Field of the Invention
This invention relates to the development and characterization of nano-carrier modules for
macrophage targeted anti-tubercular multidrug therapy. This invention also relates to the high
concentration of anti-tubercular drugs (ATDs) in the selectively cellular tropics of infection
compared to blood plasma pool region by formulating novel ligand appended liposomal
delivery systems with increased bioavailability and site specificity.
Background of the Invention
Tuberculosis (TB) is a common and often deadly infectious disease caused by
mycobacterium, mainly Mycobacterium tuberculosis Muttil et a1.,(2007). Mycobacterium
tuberculosis is an aerobic bacterium that divides every 16 to 20 hours, an extremely slow rate
compared with other bacteria, which usually divide in less than an hour. After invasion
through inspired air Mycobacterium tuberculosis resides, survive and multiply within
macrophages (Fig 1). According to the WHO, 8.8 million new TB cases were found in the
year 2009 WH0,(2009). Most commonly TB infection begins when the mycobacteria reach
the pulmonary alveoli, where they invade and replicate within the endosomes of alveolar
macrophages Muttil et a1.,(2007). The primary site of infection in the lungs is called the Ghon
focus. TB can remain in an inactive (dormant) state for years without causing symptoms or
spreading to other organs. When the immune system of a patient with dormant TB is
weakened, the TB can become active (reactivate) and cause infection in the lungs or other
parts of the body A number of characteristics of mycobacterium makes it a chronic disease
and requires prolonged treatment. MDR-TB most commonly develops in the course of TB
treatment. To reduce resistance TB is treated with drug regimen consisting Isoniazid,
Rifampicin, Ethambutol, and Pyrazinamide. Four drugs'are often taken for the first two
months then the number is reduced to two drugs, usually Isoniazid and Rifampicin for next
four months Streptomycin, a drug that is given by injection, may be used as well, particularly
when the disease is extensive andlor the patients do not take their oral medications reliably
(termed "poor compliance"). Treatment usually lasts for many months and sometimes for
years. MDR-TB is most commonly due to doctors giving inappropriate treatment, or patients
missing doses or failing to complete their treatment. It is therefore, in bio-clinical terms
desirable that by using available optional strategies differentiated concentration should be
produced selectively higher in cellular tropics of infection compared to blood plasma pool
Pulmonary administration of anti-tubercular drugs (ATD) via appropriate delivery system can
avoid the daily dosing, would be a vast improvement. They would cause:
(i) direct drug delivery to the diseased organ;
(ii) long residential time for prolonged release of Anti-T.B. drug(s);
(iii) targeting to macrophages through receptor mediated phagocytosis resulting into many
folds higher intracellular concentration;
(iv) reduced systemic toxicity of the drugs;
1 (v) and finally improved patient compliance. So receptor mediated nanoparticle based
inhalable drug delivery system through pulmonary route would be able to improve drug
bioavailability, reduction in dosing frequency, and may resolve the problem of non adherence
to prescribed therapy; which is one of the major obstacles in the control of TB epidemics.
This research work has achieved the high concentration of anti tubercular drugs (ATDs) in
the selectively cellular tropics of infection compared to blood plasma pool region by
formulating novel ligand appended liposomal delivery systems with increased bioavailability
and site specificity. Furthermore, drug resistance can be averted effectively by multi drug
combination, therefore this mode of therapy prove to be of great therapeutic potential
ensuring complete eradiation of cellular pathogens. Additionally, this will reduce down the
period of treatment frequency of dosage regimen as a consequence would be cost effective.
595lMUMl2000 describes the process for preparing pharmaceutical composition for the
treatment of drug resistant Mycobacterium tuberculosis infection comprises steps of making a
blend containing Isoniazid (INH) and penicillin in ratio between 1 :05 to 1 :20 by weight and
preparing a pharmaceutical composition.
1282/DEL/2003 describes a biodegradable microparticle composition useful for the target
specific drug delivery to manage pulmonary tuberculosis, said composition comprising two
anti-tuberculosis drugs, and a biodegradable polymer for drug delivery in a ratio of about 1 :2
to 2: 1, wherein the anti - tubercular drugs are in the ratio of 1 :2 to 2: 1, also, a process for the
preparation of the composition, and lastly, a method of treating pulmonary tuberculosis in a
subject, said method comprising administering by inhalation alone or in combination with
oral route, pharmaceutically effective amount of the composition to the subject in need
thereof, wherein the dosage for inhalation is ranging between 0.5 to 10 mgkg body
weightlday and that for oral route is ranging between 4 to 32 mglkg body weightlday.
2275lDELNP12006 describes method to obtain an irnrnunotherapeutic agent based on cell
wall fragments of Mycobacterium Tuberculosis comprising the steps of Culturing the
virulent MTB-C strain for a period of at least three weeks, Homogenizing the cell culture in
the presence of a non-ionic surfactant selected from the group consisiting of alkylphenol
ethoxylates and ethoxylated sorbitan esters,Centrifugating the homogenated cell mixture to
separate the cell wall fragments from non-fragmented cells and the solubilised cell
compounds,Inactivating any remaining virulent strain cells in the fraction containing the cell
wall fragments to obtain the immunotherapeutic agent, and Optionally, lyophilizing the
immunotherapeutic agent.
1428lCHENPl2012 discloses an intranasal spray-type tuberculosis vaccine, which has a high
prophylactic effect on human tuberculosis, particularly adult tuberculosis. The nebulizable
tuberculosis vaccine for intranasal administration comprises a pararnyxovirus gene
(particularly rh PIV2) having , integrated therein, a gene encoding an a antigen derived from
an acid fast bacterium (e.g., an a antigen derived from Mycobacteriurn kansasii or
Mycobacterium bovis BCG), an analogue of the gene, or a variant of the gene which has an
equivqlent function to that of the gene.
