ISSN: 2322 - 0902 (P)

ISSN: 2322 - 0910 (O)

International Journal of Ayurveda
and Pharma Research
Research Article

PREPARATION AND EVALUATION OF CYCLODEXTRIN COMPLEXES OF ANTI-TUBERCULAR

DRUG RIFAMPICIN FOR IMPROVED BIOAVAILABILITY
B. Prakash Rao1*, Sarasija Suresh2, Suresh Sah3, Narendra C4, Manuka Angbo5
*1Professor, Department of Pharmaceutical technology, Karnataka College of Pharmacy, Bangalore, Karnataka,
India.
2Director, Research and development, M. S. Ramaiah College of Pharmacy, Bangalore, Karnataka, India.
3PG Scholar, Department of Pharmaceutics, Karnataka College of Pharmacy, Bangalore, Karnataka, India.
4Assistant professor, Department of Pharmaceutics, Vishveshpura Institute of Pharmaceutical Science, Bangalore,
Karnataka, India.
5Deapartment of Pharmaceutical technology, Karnataka College of Pharmacy, Bangalore, Karnataka, India

Received on: 22/07/2015 Revised on: 09/08/2015 Accepted on: 14/08/2015

ABSTRACT
The aim of the study was to increase the aqueous solubility, dissolution rate, stability, in vitro anti-
tubercular activity and bioavailability of rifampicin by the way of inclusion complexation. Methyl β-
cyclodextrin in case of rifampicin were used. Based on phase solubility studies that stoichiometry
of complex of with respect to β-cyclodextrin for rifampicin was found to be 1:1 molar ratio.
Different methods of preparation such as kneading and common solvent were employed to
prepare the complexes. Formation of complexes In case of rifampicin, interaction of 4-methyl
piperazin-1-ylimino-methyl (side chain) of rifampicin with the cyclodextrin molecule was
confirmed by FTIR and 1H-NMR. The complexes prepared by different methods were subjected to
solubility and in vitro dissolution studies. In case of rifampicin, in vitro anti-tubercular activity was
found to be enhanced for the complexes of rifampicin indicated by a reduction in MIC of rifampicin.
The oral bioavailability of rifampicin-Mβ-CD complex prepared by common solvent method was
improved significantly. The results of stability studies revealed that stability of the drugs in
solution and solid state were improved significantly due to complexation. Photostability of
rifampicin is enhanced significantly by the way of complexation. Thus inclusion complexation of
rifampicin with β-cyclodextrin, β-cyclodextrin derivatives and γ-cyclodextrin improved its physical
properties, bioavailability and in vitro activity.
KEYWORDS: Rifampicin, β-cyclodextrin derivatives, γ-cyclodextrin, Dissolution, Characterization,
Bioavailability, Thermal Stability, Photostability, in vitro anti-tubercular activity.
INTRODUCTION
Pyrazinamide (Pyr) and rifampicin (Rif) are treatments yield resistant mutants in which the RNA
two of the first-line anti-tubercular drugs. They are polymerase is still highly sensitive to the drug, but the
poorly soluble in water. It is known to cause serious rate of rifampicin uptake is reduced. The mechanism of
side effects such as hepatotoxicity, anorexia, nausea, this permeability mutation is not yet clear. 6 Any
vomiting and dysuria. The treatment for tuberculosis attempt to increase the permeability would be
requires long time therapy, which may aggravate advantageous in the treatment of resistant mutant.
gastric adverse effects. Rif is prone to acid hydrolysis Cyclodextrin (CD) has been reported to increase
giving rise to 3-formylrifampicin SV where the side permeability. Main adverse effects of rifampicin are flu-
chain i.e. 4-methyl piperazin-1-ylimino-methyl is like syndrome and cutaneous syndrome, nausea
detached. Rifampicin is also prone to air oxidation of vomiting, aneroxia, diarrhoea and epigastric distress.
the paraphenolic groups in the naphthalene ring to give Thus it was felt that there is a need to reduce the
the p-quinone. It was reported that its oral absorption gastric distress, increase the uptake of rifampicin in
is decreased when side chain, 4-methyl piperazin-1- order to reduce the MIC, increase the stability and
ylimino-methyl is detached. The mechanism of improve bioavailability.
antibacterial action of rifampicin is inhibition of the Material and methods
activity of the enzyme DNA-directed RNA polymerase
(DDRP). Although the main mechanism of resistance is β-CD (HiMedia Laboratories Pvt Limited,
the modification of the target enzyme, some mutagenic Mumbai, India) and all other chemicals used were
IJAPR | August 2015 | Vol 3 | Issue 8 65
 B. Prakash Rao et al. Preparation and Evaluation of Cyclodextrin Complexes of Anti-Tubercular Drug Rifampicin for
Improved Bioavailability
A.R.Grade (Merck, Limited, Mumbai, India).β-CD oven at RT for one week. The obtained mass was then
derivatives that is methyl-β-CD were obtained as gift pulverized, passed through sieve no. 85 and stored in a
