(E Merk), HCl (E Merck),

NaOCl 5% (E Merk), talcum (Yuanfen), asam

asetate glasial (E Merck), trichoroasetat acid (TCA)

20% , Alkohol 96%, heparin, Metanol for HPLC

(E.Merck), Aquabidestilata (PT. Ikapharmando

Putramas), Metronidazol BPFI (Badan POM RI)

metronidazole tablet (indofarma), all of materials

used in the study are in pro analysis standard (Ahuja,

et al, 2005).

Spectrophotometer (Shimadzu UV-1800),

disintegration tester (Erweka), disolution tester,

HPLC (Agilent 1120 Compact LC), Colom ODS C-

18, solvent container (oberol), vial (agilent), animal

box, vakum pump (Gast DO), sonicator (branson),

paper membrane filter cellulosa nitrate 0,45 μm

(whatman), paper membrane filter nylon 0.45 μm

(whatman), PTFE 02 μm (whatman) ( Muchlisyam,

2014).

2.2 Dissolution Test

Dissolution test was performed using a dissolution

apparatus type 2 (paddle), with 900 mL medium pH

1.2, pH 7.4and pH 8 and temperature of 37 ± 0.5 ° C

with a rotation speed of 100 rpm. At certain time

intervals of 5, 15, and 30 minutes until 600 minuts,

the sample solution was taken 5 mL and measured at

a wavelength of 320 nm (United State

Pharmacopeial convention. 2008; USP, 2009).

2.3 Animal Experiment

Animal test used in this study were male rabbits

weighing 1.5-2 kg, which has been conditioned to

the environment and feeded for 1 week with kale

and carrots during the study. Blood sampling time is

10 minutes after drug administration.

2.4 Plasma Preparation

Rabbits were fasted at least 8 hours prior to the

experiment. Weighed and cleaned fur ears clean.

The blood was taken from 2 male rabbits

approximately 5 ml each, divided into 4 tubes which

had contained 2 drops of heparin, added 2 ml TCA

20%, then centrifuged at 3000 rpm for 10 minutes.

Each supernatant was taken and used as a blanko

and a calibration curve (Kemenkes RI, 2014).

2.5 In vivo Test

The test was conducted using six rabbits. The

administration of metronidazol rabbits with this

method can be seen in Table 1.

Table 1: Microparticle Metronidazole and conventional metronidazole capsules were administered to rabbits using the

cross-over design method (Chilukuri, et al. 2007).

Treatment I

2 Weeks

rea

Treatment II

Rabbit Dosage

Form

Rabbit Dosage

Form

1 A 1 B

2 A 2 B

3 A 3 B

4 B 4 A

5 B 5 A

6 B 6 A

A= Metronidazole microcapsule capsules, B = Metronidazole capsules (conventional)

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

562

At first, the rabbits were fasted for about 12

hours, then the metronidazole microparticles and the

conventional metronidazole capsules were

administered orally, the dosage forms were

administered based on the procedure according to

Table 3.3. Furthermore, rabbit blood was taken

through the marginal vein at certain time intervals,

namely: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and

15, hours using a 1.0 mL syringe. The syringe was

initially rinsed with heparin. The blood was put into

a centrifuge tube containing 2 drops of heparin.

Then 1.0 mL of 20% TCA was added to the tube and

vortexed until homogeneous. The tube was put into a

centrifuge and centrifuge at 3000 rpm for 10 minutes

and the supernatant was taken. Each supernatant was

filtered using a 0.2 µm PTFE filter membrane and

the levels were measured using a HPLC device by

injecting 10 µL of supernatant (Ahuja et al, 2005

and Kemenkes RI. 2014).

Rabbits were fasted for 12 hours before orally

administered with FCL-6, the design could be in the

Table 1. After shaving the hair around rabbits ears,

the blood was taken through the marginal vein at

specified time intervals are: 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14 dan 15, hours using 1.0 mL

syringe. Syringes rinsed beforehand with heparin.

Blood inserted into the centrifuge tube which

already contains 2 drops of heparin. Then TCA 20%

as much as 1.0 mL tubes were added and shaked

using vortex apparatus until homogeneous. The tube

was centrifuged at 3000 rpm for 10 minutes and the

supernatants were collected. Each supernatant was

filtered using a 0.2 μm PTFE membrane filter and

the metochlopramide concentrations measured using

HPLC instrument by injecting as much as 10 uL

supernatant (Kemenkes RI, 2014).