None of the above, cited references disclose or teach, what the present invention discloses or
teaches. The present invention distinguishable over these cited prior art references.It is
therefore desirable that by using available optional strategies differentiated concentration
should be produced selectively higher in cellular tropics of infection compared to blood
plasma pool. This can be achieved using inhalable or aerosolized pH sensitive or gel cored
pH sensitive nano-carriers systems delivered to macrophages through receptor mediated
phagocytosis resulting into many folds higher intracellular concentration of drug candidate
into the cellular tropics. The ligands could be Fc fragments of IgG; or murarnyl dipeptide; or
polylysinalized polymers; mannan or ligands for TOLL like receptor etc. Furthermore, when
multi drug combination as cocktail is delivered all possible multi drug resistance could be
averted effectively; therefore this mode of therapy may prove to be of great therapeutic
potential ensuring complete eradiation of cellular pathogens. Additionally, this will reduce
down the period of treatment frequency of dosage regimen as a consequence would be cost
effective. The system so developed can be used through various routes for site specific
mycobacterium infection, i.e. insufflators for pulmonary T.B, IM inj. for lymphatic
tuberculosis & similarly IV inj. for brain and organ specific tuberculosis.
Summary of the Invention:
This invention has achieved the high concentration of anti tubercular drugs (ATDs) in the
selectively cellular tropics of infection compared to blood plasma pool region by formulating
novel ligand appended liposomal delivery systems with increased bioavailability and site
specificity. Furthermore, drug resistance can be averted effectively by multi drug
combination, therefore this mode of therapy prove to be of great therapeutic potential
ensuring complete eradiation of cellular pathogens. Additionally, this will reduce down the
period of treatment frequency of dosage regimen as a consequence would be cost effective.
.
This invention can be described as follow:
1. A pharmaceutical formulation for macrophage targeted anti-tubercular multidrug therapy
comprising ligand appended liposomal delivery systems.
2. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems comprising a gel cored liposomal formulations having PC: CHOL
in the molar ratio of 7:3 of rifampicin; 5mg, isoniazid; 2mg and pyrazinamide; 2mg
respectively.
3. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems comprising a plain liposomal formulations.
4. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems comprising a pH sensitive liposomal formulations having
D0PE:CHEM in the molar ratio of 4:O 1.
a 5. The pharmaceutical formulation as claimed in claim 2, wherein said gel cored formulation
having half-life (tllz) of Dry Powder Inhalers(DP1) is found to be 18.12 1.9 h, 19.14 k 1.8 h
and 28.06 * 0.7 h for rifampicin, isoniazid and pyrazinamide respectively.
6. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems is coated by Mannan.
7. The pharmaceutical formulation as claimed in any one of claims 1-6, wherein said
pharmaceutical formulation is used in site specific and multidrug therapy of tuberculosis.
The most common route of drugs for treatment of TB is by oral route. Nowadays the
researchers are concentrating on other route like pulmonary route due to drawbacks of
conventional oral administration therapy over long period of time, lower drug concentration
in cellular tropics of infection compared to blood plasma pool, more toxicity and poor patient
compliance. A convenient way of delivering drugs to the lungs is aerosolization of the drugs
as fine powder with the aid of dry powder inhalers (DPIs). Different carrier systems like
liposomes, microparticles, nanoparticles and solid lipid nanoparticles have been reported for
encapsulation of ATD(s) and as inhalable ATD(s) drug delivery systems. The system is
essentially based on conventional (phosphatidylcholine/ cholesterol) liposomes with median
aerodynamic diameter 0.96pm, encapsulating rifampicin and isoniazid for nebulization. They
showed duration of drug release for 2 days in plasma and 5 days in lungs. Another
Chemotherapeutic system in tuberculosis has also been reported by Pandey et a1.,(2003). The
system is essentially based on nanoparticles made up of Poly lactic-co-glycolic acid (PLGA)
and they were found by nebulized administration with many fold better result compared to
conventional drug therapy. They concluded that nebulization of nanoparticle-based ATDs
form a sound basis for improving drug bioavailability and reducing the dosing frequency for
better management of pulmonary tuberculosis. In another report Vyas et a1.,(2004) liposomal
aerosols were successfully tested with notably improved delivery of rifampicin to alveolar
macrophages Further to consolidate the concentration of drug and carrier within
macrophages, alveolar macrophage specific ligands like mannosylated bovine serum albumin
(MBSA) and 0-steroyl amylopectin were used (0-SAP). In-vivo studies in albino rats
demonstrated a higher pulmonary delivery and better localization of ligand-appended
liposomes to alveolar macrophages compared with conventional liposomes or free rifampicin.
Ahrnad et a1.,(2005) have reported the inhalable alginate nanoparticles as antitubercular drug
carriers. They showed that chemotherapeutic efficacy of three doses of drug-loaded alginate
nanoparticles nebulized 15 days apart were comparable with 45 daily doses of oral free
drugs.Mutti1 et a1.,(2007) found that isoniazid and rifabutin loaded L-PLA microparticles can
efficiently target alveolar macrophages when used as dry powder inhalations. They found that
drug concentrations in macrophages were approx. 20 times higher when microparticles were
inhaled rather than drug solutions administered orally.