sample. Rifampicin was obtained as gift sample from desiccator.
KAPL, Bangalore, India. All the chemicals (Sigma Common Solvent (CS): Similarly drug and complexing
Aldrich Chemie Gmbh, Steinheim, Germany) were agents were dissolved in suitable solvent (chloroform
purchased for the preparation of Lowenstein-Jensen or DMF are used in case of rifampicin). The solutions
medium (L.J. medium). HPLC grade water (Merck were mixed in ratio as mentioned in physical, and
Limited, Mumbai, India) and acetonitrile (Qualigens kneading method and evaporated to dryness at
Fine chemicals, Mumbai) were purchased. ambient temperature. The dry mass obtained was
Dichloromethane and diethyl ether are analytical grade pulverized, passed through the sieve no. 85 and stored
were freshly distilled before use; hydrochloric acid; in a desiccator.
water was HPLC grade (Merck Limited, Mumbai, India),
acetonitrile (Qualigens Fine chemicals, Mumbai) and Content uniformity
paracetamol. The percentage of rifampicin in each of
Phase Solubility: Phase solubility studies were carried complexes was determined by using the complex
by taking excess of rifampicin is added to 10 ml of an containing 100 mg of the drug. The sample that is
aqueous solution of β-CD ranging from 5 mM to 15 mM rifampicin after suitable dilution with pH 7.4-
and different concentrations 100mM of β-CD phosphate buffer was determined at 475 nm by using
derivatives in a series of 25 mL stoppered conical flasks Shimadzu double beam UV spectrophotometer model
and the mixtures were shaken for 48 hours at room UV-1601 116.
temperature on a rotary flask shaker. After 48 hours CHARACTERIZATION STUDIES
shaking to achieve equilibrium, 2-mL aliquots were
withdrawn at 12-hour intervals and filtered β-CD complexes were investigated to establish
immediately using a 0.45 µl nylon disc filter. The formation of complex and the intactness of the drug in
filtered samples were diluted suitably and assayed for the complex by FAB mass spectrometry, FTIR, DSC, 1H-
pyrazinamide by measuring absorbance at 475 nm by NMR, SEM and powder x-ray diffraction methods.
using Shimadzu double beam UV spectrophotometer. Fourier Transform Infrared (FTIR Studies)
Shaking was continued until 3 consecutive estimations
IR spectroscopy was carried out for the
were the same (96 hours). The phase solubility
following a) pure drugs, b) β-CD and derivatives, c)
experiments were conducted in triplicate. The blanks
complexes using Shimadzu FTIR model 8700 by taking
were performed on the same concentrations of CDs in
KBr disc.
water so as to cancel any absorbance that may be
exhibited by the CD molecules. Differential Scanning Calorimetry (DSC) Studies
Individual coils that are heated and cooled at
the same rate heat DSC in which sample and reference
containers are not contiguous and heated them
separately. Platinum resistance thermometers monitor
the temperature of the sample and reference holders
and electronically maintain the temperature of the two
Where So is the intrinsic solubility of rifampicin. holders constant. Differential scanning calorimetry of
Preparation of the complexes (Solid Binary pure drug, its physical mixture of β-CD, all the
System): Different methods were used for the complexes were carried out (Perkin Elmer Pyris) with a
preparation of β-CD complexes of rifampicin. The temperature increase of 5 o C /min. The scanning
methods include common solvent (cosolution) and temperature range was from 50o C to 250oC.
kneading. In case of rifampicin 1:1 molar ratio of drug Temperature and heat flow calibration were performed
with methylβ-CD, were prepared. The optimum ratio using indium as a standard.
was determined by phase solubility studies. Physical Powder X-ray diffraction Studies
mixtures (PM) of drugs and cyclodextrin were
prepared in the same ratios. X-ray diffraction study was carried out for
pure drug, β-CD, and their complexes (Philips
Physical Mixture (PM): 1:1 molar ratio in case of Diffractometer Model PW 17291 and Model PW
rifampicin to β-CD and its derivatives was taken in 1050/37) with a vertical goniometric using a Nickel
mortar in geometrical ratio and triturated for one hour. filter cu k α radiation operating at 30 KW and 20 milli
The dry powder obtained was passed through the sieve amps in the range from 5o-40o angles. The scanning
no. 85 and stored in a desiccator. rate was 1 o/min.
Kneading complex (KN):1:1 molar ratio of rifampicin Proton Nuclear Magnetic Resonance (1H-NMR)
to β-CD was taken in mortar and adequate distilled Spectrometry: 1H-nuclear magnetic resonance (1H-
water was added to make paste-like consistency. The NMR) spectroscopic experiments were performed on a
paste was kneaded for half an hour and dried in an Varian 500 MHz with dual full-band channels and z-axis