2.6

Correlation

of In vitro and In vivo

Correlation of in vitro and in vivo was determined

by using a level A correlation that explains the

relationship between the rate of drug release (%

cumulative drug apart) in vitro and speed of drug

release in vivo (plasma drug concentration).

2.7

Analysis

of Blood Plasma Level

Rabbits that have been granted in accordance with

the oral drug bioequivalence trial design that can be

seen in Table 1. At intervals; 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14 dan 15, hours, rabbit blood drawn

with the help of 1.0 mL syringe that has been rinsed

with heparin, was transferred to a centrifuge tube

which already contains heparin, and add 2 drops of

20% TCA 1 mL, centrifuged at 3000 rpm for 10

min, the supernatant was taken, filtered with a 0.2

μm PTFE membrane filter and assayed using HPLC.

3 RESULTS AND DISCUSSION

3.1

Correlation

Test

Chilukuri, et al. (2007) stated the correlation

between in vitro assay with in vivo assay can be

explained by using the correlation IVIVC level A

which is a relation between the cumulative percent

of the drug released of in vitro assay and the percent

amount of absorbed drug in blood plasma of in vivo

assay. The release of metronidazol mikroparticle and

konventional metronidazol for the in vitro test.

In vitro release of metronidazole and the average

level of metronidazole absorbed in plasma (in vivo)

can be seen in Figure 1. The presence of

metronidazole measured in artificial colon and in the

plasma showed a sustain release pattern. The

optimal in vivo measurement was depicted at 7

hours after administration.

Figure 1: Cumulative percent of metronidazole

microparticles (in vitro) and average percent of

metronidazole microparticles in plasma (in vivo).

Figure 2: In vitro and in vivo correlation of metronidazole

microparticles.

In vitro - In vivo Correlation Study of Colon-targeted Metronidazole Microparticle in Corncob Hemicellulose Capsule

563

Based on the plotted graph (data retrieval was

started from the drug released in the colon because

the drug began to be absorbed at that time), a

correlation value was obtained (R

= 0.8785). It can

be stated that there is a correlation between the

formulations tested in vitro and in vivo because the

correlation value was greater than 0.8, it is assumed

to have a correlation (Dalimunthe et al. 2019,

Shargel 1988).

The in vivo test was carried out for 15 hours, but

the new drug was released at 7 hours, which was

about 0.1888 µg / ml, this means that it can be

proven that the capsule is not destroyed in the in

vivo test either in the stomach or in the intestine, but

the intestines have begun to expand, and you can see

that the drug begins to release and there is an

increase in the percentage of drug release at 8 hours.

Based on the reaction kinetics, the in vitro test

(dissolution) shows that the drug follows order 1,

order zero, and higuchi but it tended to be order zero

because the release is relatively constant.

4 CONCLUSION

Corncob hemicellulose capsule considered as

altenate carrier for colon-targeted drug, it was

recorded that the drug release was occurred at pH 8

and 7 hours post oral administration. Therefore, it

has distinctive properties with conventional gelatin

capsule which is vulnerable to stomach acidic

conditions. In vitro in vitro correlation (IVIVC)

study also exhibites a strong relationship which was

indicated by the value of R

= 0.8785. It

demonstrated that in vitro dissolution test of

metronidazole microparticle in corncob

hemicellulose capsule is of high relevance for in

vivo assay.

REFERENCES

Ahuja, S., Dong, M., W, Eds. 2005. Handbook of

Pharmaceutical Analysis HPLC edisi 1 United

Kingdom: Elsevier, Inc.,.p. 191-217, 401- 412.

Amidon, S., Brown, J.E., Dave, VS. 2015. Colon-targeted

oral drug delivery systems: Design Trends and

approaches. American Association of Pharmaceutical

Scientist Pharm. Sci.Tech. 16(4) 731-741.

Chilukuri, D.M., Suskara G., Young, D. 2007.

Pharmaceutical Product Development in vitro - in vivo

Corelation, Informa USA, New York. (165):110-

165.

Chow, Shein-Chung, Liu-Pei. 2003. Design and Analysis

of Clinical Trials: Concepts and Methodologies, 3

Edition. Wiley.