Of late, the focus of research in the area of liposomes has been the development of strategies
to increase the ability of liposomes to mediate intracellular delivery of biologically active
molecules. This resulted in the emergence of a modified form of liposomes called pHsensitive
liposomes. The theory of pH-sensitive liposomes emerged from the reality that
4
certain enveloped viruses developed strategies to benefit from the acidification of the
endosomal lumen to infect cells, as well as from the observation that some pathological
tissues (tumours, inflamed and infected areas) exhibit an acidic environment as compared
with normal tissues Torchilin et a1.,(1993). Three hypothetical mechanisms have been
proposed : firstly, destabilization of pH-sensitive liposomes triggers the destabilization of the
endosomal membrane, presumably through pore formation, leading to cytoplasmic delivery
of their contents; secondly, upon liposome destabilization, the encapsulated molecules diffuse
to the cytoplasm through the endosomal membrane; thirdly, fusion between the liposome and
the endosomal membranes leads to cytoplasmic delivery of their contents Collins,(1995;
Ropert et a1.,(1995) Antisense oligonucleotides can be delivered into cells by anionic pHsensitive
phosphatidyl ethanolamine (PE)-containing liposomes that are stable in blood but
undergo phase transition at endosomal acidic pH Fattal et a1.,(2004) Pressurized packed or
aerosolized liposomes for pulmonary targeting of drugs have been well-documented Vyas et
a1.,(2005) liposomes are unstable and release from them cannot be controlled over a
prolonged period of time. To overcome this limitation of liposomes, Tiwari et a1.,(2009)
developed gel core liposomes in which a core of polymer was incorporated inside the
liposomal vesicles, which serve the function of skeleton and provide mechanical strength to
vesicles.
The review of literature reveals that structuring of microparticles or novel vesicular system
with aerodynamic properties could be a critical determinant in the preparation of
compartment targeted inhalable cell specific sustained release therapy. It is also conclusive
that simultaneously other drugs can be administered in order to cover the possibilities of drug
resistance which is responsible for latent phase tuberculosis and secondary progressive
tuberculosis, therefore there is a need of colloidal carrier based multidrug cocktail therapy
which could be based on appropriate cytosolic delivery system including aerodynamic
microparticles, surface decorated macrophage targeted nanoparticles or polar core ternplated
liposomes for better entrapment of drug. Moreover, the surface ligands can be used to further
specify the target ability of the system to the macrophagic tropics for better therapeutic
performance.
It therefore, becomes necessary that by using available optional strategies differentiated
concentration should be produced selectively higher in cellular tropics of infection compared,
to blood plasma pool. This can be achieved using inhalable ligand mediated pH sensitive or
ligand mediated gel-cored nano-carriers systems delivered to macrophages through receptor
mediated phagocytosis resulting into many folds higher intracellular concentration of drug
candidate into the cellular tropics. The proposed work comprising of multidrug strategy
against tuberculosis have not been reported earlier.
Brief description of the drawings:
Figure 1 : life cycle of M Tuberculosis
Figure 2: Percentage cumulative release of drugs from plain (A), pH sensitive (B) and gel
cored (C) liposomes
@
Figure 3: TEM photographs of Mannan Coated Plain, Gel Cored and pH Sensitive
Liposomes
Figure 4: Percentage cumulative release of drugs from mannan coated plain (A), pH sensitive
(B) and gel cored (C) liposomes
Brief description of the Invention:
PLAN OF WORK
8 Selection and Identification of drugs
a. Identification using official compendia methodologies with the help of
UV spectroscopy, IR analysis, melting point and Solubility studies.
b. Preparation of standard curve of different ATDs (RIF, INH & PYZ)
c. Preparation of standard curve of different ATD in organ homogenates
¤ Preparation of drug containing vesicular systems
a. Preparation of conventional liposomes
b. Preparation of pH sensitive liposomes
c. Preparation of Gel-cored Vesicles
d. Preparation of ligand coated conventional liposomes, pH
sensitive liposomes and gel-cored Vesicles
8 Characterization of different vesicular carrier systems
• Vesicle shape and morphology using transmission
electron microscopy (TEM).
• Vesicle size and size distribution determination using
particle size analyzer
Percent drug entrapment
• In-vitro pH sensitivity using florescent dyes
In-vitro release studies
I Development of DPI of formulations
8 Characterization of DPI of formulations
8 In vivo studies
• Tissue distribution
Plasma Pharmacokinetic Analysis
8 Compilation of data
MATERIALS AND METHODS:
Pure drugs were provided as a gift samples by BV Pate1 Perd Centre, Ahmadabad. Lipid
Dioleoylphosphatidylethanolamine was also provided as a gift sample by Lipoid, Germany.
Acetonitrile, Chloroform, Heparin, Potassium bromide,LactoseD-(+ and Trehalose were
purchased from CDH Laboratory reagents, New Delhi. Cholesterol, Dialysis tubing, Egg
phosphtidylcholine, Mannan and Sephadex G-50 were purchased from Sigma chemical Co.,
USA. Dimethylsulfoxide, Disodium hydrogen phosphate, D- mannitol, Methanol, Potassium
dihydrogenphosphate, Sucrose and Sodium chloride were purchased from Rankem
m
Laboratory reagents, New Delhi. Ketamine was purchased from Neon Pvt. Ltd. India. Triton
X-100 was purchased from HiMedia Laboratories Pvt. Ltd. Mumbai.
Animals: Healthy Wistar rats (170-230 g) of either sex, bred in animal house of I.S.F.
College of pharmacy- Moga (Pb.), were used in various animal studies. All animal
experimentations were carried out as per the approved protocol no.
ISF/IAEC/CPCSEA/44/2011 by the Institutional animal ethical committee (IAEC) formed as
per the norms of committee for prevention, control and supervision of experiments on
animals (CPCSEA). Rats were subjected to standard laboratory conditions (i.e. room
temperature, 23k2"C; relative humidity, 55&5%; 12/12 hr lightldark cycle) with free access to
a commercial rodent diet and water before experimentation. In vivo studies of promising
formulations were performed by administering the formulations to the lungs with the help of
a suitable delivery device (canula). Rats were anaesthetized using ketarnine solution
(50mg/kg) by intra-peritoneal administration. The canula was inserted up to the bifurcation of
the trachea.