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 Int. J. Ayur. Pharma Research, 2015;3(8):65-76

gradients using a Varian. The spectra obtained were randomly selected. All rabbits were fasted over night
measured at 298 K with an operating frequency of with ad libitum access to water. All rabbits received
499.742 MHz. The 90 o pulse width for 1 H was 10.8 at rifampicin solution by intravenous administration into
a transmitter power of 50. Solutions were purged prior marginal vein at a dose of 30 mg/kg. Rabbit’s in-group
to data collection under a stream of argon for 1 hour to 1 received pure rifampicin plain powder suspension in
reduce the amount of dissolved oxygen. 1% sodium carboxy methyl cellulose while rabbit’s in-
Scanning Electron Microscopy (SEM) group 2 received rifampicin complex powder
suspension in 1% sodium carboxy methyl cellulose by
The surface morphology of pure materials, oral feeding tube at a dose of 30 mg/kg, followed by 1
their treated counterparts, and all binary systems were ml of deionized water. Rabbit’s in-group 3 received
examined by scanning electron microscope. The pure rifampicin intravenous injection. Blood samples
samples were fixed on a brass stub using double-sided were collected from the marginal vein at 0, 0.25, 0.5, 1,
tape and then gold coated in vacuum by a sputter 2, 3, 5, 7 and 24 h. Immediately the samples were
coater. The pictures were taken at an excitation voltage precipitated the plasma proteins by adding methanol
of 20 Kv. JSM-840A Scanning Microscope, Jeol-Japan (200 µL), followed by 1 M potassium dihydrogen
with JFC-1100E Ion Sputtering Device was used. phosphate in 0.2% ascorbic acid (1mL) to adjust the pH
Fast Atomic Bombard (FAB) Mass Spectra: were to 4.2.
recorded on a JEOL SX 102/DA-Mass Extraction of Rifampicin: To 2 ml of plasma in a 15-ml
Spectrometer/Data system using argon/xenon (6kV, test-tube 0.4 ml of 10 % (v/v) aqueous acetic acid was
10mA) as the FAB gas. The accelerating voltage was added to adjust the pH to 4.2. Rifampicin was extracted
10kV and the spectra were recorded at room by shaking with 7 ml of diethyl ether:dichloromethane
temperature and m-nitrobenzyl was used as the matrix. (2:1 v/v) that, after centrifugation for 10 min at 2059 g,
Solubility Studies was transferred to a tapered test-tube and evaporated
under nitrogen at 40o C. The residue was dissolved in
The solubility of pure rifampicin and the 200 µl of methanol of which 20 µl aliquot was injected
complexes was determined as reported. Excess of pure into the HPLC column and eluted with system. The
drug or the complex was added in 25 ml conical flask elute was detected at 248 nm. A simple, specific and
and the mixture was shaken for 24 hours in a sensitive high-performance liquid chromatography
thermostatic shaker water bath (REMI Model) at RT at (HPLC) method was developed for the determination of
the rate of 110-120 strokes/min, till a saturated rifampicin in human plasma. Rifampicin and sulindac
solution in distilled water was obtained. The sample (internal standard) are extracted from human plasma
was filtered using Whatman filter paper No 44. The using a C 2 Bond Elutextraction column. A 100-µl
filtrate was suitably diluted and the concentration of volume of 0.1 M HCl is added to the plasma before
drug was determined spectrophotometrically. extraction to increase the retention of the compound
Bioavailability Studies on the extraction column. Methanol (1ml) is used to
elute the compounds and 0.5 ml of 3-mg/ml ascorbic
Eighteen male New Zealand rabbits weighing
acid in water is added to the final elute to reduce the
1.5 to 2.5 kgs were used. The following formulation was
oxidation of rifampicin. Separation is achieved by
tested (1) I.V. Injection of pure rifampicin, (2)
reversed-phase chromatography on a Zorbax Rx C 8
rifampicin-M ethyl-β-cd inclusion complex (CS)
column with a mobile phase composed of 0.05 M
suspension in 1% sodium carboxymethylcelulose, (10
potassium dihydrogen phosphate-acetonitrile (55: 45,
mg rifampicin/kg) in 1%, (3) rifampicin suspension in
v/v). Detection is by ultraviolet absorbance at 340 nm.
1% sodium carboxy methyl cellulose (low viscosity)
The retention times of rifampicin and internal standard
given orally.
are approximately 4.4 and 7.8 min, respectively. The
High Performance Liquid Chromatography (HPLC): assay is linear in concentration ranges of 50 to 35 000
The liquid chromatography consisted of a Waters 6000 mg/ml. The quantitation limit is 50ng/ml. Both intra-
A pump, a U6K injector with a 25 loop (Waters Assoc., day and inter-day accuracy and precision data showed
Milford, MA, U.S.A.) and a variable-wavelength Hitachi good reproducibility.
220-S UV detector with a chart recorder (Hitachi, STABILITY STUDIES
Tokyo, Japan). Analyses were performed on a reversed-
Solid-state Stability studies: Accelerated Thermal
phase C 8 Column (Hibar, LiChroCart RP-8, 250 mm x
Stability studies (ICH): For rifampicin the 25o C ± 2o C
4.6 mm I.D., Merck).
/60% RH ± 5% are used as it is stored at cool
The operating conditions for the HPLC system temperature.
were: a mobile phase of a freshly prepared mixture of
Photostability Studies: Photostability studies are
acetonitrile and 10 mM phosphate buffer at pH 3.5 (1:9
carried out in Newtronic chamber. Studies are carried
v/v); flow-rate 1.5 ml/min; temperature, ambient (25
in UV light and tube light 1.2 million lux hours and an
±1o C); UV detector wavelength, 340.
integrated near ultraviolet energy of not less than 200-
Design: The rabbits were divided into three groups of watt hours/square meter to allow direct comparisons
six rabbits (n = 6). The order of administration was