Cliceri, D., Spinelli, S., Dinella, C., Prescott, J.,

Monteleone, E. 2018. The influence of psychological

traits, beliefs and taste redponsiveness on implicit

attitudes toward plant-and animal-based dishes among

vegetarians, flexitarians and omnivores. Food Quality

and Preference. Vol 68. 276-291.

Dalimunthe, G.I., Muchlisyam, Urip Harahap, Azizah

Nasution. 2020. The Effect of Variation In The

Amount of Hemicellulose from Corncob (Zea mays L)

and pH On Metronidazole Microparticle Release. IOP

Conf. Series: Journal Of Physics:

Conf.Series.1462.01203.

Dalimunthe, G.I., Muchlisyam Bachri, U. Harahap, A.

Nasution. 2019. Formulation of capsules shell from

corncob hemicellulose combined with isolated sodium

alginate. rasayan Journal of Chemistry: ISSN: 0974-

1496 Rasayan J Chem. Vol. 12(3): 1668-1675.

Emami, J. 2006. In vitro- in vivo correlation: From the

theory to applications. Journal of Pharmacy and

Pharmaceutical Sciences. 9(2): 169-189.

Kemenkes RI. 2014. Farmakope Indonesia. Edisi V.

Jakarta.Departemen Kesehatan RI

Lee, S.H., Bajracharya, R., Min. J.Y., Han, J.W., Park,

B.J., han, H.K. 2020. Strategic approaches for colon

targeted drug delivery: an overview of recent

advancements. Journal of Pharmaceutics. 12(1) 68.

Maity, S., Kundu A., Karmakar S., Biswanath, 2016, In

Vitro and In vivo Correlation of Colon-Targeted

Compression-Coated Tablets, Hindawi Publishing

Corporation. Journal of Pharmaceutics. vol. 2016,

Article.

Muchlisyam, 2014. Corn Cobs Hemicelluloses Isolation

Method Comparison and its Characterization with

Infra Red Spectrophotometry (FTIR) and High

Perfomance Liquid Chrromatography (HPLC).

International Journal of Chem Tech Research CODEN

(USA): IJCRGG ISSN: 0974-4290 vol.6 No.5.pp

3062-3070.

Muchlisyam, Sumaiyah, 2016. Application of corn cobs

hemiselulose microparticle, carrier with insulin as

model. Der Pharma Chemica 8(19): 632: 641.

Muhaimin. 2013, Study of Microparticle Preparation by

The Solvent Evaporation Method Using Focused

Beam Reflectance Measurement (FBRM) Disertation

Nicholas, E.M., Panaganti, S., Prabakaran, l., Jayveera,

KN. 2011. Novel colon spesific drug delivery system:

a review. International Journal of Pharmaceutical

Sciences and Research. Vol 2(10): 2545-2561.

Oladzadabbasabadi, N., Ebadi, S., Nafchi, A.M., Karim,

A.A., Kiahosseini, SR. 2017. Functional properties of

dually/K-carrageenan films: an alternative to gelatin in

pharmaceutical capsules. Carbohydrate Polymers. Vol.

160. 43-51.

Qiu, Y., Duan J.Z. 2017. Chapter 16-In vitro/ in vivo

correlations: Fundamentals, development

considerations and applications. Developing Solid

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

564

Oral Dosage Forms. Pharmaceutical Theory and

Practice. 415-452.

Shargel, L. 1988. Biopharmaceutical and

pharmacokinetics Application., Second Edition.

Interpreter: Sitti Sjamsiah. Surabaya: Airlangga

University Press. p.137, 160-186.

United States Pharmacopoeia. 2007. The National

Formulatory. Edisi Keduapuluh Lima. The United

States Pharmacopoeia Convention XXX. Hal. 277

United State Pharmacopeial convention. 2008. The United

States Pharmacopeia 32

. Mack publ.co.easton.

Halaman: 100-103.

USP. 2009. The National Formulary. Edisi ke-32.

Rockville: The United State Pharmacopeia

Convention. .p 3558.

Yang, C., Zhang, Y., Cai, P., Yuan, S., Ma, Q., Song, Y.,

Wei, H., Wu, Z., Wu, Z., Qi, X., 2020. Highly specific

colon targeted transformable capsule containing

indomethacin immediate-release pellets for colon

cancer therapy.

In vitro - In vivo Correlation Study of Colon-targeted Metronidazole Microparticle in Corncob Hemicellulose Capsule

565