PREFORMULATION STUDIES
1. RIFAMPICIN (RIF)
a) Determination of melting point
Differential scanning colorimetry (DSC) was performed to determine the melting point of the
Rifampicin. Accurately weighed samples (2 mg) were transferred to aluminium pans and
sealed. All samples were run at a heating rate of 20°C/min over a temperature range 40-300°C
using Shimadzu DSC-60 Thermal Analyzer.
b) Determination of absorption maxima in methanol ( h a x )
10 mg of Rifampicin was accurately weighed and transferred to 100 ml volumetric flask. The
drug was dissolved in lOml of methanol and the volume was made up to 100 ml to obtain a
stock solution of 100pg/ml. This solution was scanned between 300 nrn to 500 nm in a
double beam UV/ Visible spectrophotometer (Shimadzu 1700).
c) Determination of absorption maxima in PBS (pH 7.4)
5 mg of Rifampicin was accurately weighed and transferred to 100 ml volumetric flask. The
drug was dissolved in 10ml of PBS and the volume was made up to 100 ml to obtain a stock
solution of 50pg/ml. This solution was scanned between 300 nm to 500 nm in a double beam
UV/ Visible spectrophotometer (Shimadzu 1700).
d) Infra Red Spectroscopy
The Infra red spectroscopy of the sample was carried out to ascertain the chemical identity of
the drug. A pellet of approximately 1 mm diameter of drug was prepared by compressing 5
mg of the drug with 200 mg of potassium bromide in KBr press. The pellet was mounted in
1 .
IR compartment and scanned between wave number 4000-' - 600 cm'l using a Thermo
Nicolet 380, USA.
e)Determination of solubility
An excess of drug was dissolved in 10 ml of respective solvents in conical flask and was
continuously shaken for 24 hours at room temperature with the help of conical flask shaker.
After 24 hours sample was filtered through Whatman filter paper no. 1, diluted appropriately
and the drug was estimated using UV spectroscopy (Shimadzu 1700).
f) Drug partition studies
The partition coefficient of the drug was determined in n-octanol : PBS (pH 7.4). Accurately
weighed drug (20mg) was transferred into a glass stoppered test tubes containing lOml of noctanol
and lOml of PBS (pH 7.4). The mixture was shaken on wrist action shaker for 4hrs.
Both the phases were separated using separating funnel and drug concentration in both phases
was determined using UV-visible spectrophotometer against respective blank,
g)Drug-Excipients Compatibility Studies
FTIR spectra were recorded to assess the compatibility of the drugs and excipients. Drug(s)
and excipients in the ratio of 1 : 1 were mixed thoroughly and stored at 40 OC and 75% RH and
in ambient conditions for 1 month Sinha et a1.,(20 10).
h) Calibration curve of RIF in methanol
Accurately weighed 50 mg of RIF was taken in a clean and dry flask and was dissolved in
methanol 50 ml. 1 ml of the above solution was diluted to 10 ml with methanol to produce a
stock solution of 100pg/ml. From this solution, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5ml
were withdrawn separately in different 10 ml volumetric flasks and volume made in each
case up to 10 ml with methanol to produce the concentrations 5, 10, 15,20,25, 30,35,40,45
and 50 pglml. Absorbance of these solutions was recorded at hmax 475 nm against methanol
as blank using UV-visible spectrophotometer.
i) Calibration curve of RIF in PBS (pH 7.4)
The above procedure was adopted except the aliquots in 10 ml volumetric flasks was made in
each case up to 10 ml with PBS (pH 7.4) to produce the concentrations 5, 10, 15, 20, 25, 30,
35,40,45 and 50 pglml. Absorbance of these solutions was recorded at hmax 475 nrn against
PBS as blank using UV-visible spectrophotometer.
2. ISONIAZID (INH)
a) Determination of melting point(s)
Differential scanning colorimetry (DSC) was performed to determine the melting point of the
isoniazid. Accurately weighed samples (2 mg) were transferred to aluminium pans and
ilD
sealed. All samples were run at a heating rate of 20'~lmin over a temperature range 40-300'~
using Shimadzu DSC-60 Thermal Analyzer.
b) Determination of absorption maxima in PBS (pH 7.4)
2 mg of isoniazid was accurately weighed and transferred to 100 ml volumetric flask. The
drug was dissolved in 10ml of PBS and the volume was made up to 100 ml to obtain a stock
solution of 20 pglml. This solution was scanned between 200 nm to 400 nm in a double beam
UVI Visible spectrophotometer (Shimadzu 1 700).
c) Infra Red Spectroscopy
The Infra red spectroscopy of the sample was carried out to ascertain the chemical identity of
the drugs. A pellet of approximately 1 mrn diameter of drug was prepared by compressing 5
mg of the drug with 200 mg of potassium bromide in KBr press. The pellet was mounted in
IR compartment and scanned between wave number 4000-' - 600 cm-' using a Thermo
Nicolet 380, USA.
d) Determination of solubility
An excess of drug was dissolved in 10 ml of respective solvents in conical flask and was
continuously shaken for 24 hours at room temperature with the help of conical flask shaker.
After 24 hours sample was filtered through Whatman filter paper no. 1, diluted appropriately
and the drug was estimated using UV spectroscopy (Shimadzu 1700).
e) Drug partition studies
The partition coefficient of the drug was determined in n-octanol : PBS (pH 7.4). Accurately
weighed drug (20 mg) was transferred into a glass stoppered test tubes containing 10ml of noctanol
and lOml of PBS (pH 7.4). The mixture was shaken on wrist action shaker for 4hrs.