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 B. Prakash Rao et al. Preparation and Evaluation of Cyclodextrin Complexes of Anti-Tubercular Drug Rifampicin for
Improved Bioavailability
to be made between the drug substance and drug
product. Where S is the intrinsic solubility of rifampicin. Slope
o
HPLC System: The liquid chromatography consisted of of the phase solubility line.
a Waters 6000 A pump, a U6K injector with a 25 loop Content Uniformity The actual drug content in each
(Waters Assoc., Milford, MA, U.S.A.) and a variable- binary mixture was determined. The results are
wavelength W 2487 UV detector. Analyses were reported (Table 22). The physical mixture, kneaded
performed on reversed-phase Symmetry C 18 5 µm and common solvent products showed a good
W032815 4.6 x 150 mm. agreement between theoretical and actual drug
The operating conditions for the HPLC system content.
were: Composition of mobile phase is methanol and Solubility
0.015 disodium hydrogen phosphate (75+25, v/v), pH
adjusted to 4.5 with 85% phosphoric acid. Flow rate is The aqueous solubility of rifampicin was found
(isocratic) 1 ml/min, temperature, ambient (25 ±1o C); to be maximum in Mβ-CD complexes, (Table 3, Fig.2)
UV detector wavelength 254 nm; sensitivity scale, 0- while the physical mixture showed a 2.4 fold increase
0.01 a.u.f.s. in solubility. The increase in solubility was 2 fold when
compared to that of pure drug in case of CS complex of
PROTOCOL OF HPLC Mβ-CD. In case of β-CD complexes, CS complex showed
Sample preparation: Suitable quantity of composite 2 fold increases in solubility while KN complex showed
sample was extracted with methanol and filter. The the least solubility, less than that of the pure drug.
filtrate was suitably diluted with MP to get Fourier Transform Infrared Spectroscopy (FTIR
concentrations as described under standard solution to Spectroscopy): Considerable information can be
establish linearity range. HPLC System: Waters LC obtained from FTIR spectral interpretation, regarding
systems equipped with dual piston. 510 reciprocating the involvement of various functional groups in
pump and 7125 Rheodyne injector with 20 µl fixed hydrogen bonding. This generally shifts the absorbance
loop. bands to the lower frequency increases the intensity
Column: Novapak RP C – 18 (5 µm) column. 150 x 3.9 and widens the band caused by stretching vibration of
mm the group involved in the formation of the hydrogen
Temperature: Ambient bond. In case of FTIR of M β-CD complexes (Fig. 3), the
stretch of >CH=N of rifampicin from 1647.1 cm -1 to
Chromatographic conditions: 1625.9 cm -1, the stretch of -C-H, from 2883.4 cm -1 to
1. Composition of mobile phase and its pH: Methanol- 2844.8 cm -1 were shifted, and 1436.9 cm -1 of C-H was
0.01 to 0.02 M disodium hydrogen phosphate (75+ 25, disappeared in the KN further confirmed the formation
v/v) or (70+30, v/v), pH adjusted to 4.5 with 85% of complex with part of molecule i.e. 4-methyl
phosphoric acid. piperazin-1-ylimino-methyl side chain was included in
2. Flow rate isocratic: 1 ml/min the complex. Breakage of hydrogen bond at 1023 cm -1
frequency of secondary alcoholic group was observed
3. Volume injected: 20 µl
in all the cases. But no changes were observed in case
4. Type of detector, detector mode and wavelength: UV- of PM. The reduced intensities of peaks of rifampicin
254 nm were observed in CS complex. Thus complexation of
5. Retention time for Rifampicin- 2.91 and rifampicin could be obtained in M β-CD complexes
Degradation product of Rifampicin- 5.95 where a part of drug molecule might have included
6. Linearity range: Rifampicin – 10- 100 µg/ml within the cyclodextrin molecule.
7. Recoveries (%): 97.50- 100.45 for drugs. Differential Scanning Calorimetry (DSC)
RESULTS M-β-CD Complexes
Rifampicin with β-cyclodextrin derivatives In DSC thermograms of M β-CD complexes
(Fig. 4), endothermic melting peaks of pure drug at
Phase Solubility
185.020o C and 229.110o C were reduced in area
Phase solubility studies of rifampicin with β- significantly or absent in the complexes obtained by
CD derivatives showed that it followed A L- type of different methods. New exothermic peak was observed
phase solubility (Fig. 33 and Fig 34). 41 The slope of at 214.230oC in kneading complex and endothermic
the line of phase solubility is less than unity indicating peaks of rifampicin were almost disappeared, which
that stoichiometry is 1:1 for drug to complexing agent. indicated maximum interaction. Similarly in case of CS
The apparent 1:1 stability constants of all complexes complex the endothermic peaks of rifampicin were
were calculated according the equation given below. absent, which indicated complete interaction.
Powder X-ray diffraction
X-ray diffraction reveals that the crystalline
nature and change in the crystalline structure. There