Both the phases were separated using separating funnel and drug concentration in both phases
was determined using UV-visible spectrophotometer against respective blank.
f) Drug-Excipients Compatibility Studies
FTIR spectra were recorded to assess the compatibility of the drug and excipients. Drug(s)
and excipients in the ration of 1:l were mixed thoroughly and stored at 40 OC and 75% RH
for and room temperature for 1 month.
g) Calibration curve of INH in PBS (pH 7.4)
Isoniazid (10 mg) was accurately weighed and was taken in a clean and dry 100 ml
volumetric flask. It was dissolved in lOOml PBS (pH 7.4) to produce a stock solution of
100pg/ml. From this solution 0.2,0.4,0.6,0.8, 1 .O, 1.2, 1.4, 1.6, 1.8 and 2ml were withdrawn
separately in different 10 ml volumetric flasks and volume were made in each case upto 10
ml with water to produce concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 pglml.
Absorbance of these solutions was recorded at hmax 269 nm against water as blank using
UV-visible spectrophotometer (Shimadzu, Japan).
3. PYRAZINAMIDE (PYZ)
a) Determination of melting point(s)
Differential scanning colorimetry (DSC) was performed to determine the melting point of the
pyrazinamide. Accurately weighed samples (2 mg) were transferred to aluminium pans and
sealed. All samples were run at a heating rate of 20°C/min over a temperature range 40-300'~
using Shimadzu DSC-60 Thermal Analyzer.
b) Determination of absorption maxima in PBS (pH 7.4)
2 mg of pyrazinamide was accurately weighed and transferred to 100 ml volumetric flask.
The drug was dissolved in lOml of PBS and the volume was made up to 100 ml to obtain a
stock solution of 20 pglml. This solution was scanned between 200 nm to 400 nm in a double
beam UV/ Visible spectrophotometer (Shimadzu 1700).
c) Infra Red Spectroscopy
The Infra red spectroscopy of the sample was carried out to ascertain the chemical identity of
the drugs. A pellet of approximately 1 mm diameter of drug was prepared by compressing 5
mg of the drug with 200 mg of potassium bromide in KBr press. The pellet was mounted in
IR compartment and scanned between wave number 4000-' - 600 cm" using a Thermo
Nicolet 380, USA.
d) Determination of solubility
An excess of drug was dissolved in 10 ml of respective solvents in conical flask and was
continuously shaken for 24 hours at room temperature with the help of conical flask shaker.
After 24 hours sample was filtered through Whatman filter paper no. 1, diluted appropriately
and the drug was estimated using UV spectroscopy (Shimadzu 1700).
e) Drug partition studies
The partition coefficient of the drug was determined in n-octanol : PBS (pH 7.4). Accurately
weighed drug (20mg) was transferred into a glass stoppered test tubes containing 10ml of noctanol
and lOml of PBS (pH 7.4). The mixture was shaken on wrist action shaker for 4hrs.
Both the phases were separated using separating funnel and drug concentration in both phases
was determined using UV-visible spectrophotometer against respective blank.
f) Drug-Excipients Compatibility Studies
FTIR spectra were recorded to assess the compatibility of the drug and excipients. Drug(s)
and excipients in the ration of 1: 1 were mixed thoroughly and stored at 40 O C and 75% RH
for and room temperature for 1 month.
i 0 g) Calibration curve of PYZ in PBS (pH 7.4)
Pyrazinamide (10 mg) was accurately weighed and was taken in a clean and dry 100 ml
volumetric flask. It was dissolved in lOOml volume of water to produce a stock solution of
100pg/ml. From this solution 0.2, 0.4, 0.6, 0.8, 1 .O, 1.2, 1.4, 1.6, 1.8 and 2ml were withdrawn
separately in different 10 ml volumetric flasks and volume were made in each case upto 10
ml with water to produce concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20pg/ml.
Absorbance of these solutions was recorded at hmax 254 nm against water as blank using
UV-visible spectrophotometer (Shimadzu, Japan).
RESULT AND DISCUSSION OF PREFORMULATION STUDIES
Rifampicin, pyrazinamide and isoniazid were identified using different methods viz. melting
point determination, determination of absorption maxima (hmax), solubility and FTIR
spectroscopy.
The thermogram of differential scanning colorimetry showed sharp endothermic peaks of
rifarnpicin, pyrazinamide and isoniazid at 183.23"C, 191.23"C and 171.56"C , respectively
corresponding to the melting range of rifampicin (1 82- 1 84"C), pyrazinamide (1 90- 1 94 OC)
and isoniazid (1 70- 176 "C) in the crystalline form.
Absorption maxima (hmax) of rifampicin, pyrazinamide and isoniazid were found to be at
wavelength 475 nrn, 254nm and 269 nm corresponding to the values reported in literature
(rifampicin - 475 nrn, pyrazinamide - 254nm and isoniazid - 269 nm).
FTIR spectra of the rifampicin, showed characteristic C=O, C-N, C-0 and C=C, stretching
bands at 1655 cm-', 1097 cm-', 1252 cm-' and 1566 cm-I, respectively. FTIR spectra of
isoniazid, showed characteristic C=O, C=N, N-H and C-H stretching bands at 1653 cm-I,
1602 cm-I, 1545 cm-' and 675 cm-' respectively. FTIR spectra of pyrazinamide, showed
characteristic C=O, C=N, C-0 and C-C stretching bands at 1712 cm-I, 1375 cm-', 1252 cm-'
and 1054 cm-' respectively. The infrared absorption spectrum of rifampicin, pyrazinamide
and isoniazid is found to be in concordant with the spectrum obtained from with the reference
spectrum of standard drugs respectively.