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 Int. J. Ayur. Pharma Research, 2015;3(8):65-76

were 47 peaks and 29 peaks in powder X-ray as well as complexes of rifampicin, but, in case of wild
diffraction patterns of β-CD and pure rifampicin strain No.10934, MIC was found to be 64µg/ml for pure
respectively that showed their crystalline nature. One rifampicin. However, a reduction in MIC from 64µg/ml
peak at 12 o of 2 θ angles was observed in pure M β-CD to32 µg/ml was observed in all the complexes of
and 12 peaks were observed in the range of 2 θ angles rifampicin. 28 M β-CD complexes were showing no
from 11 o to 27.5 o in pure rifampicin. As expected 13 growth at all concentration thus exhibiting maximum
peaks is their mixture, provided there is no interaction. effectiveness.
However X-ray diffraction pattern showed 4 peaks in BIOAVAILABILITY
the physical mixture and single peak each in the
complexes prepared by KN and CS. Plasma profiles are given the in Table 8. AUC
IV, 0-24 of rifampicin (Fig. was found to be 90.34 ± 1.54
Proton Nuclear Magnetic Resonance (1H-NMR) hr µg/ml (Kinetica Software was used. The AUC 0-24 of
1H-NMR studies are used to investigate rifampicin CS complex was 85.86 ± 2.98 hr µg/ml
whether the type of interaction is hydrogen bond (prepared by M β-CD (common solvent) and AUC 0-24
formation, van der Waals forces or dipole-dipole for pure rifampicin (oral) was 64.52 ± 3.6 hr µg/ml.
interaction. It also helps to interpret the geometry, The C max found to be 25 ± 0.73 µg/ml, 14.4±
stoichiometry and alignment of the guest molecule in 0.13µg/ml, and 15.7 ± 0.35 µg/mlfor IV, oral pure drug,
the CDs cavity. 1H-NMR study is used to investigate the and CS respectively. The T max was found to be 1.15
type of interaction. The changes in the chemical shift hrs in case pure rifampicin and CS complex. The half-
were positive (desheilding) and negative (shielding). In life was found to 6.2 ± 1.2 hrs, 6.5 ± 1.3 hrs, and 6.96 ±
this study the changes in chemical shifts to second 1.32 hrs for IV, oral pure drug, and CS respectively.
decimal were taken. The 1H-NMR studies revealed that Stability Studies in Solution
changes in chemical shift of group, –CH=N of rifampicin
molecule in the complexes are given in Table 23. Thermal Stability Studies: The thermal degradation
Another group, >NH group ppm of pure rifampicin has constant for pure rifampicin on the basis of first order
been changed to second decimal (Table 24). The 1H- kinetics was found out to be 8.9535 ± 0.2795 x 10 -5
NMR spectra of rifampicin, cyclodextrin complexes day -1, the KN complex of M β-CD showed the least
prepared by different methods are given in Fig. 6, Fig. 7, value, 6.101 ± 0.190 x 10 -5 day -1. The reduction in
Fig.8 degradation constant found to be statistically
significant (p <0.05).
Scanning Electron Microscopy Studies
Photostability Studies: The photostability studies
SEM studies reveal change in the morphology revealed that there is no physical changes were
of particles and homogeneity of the product. Even if observed. There is no observable colour change in
there is a clear difference in crystallization state of the samples. The reduction or decrease in purity for
raw material and the products, this study is inadequate rifampicin and the least of all rifampicin-CD in 6
to confirm inclusion complexation, but helps to assess months were observed in 6 months is 1.69%, 1.50%
the existence of homogeneity of single component in respectively.
the preparations obtained. SEM observed that SEM of
the M β-CD (Fig. 9) had shown ball shaped in pure CONCLUSION
compound and these ball shaped particles are Thus it can be concluded that rifampicin
disappeared in the complexes. Both the drug and pure complex rifampicin could be successfully prepared by
rifampicin particles were observed in the PM. KN the kneading and common solvent methods. The
complex has shown small crystals while irregular complexes were found to have enhanced solubility, and
shaped crystals were seen in the CS. It was observed dissolution rate when compared to the pure drug. FAB
that complex is single component indicating the mass spectrometry, FTIR, and 1H-NMR methods
interaction in KN and CS. confirmed the intactness of drug in the complexes. In
Mass Spectroscopy case of rifampicin a part of rifampicin molecule i.e. 4-
methyl piperazin-1-ylimino-methyl chain was
Mass spectrometry of CS complex of M β-CD interacted with cyclodextrin molecules as indicated by
and rifampicin reveals that mass of theadduct formed FTIR and 1H-NMR. Powder x-ray diffraction, DSC and
out of complexation and also supports that the SEM studies confirmed the change in crystallinity of
stoichiometry of the complexes. The FAB mass both drugs. The M β-CD complexes had shown
spectroscopy showed that adduct, with molecular maximum stability constant, solubility, and anti
weight 2126 +, which is sum of M β-CD, 1303 +and, mycobarium activity. In case of DSC of M β-CD
rifampicin, 822.95 +. This supports the stoichiometry of complexes, the endothermic peaks of rifampicin were
rifampicin and M β-CD for the formation of complex is almost disappeared. The thermal and photostability of
1:1. rifampicin was improved significantly. Increased i n
In vitro Anti-tubercular Activity vitro anti-tubercular activity of the rifampicin was
The MIC for laboratory strain (H 37 RV) was observed and it is concluded that these complexes have
found to be identical that is 4µg/ml for pure rifampicin a potential of increased anti-tubercular activity.

IJAPR | August 2015 | Vol 3 | Issue 8 69

 B. Prakash Rao et al. Preparation and Evaluation of Cyclodextrin Complexes of Anti-Tubercular Drug Rifampicin for
Improved Bioavailability
Similarly the dose of rifampicin can be reduced as MIC 13. Brahmankar DM, Sunil BL. Biopharmaceutics and
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cyclodextrin improved its physical properties, Pharmaceutical potential of cyclodextrins Indian J.
bioavailability and in vitro activity. Pharm. Sci. 1999; 61, (4): 193-198.
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Cite this article as: *Address for correspondence

B. Prakash Rao, Sarasija Suresh, Suresh Sah, Narendra C, Manuka. *For correspondence,
Preparation and Evaluation of Cyclodextrin Complexes of Anti- Dr. B. PrakashRao
Tubercular Drug Rifampicin for Improved Bioavailability. International Professor and HOD
Journal of Ayurveda and Pharma Research. 2015;3(8):65-76. Department of Pharmaceutical
Source of support: Nil, Conflict of interest: None Declared technology, Karnataka college of
Pharmacy, Thirumenanahally,
Hegdenagar Main Road,
Bengaluru-560064, India.
Fax: +91 80 28571544
Email: [email protected]
Contact no. +91 9945479891

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B. Prakash Rao et al. Preparation and Evaluation of Cyclodextrin Complexes of Anti-Tubercular Drug Rifampicin for
Improved Bioavailability
TABLES