Calibration curve data of the drugs were subjected to linear regression analysis. Beer and
Larnbert's law was found to be obeyed in the concentration range of 5-50 mcg/ml, 2-20
mcg/ml and 2-20 mcg/ml for rifampicin, pyrazinamide and isoniazid, respectively in all the
experimented media. R-values were found to be 0. 9895, 0.9877 & 0.9998 for rifampicin,
pyrazinamide and isoniazid in phosphate buffer (pH 7.4) respectively which indicate
linearity.
The solubility of the drugs was determined in different media. Rifampicin is freely soluble in
chloroform and DMSO; soluble in ethyl acetate, methanol and slightly soluble in acetone,
water and carbon tetrachloride. Isoniazid is Soluble in water, ethanol and chloroform and
insoluble in ether, benzene. Pyrazinamide is soluble in water and in chloroform; slightly
soluble in ethanol (95 percent) and very slightly soluble in ether. Solubility studies inferred
11
a that rifampicin is lipophilic in nature whereas isoniazid and pyrazinamide id hydrophilic in
nature. Further log P values calculated for rifampicin, isoniazid and pyrazinamide using the
flask stirring method were 1.075, - 1.22 and - 1.2 16 respectively. Log P values of less than 1 in
case of isoniazid and pyrazinamide are indicative of highly water soluble but poorly
permeable molecules. Whereas log P value of rifampicin indicative of the lipophilic nature of
the drug.
The drug identification studies showed that drugs supplied by BV Pate1 perd Centre and
Lupin Pharmaceutical Pvt. Ltd., Aurangabad, India (Rifampicin and Isoniazid and
Pyrazinamide respectively) concordant with the reported data for solubility, melting point,
UV and IR . The results of various preformulation studies are given in Table 1.
Table 1 : Results of preformulation studies of rifampicin, isoniazid and pyrazinamide
PREPARATION AND CHARACTERIZATION OF DRUGS LOADED LIPOSOMES
Preformulation
parameters
DSC endotherm
in deg C.
hmax in nm
FTIR peaks in
cm-'
Solubility
(mglml)
Partition
coefficient
1. Preparation of Rifampicin loaded liposomes
The liposomes were prepared using the hand shaking method as described by Azmin et
a1.,(1985). Rifarnpicin was dissolved in 5ml of chloroform in a round bottom flask.
Phosphatidylcholine and cholesterol in different molar ratio were dissolved in the above
Standard
RIF
182-1 84
"C
475
C = 0
stretch -
1650
C-N
stretch -
1098
C - 0
stretch -
1250
C=C
stretch -
1567
Freely
soluble in
chloroform
and
slightly
soluble
water
1.086
Observed
RIF
183.23 "C
475
C = O
stretch -
1655
C-N
stretch -
1097
C - 0
stretch -
1252
C=C
stretch -
1566
5.71mg/ml
in
methanol
and
0.08mg/ml
in water
1.075
values
rNH
170-176
"C
269
C=O
stretch -
1652
C=N
stretch -
1621
N-H
bending -
154 1
=C-H
bending -
674
Soluble in
water and
insoluble
in ether,
benzene.
- 1.2
PYZ
190-194 "C
254
C=O
stretch -
1714
C=N
stretch -
1378
C - 0
stretch -
1250
C-C
stretch -
1054
soluble in
water and
slightly
soluble in
ether
- 1.17
values
INH
171.56"C
269
C = 0 stretch
- 1653
C=Nstretch
- 1602
N - H
bending -
1545
=C - H
bending -
675
2.8mg/ml in
water and
0.3mglml in
ether
- 1.228
PYZ
19 1.23"C
254
C = 0
stretch -
1712
C=N
stretch -
1375
C - 0
stretch -
1252
C-C
stretch -
1054
1.9mg/ml in
water and
0.3mglml in
ether
- 1.168
a
mixture. The organic solvent was removed by rotating the flask at a temperature of 37°C
under reduced pressure. The dried lipid film was then hydrated using PBS pH (7.4). The
suspension of liposomes was sonicated for 2 min to reduce the size of liposomes.
2. Preparation of Isoniazid and pyrazinamide loaded liposomes
The liposomes were prepared using thin film hydration method as described by.Azmin et
a1.,(1985). Phosphatidylcholine (PC) and cholesterol (CHOL) in different molar ratio were
dissolved in 5ml of chloroform. The organic solvent was removed by rotating the flask at a
temperature of 37°C under reduced pressure. The dried lipid film was then hydrated using
PBS pH (7.4), containing drug (isoniazid and pyrazinamide) for about 3 hrs using vortex
mixer to get uniform suspension. The suspension of liposomes was sonicated for 2 min to
reduce the size of liposomes. The composition of various liposomal formulations are given in
Table 2
Table 2: Compositions of various drug loaded plain liposomal formulations
S. No Formulation Molar ratio DRUG (mg)
Code PC:CHOL RIF INH PYZ
1 A1 6:4 5 - -
2 A2 7:3 5 - -
3 A3 8:2 5 -
4 A4 6:4 2
5 A5 7:3 2
6 A6 8:2 2
17 1 ~ 7 1 6:4 1 I - I - 12
3. Preparation of pH sensitive liposomes
pH sensitive liposomes were prepared by thin film hydration method. Drug (Rifampicin) and
the different molar ratio of CHEMS and DOPE were dissolved in chloroform Chen et
a1.,(2011). Chloroform was evaporated using rotary vacuum evaporator and kept overnight
under vacuum. Nitrogen gas was passed over the thin film. The film formed was hydrated
with using PBS pH (7.4). The suspension of liposomes was sonicated for 2 min to reduce the
size of liposomes. The composition of various pH sensitive liposomal formulations are given
in Table 3
Table 3: Compositions of drug loaded pH sensitive liposomal formulations
S. No Formulation Molar ratio DRUG (mg)
Code D0PE:CHEM RIF INH PYZ
1 A 10 3.5: 1.5 5
4. Preparation of gel cored liposomes
Gel cored liposomes were prepared by reverse phase evaporation technique as reported by
Szoka and Papahadjopoulos,(l978) with slight modifications. Briefly, PC and CHOL were
dissolved in 5ml diethyl ether to which 2ml of aqueous phase, i.e. phosphate-buffered saline
(PBS, pH 4.2, since at this pH polymer remain in sol state) containing drugs with 0.3%
concentration of poly acrylic acid was added and sonicated (MISONIX, Ultrasonic Liquid
Processsors) for 3 min at 4OC. The resultant mixture was placed in a rotary evaporator until a
thick gel formed and kept over a vortex mixer (SPINIX) in order to remove any residual
ether. Un-entrapped drug and polymer from the gel core liposomes was removed by ultracentrifugation
at 50,000 g for 15 min and fkther, dialysed against PBS pH 7.4 to induce
gelation of entrapped polymer. The composition of various gel cored liposomal formulations
are given in Table 4
Table 4: Compositions of drug loaded gel cored liposomal formulations
I S. I Formulation I Molar ratio ( Conc. Of PAA ( DRUG (mg) I No I Code I PC:CHOL I in % (w/v) 1 RIF I INH / PYZ
1 I I I I I I I
5.5. Preparation of Mannan Coated Liposomes
Mannan coating of optimized liposomal formulations were carried out by using incubation
method. Coating solutions were prepared by dissolving mannan (1:l) wlw in hot water.