Table: 1 Comparison of the stability constants (K s) of complexing agents with the rifampicin
Types of cyclodextrin Stability constant(Ka)
By solubility method(m-1)
M β-CD 294.27±21.4
Each value is an average of three determinations with SE
Table: 2 Drug content in complexes of rifampicin (Binary Mixtures) (% molar ratio ± SE)
Complexing agent Methods of preparation Theoretical (%) Actual (%)
PM 50 49.78 ± 0.18
Mβ-CD KN 50 48.89 ± 0.12
CS 50 49.70 ± 0.14
Each value is an average of three determinations with SE
Table: 3 Solubility of rifampicin and its complexes in water
S.N. Complexing agent Methods of preparation Solubility (mg/ml)
1 Methyl-β-cyclodextrin Physical mixture 3.200 ± 0.058
2 kneading 2.760 ± 0.058
3 Common solvent 2.960 ± 0.048
Each value is an average of three determinations with SE
Table 4: 1H-Chemical Shifts corresponding to CH=N group of rifampicin (δfree = 8.826) in the Presence and
Absence of complexing agents
Complexing agent Cmplex δcomplex Δδa
PM 8.834 +0.008
M-β-CD KN 8.862 +0.036
CS 8.866 +0.040
Table 5: H-Chemical Shifts corresponding to >NH group of rifampicin group (δ free = 12.533) in the presence
1

and absence of complexing agents

Complexing agent Complex δcomplex Δδa
PM 12.529 -0.010
M-β-CD KN 12.516 -0.017
CS 12.519 -0.014
Table: 6 Anti-tubercular activities of rifampicin and its cyclodextrin complexes Strain: H 37 RV
Complexing agent Methods of Drug c. Drug c. Drug c.
preparation 1μg/ml 2μg/ml 4μg/ml
Pure rifampicin 2+ 1+ NG
M-β-CD KN 3+ 1+ NG
CN 3+ 5C NG
Note: 1+ = >100 colonies &<200 2+ = >200 colonies &<300 3+ = >300 colonies NG = No growth C = Colonies
Table-7 Anti-tubercular activities of rifampicin and its cyclodextrin complexes Strain 10934
Complexing agent Prepn method Drug concn Drug concn Drug concn
16µg/ml 32μg/ml 64μg/ml

Pure rifampicin 1+ 25C NG

M-β-CD KN 20C NG NG
CS NG NG NG
Note: 1+ = >100 colonies &<200 C = Colonies NG = No growth
Table: 8 Bioavailability rifampicin of IV, oral, and CS of M- β-CD
S No. Time Plasma concentration (μg/ml)
(hrs) I.V. Oral Pure drug Oral CS
1 0 25.00±2.00 0 0
2 0.5 18.00±1.26 5.8±0.79 7.00±0.57
3 1 13.60±1.77 11.3±0.88 12.10±1.51
4 1.5 10.30±0.96 14.4±0.23 15.70±0.61
5 2 8.8±0.17 9.3±1.07 12.10±1.03
6 3 6.6±1.51 8.6±0.99 8.60±0.44
7 4 4.1±0.84 5.4±1.32 6.90±0.35

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 Int. J. Ayur. Pharma Research, 2015;3(8):65-76

8 6 3.8±0.62 3.1±1.10 3.60±0.44

9 8 2.4±0.62 2.2±0.30 1.98±0.18
10 24 0.8±0.11 0.8±0.14 0.80±0.18
*Each value is an average of three determinations with SD
Table 9: Degradation constants complexes of rifampicin, and β-CD derivatives in aqueous solutions at
different temperatures
Complexing Methods of 100oC 450c RT
agent prepn -k x 10-2 -k x 10-2 -k x 10-2
(Min-1) (Min-1) (Min-1)
Pure drug 1.126±0.016 0.136±0.021 0.0005±0.00002
PM 0.8370±0.120 0.0721±0.002 0.0004±0.00002
M-β-CD KN 0.5087±0.050 0.0858±0.001 0.0003±0.00002
CS 0.5409±0.061 0.0328±0.002 0.0004±0.00002
*Each value is an average of three determinations with SE
k = first order rate constant,
- ve sign indicate the decrease in the value
Table10: Accelerated stability study of rifampicin and M β-CD complex
S.No. Time in days Concentration in mg
R PM KN CS
1 0 9.997±0.0058 9.997±0.0033 9.996±0.0033 9.983±0.0033
2 30 9.975±0.0022 9.979±0.0010 9.977±0.0033 9.968±0.0017
3 60 9.947±0.0025 9.965±0.0050 9.957±0.0033 9.950±0.000
4 90 9.921±0.0047 9.942±0.0072 9.936±0.0033 9.931±0.0007
5 120 9.889±0.0003 9.916±0.0088 9.924±0.0031 9.910±0.0000
6 150 9.870±0.0058 9.892±0.0017 9.903±0.0033 9.893±0.0033
7 180 9.841±0.0058 9.873±0.0012 9.886±0.0033 9.860±0.0058
*Each value is an average of three determinations with SE
Table 11: Degradation constants of rifampicin and its cyclodextrin complexes (Thermal)
S.No. Complexing agent Methods of prepn Degredatation constant -k x 10-5
1 Pure drug 8.954 ± 0.280
2 PM 7.088 ± 0.924
3 M-β-CD KN 6.101 ± 0.190
4 CS 7.719 ± 0.209
*Each value is an average of three determinations with SE
Table 12: Photo stability study of rifampicin and M β-CD complexes
S.No. Time in days Concentration in mg
R PM KN CS
1 0 9.997±0.0033 9.990±0.0058 9.983±0.0033 9.985±0.0029
2 30 9.971±0.0058 9.972±0.0061 9.963±0.0033 9.967±0.0015
3 60 9.942±0.0015 9.960±0.0029 9.942±0.0043 9.944±0.0031
4 90 9.917±0.0015 9.938±0.0017 9.921±0.0017 9.920±0.0006
5 120 9.897±0.0033 9.921±0.0010 9.987±0.0033 9.908±0.0044
6 150 9.869±0.0021 9.903±0.0017 9.875±0.0050 9.888±0.0044
7 180 9.841±0.0067 9.891±0.0017 9.853±0.0067 9.985±0.0029
*Each value is an average of three determinations with SE
Table 13: Degradation constants of rifampicin and its cyclodextrin complexes in photostability studies
S.No. Complexing agent Methods of prepn Degratation constant -K X 105
1 Pure drug 8.734 ± 0.107
2 PM 5.646 ± 0.296
3 M-β-CD KN 7.351 ± 0.327
4 CS 0.222 ± 0.050
*Each value is an average of three determinations with SE

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B. Prakash Rao et al. Preparation and Evaluation of Cyclodextrin Complexes of Anti-Tubercular Drug Rifampicin for
Improved Bioavailability

Fig: 1 Phase solubility studies of rifampicin and M Fig. 4: DSC Thermograms of rifampicin, M- β-CD
β-cyclodextrin. their inclusion complexes.
mcd is M –β-CD, y = 0.2216 x + 1.5573, R 2 = 1 DSC thermogram of (A) pure rifampicin; (B) M β-CD;
(C) M β-CD-rifampicin physical mixture; (D) M β-CD-
rifampicin prepared by kneading; (E) M β-CD-
rifampicin prepared by common solvent method.

Fig: 2 Solubility of complexes of rifampicin and β-

CD derivatives in water.
Solubility of (A) pure rifampicin, (B) Mβ-CD-rifampicin
prepared physical mixture, (c) M β-CD-rifampicin
prepared by kneading, (d) M β-CD-rifampicin prepared
by common solvent method. Fig. 5: Powder x-ray diffraction pattern of
rifampicin, M-β-CD and their inclusion complexes.
Powder X-ray diffraction pattern of (A) pure
rifampicin; (B) Mβ-CD; (C) M β-CD- rifampicin physical
mixture; (D) M β-CD-rifampicin prepared by kneading
and (E) M β-CD-rifampicin prepared by common
solvent.

Fig: 3 FTIR Spectra of rifampicin, M- β-CD and their

inclusion complexes.
FTIR Spectra of rifampicin (A) pure rifampicin; (B) M-
β-CD; (C) M- β-CD-rifampicin physical mixture; (D) M-
β-CD-rifampicin prepared by kneading (E) M-β-CD-
rifampicin prepared by common solvent. Fig. 6: 1H-NMR spectrum of rifampicin-M- β-CD
(PM) in DMSO (D 6)

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 Int. J. Ayur. Pharma Research, 2015;3(8):65-76

Fig. 7: 1H-NMR spectrum of rifampicin-M- β-CD Fig. 8: 1H-NMR spectrum of rifampicin-M-β-CD (CS)
(KN) in DMSO (D 6) in DMSO (D 6)

Fig 9: Scanning electron microscopy of rifampicin, M- β-CD and their inclusion complexes
Scanning electron microscopy of rifampicin (A) pure rifampicin; (B) M β-CD; (C) M β- CD-rifampicin physical
mixture; (D) M β-CD-rifampicin prepared by kneading and (E) Mβ-CD-rifampicin prepared by common solvent

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B. Prakash Rao et al. Preparation and Evaluation of Cyclodextrin Complexes of Anti-Tubercular Drug Rifampicin for
Improved Bioavailability

Fig. 10: Mass spectrum of rifampicin-M- β-CD at 10 kV, m-nitrobenzyl was used as matrix

Fig. 11 : Bioavailbility of rifampicin IV, its oral and CS of M - β-CD I.V. - Plasma profiles Intravenous Injection
Pure drug oral- Plasma profiles of rifampicin Oral CS- Plasma profiles of CS complex of M β-CD

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