Coating was done by depositing this Chen et al.,(20ll)coating solution on the surface of
liposomes by mixing of the preformed liposomes with mannan solution and stirring this
mixture overnight at room temperature. Free mannan was removed by passing through a
sephadex G-50 column Vyas et a1.,(2005). The composition of various ligand coated
liposomal formulations are given in Table 5
Table 5: Compositions of mannan coated liposomal formulations
S. No Formulation Molar ratio Conc. of DRUG(mg)
Code PC:CHOL mannan RIF j INH I PYZ
I 0%) 1 MANNAN COATED DRUG LOADED PLAIN LIPOSOMES 1
3 A3 0 7:3 6 5
4 A3 1 7:3 2 - 2
5 A32 7:3 4 - 2
6 A3 3 7:3 6 2
7 A34 7:3 2 2
8 A3 5 7:3 4 - - 2
9 1 A36 7:3 6 - 2
I
I MANNAN COATED DRUG LOADED pH SENSITIVE LIPOSOMES
S. No Formulation Molar ratio Conc. of DRUG(mg)
Code D0PE:CHEM mannan RIF
(mg )
INH PYZ
1 A3 7 4: 1 2 5 -
2 A3 8 4: 1 4 5
3 A3 9 4: 1 6 5
4 A40 4: 1 2 2
5 A4 1 4: 1 4 - 2
6 A42 4: 1 6 - 2
7 A43 4: 1 2 2
1 8 A44 4: 1 4 2
9 A45 4: 1 6 2
MANNAN COATED DRUG LOADED GEL CORED LIPOSOMES
S. No Formulation Molar ratio PAA Conc. of DRUG(mg)
Code PC:CHOL (wlv) mannan RIF INH PYZ
(mg)
1 A46 7:3 0.3 2 5
2 A47 7:3 0.3 4 5 -
3 A48 7:3 0.3 6 5
4 A49 7:3 0.3 2 - 2
5 A5 0 7:3 0.3 4 2
6 A5 1 7:3 0.3 6 2 -
7 A52 7:3 0.3 2 2
8 A53 7:3 0.3 4 2
9 A54 7:3 0.3 6 - 2
Purification
The liposomes were purified using the gel permeation technique as described by Vyas et
a1.,(2005). Briefly the liposomal formulations were centrifuged through sephadex G-50 minicolumn
at 2000 rpm for 3 min for the separation of unentrapped drug. Sephadex G-50 (1 g) in
100ml of 0.9% sodium chloride solution at room temperature for overnight. The dispersion so
formed was stored at 4'~. The liposomal fraction was added with minimum amount of Triton
e X-100 (0.1%, vlv), drug content was determined spectrophotometrically and percent drug
entrapment was calculated.
CHARACTERIZATION OF DRUG LOADED VARIOUS LIPOSOMAL
FORMULATIONS
The liposomal preparations containing drug were characterized for following attributes:
1. Particle size measurement by TEM
Various liposomal formulations were evaluated for vesicle shape and morphology using
transmission electron microscopy (TEM). Phosphotungustic acid (1%) was used as a negative
stain. Samples were placed over a copper grid and subjected to TEM analysis (JEM-200 CX,
JEOL, Tokyo, Japan). Vesicle size and size distribution were determined using particle size
analyzer (DelsaTMN ano C Particle Analyser), which works on a laser diffraction principle.
2. Particle size measurement by zetasizer
The mean particle size and size distribution of liposomes were measured by dynamic light
scattering (DLS) using DelsaTM Nano C Particle Analyser. The sample was placed in an
automated dispersion unit and subjected to particle size analysis. The collimated laser beam is
made incident to the suspended sample particles. The intensity signals of the different bar
scattered light are processed into particle size distribution. The average particle size was
measured after performing the experiment in triplicates.
3. Entrapment efficiency
The fiee un-entrapped drug was removed by passing the dispersion through a Sephadex G-50
column. The liposomes were dissolved in methanol to extract rifampicin from the
phospholipids structure, and the drug was determined spectrophotometrically at 475nm. To
accesses the entrapment efficiency of isoniazid and pyrazinamide, the vesicles were disrupted
using 1.0 ml of 0.1% (v/v) Triton X-100 and the liberated drug was estimated
spectrophotometrically using a UV-VIS Spectrophotometer. All samples were prepared in
triplicate.
Entrapment efficiency (%) = [(A-B)/A] * 100
Where A is concentration of total drug and B is concentration of free drug.
4. DSC studies
Differential scanning calorimetry (DSC) was used to determine the phase transition
temperatures of phospholipid samples in order to characterize the physical state of drug. The
transition temperatures correspond to the peaks of the endotherms during the heating scans.
Samples consist of known mass of liposomal formulation were placed in aluminium pans and
thematically sealed. The heating rate was 10°C per minute using nitrogen as the purge gas.
The DSC instrument was calibrated for temperature using Indium. In addition, for enthalpy
calibration Indium was sealed in aluminium pans with sealed empty pan as a reference.
5. In- Vitro drug release
The in vitro release of conventional, pH sensitive and stealth gel core liposomes was studied
by using simple difhsion cell apparatus. The diffusion cell apparatus consists of a glass tube
with an inner diameter of 2.5cm, open at both ends, one end of the tube is tied with Sigma
dialysis membrane, which serves as a donor compartment. Liposomal suspension was taken
in a dialysis tube and placed in 200ml PBS pH 7.4, stirred with a magnetic stirrer and the
16
e temperature was maintained at 37k 0.5'~ and samples were withdrawn at specific time
intervals and after each withdrawn same volume of medium were replaced.
6. Zeta potential
Particle size and zeta potential of liposomes was determined using a DelsaTM Nano C Particle
analyser. Samples were prepared by dispersing 50 pl of liposome suspension in 1 ml
phosphate buffered saline (pH 7.4). Samples were loaded into clear, disposable, folded
capillary cells and analysed for both particle size and zeta potential at 25oC. For particle size,
the instrument was set to automatically determine attenuator level, measurement position and
number of sub-runs based on correlation data and all measurements were performed in
triplicate.
7. Calcein leakage test
The quantity of drug released fiom the liposomes was measured using carboxyfluorescein as
an example of a hydrophilic drug. carboxyfluorescein is ideal for the measurement of drug
release from liposomes, as concentrations entrapped within the liposome core are high and
result in self-quenching of the fluorescence of carboxyfluorescein Torchilin and
Weissig,(2003). Liposomes hydrated with 100 mM carboxyfluorescein at pH 7.4 and were
separated from free carboxyfluorescein through gel filtration using Sephadex G50 (Sigma-
Aldrich, St Louis, USA) hydrated in PBS pH 7.4. Liposome dispersion separated from free
carboxyfluorescein was immediately transferred to the well of a 96 well black microtitre plate
along with 50 pl PBS pH 7.4 and 50 pl foetal calf serum. The fluorescence was measured at
an excitation wavelength of 485 nrn. Measurements were taken every 30 minutes for four
hours, after which, 50 pl of a 10% Triton X-100 solution in PBS was added to each well in
order to digest the liposome membranes and release the remainder of the entrapped
carboxyfluorescein. The fluorescence was measured once more in order to determine the total
carboxyfluorescein present and the % carboxyfluorescein released calculated at each time
point as a percentage of the total.
8. Confirmation of gelling in inner compartment of liposomes: Confirmation of gelling in the
inner compartment of liposomal vesicle was done with Triton X 100. Briefly the gel core
liposomal preparation was centrifuged to remove un-entrapped polymer. Then it was divided
into two parts, one remained as such and the other was dialyzed against PBS (pH 7.4) for
gelation in the core Tiwari et a1.,(2009). Both of the preparations were treated with Triton
X100and evaluated for morphological characterization by a light microscope (Nikon, Japan).
9. Statistical analysis
The data were statistically processed to determine the level of significance. Standard
deviation (S.D.) was calculated, and values are given as mean k S.D. Student's t-test was
used to compare mean values of different groups. Statistical significance was designated as P
< 0.05.
* In the present studies various liposomal formulation were made with different compositions
of PC and CHOL by using constant amount of drug (Rifampicin; 5mg, Isoniazid; 2mg and
Pyrazinamide; 2mg). The results of different parameters such as entrapment efficiency (EE),
particle size (PS), zeta potential (ZP) and polydispersity index (PDI) are tabulated in table 6.
In this study we observed that the size of the vesicles plays a significant role in the
encapsulation efficiency of both hydrophilic and lipophilc drugs. Increasing the PC: CHOL
ratio, the size of vesicle was increased with subsequent decrease in entrapment efficiency Al-
Angary et a1.,(1996). Potential drop in entrapment efficiency was also observed on increasing
the PC level. Entrapment of rifampicin in the liposomes could be attributed to the lipophilic
nature of the drug represented by lipid: aqueous phase ratio and acyl chain length of
phospholipid. An increase in chain length of fatty acid and inclusion of cholesterol results in
an increase in the encapsulation efficiency Puglisi et a1.,(1995; Al-Angary et a1.,(1996).
This clearly indicates that RIF molecules intercalate into the lipid bilayers, as anticipated by
its lipophilic nature. The bulky size of RIF is thus responsible for the increased mean
diameter of the RIF-liposomes compared to INH and PYZ loaded liposomal formulations
Deol and Khuller,(1997). Also, 'from this study we found that entrapment of isoniazid is
higher than rifampicin. This is due to hydrophilic and lipophilic nature of the drug molecules.
In this case, the encapsulation of drug is completely dependent upon the volume of aqueous
phase encapsulated during liposome formation. However the encapsulation of rifampicin is
better than pyrazinamide, this may be attributed to poor aqueous solubility of pyrazinamde.
Entrapment of Isoniazid and pyrazinamide in the liposomes could be attributed to the
hydrophilic nature of the drug represented the aqueous core of the vesicular carriers. Further
entrapment efficiency found to be decrease with increase in the molar ratio of PC : CHOL,
this may attributed that with changing in the molar ratio from 7:3, results leaky membrane
structure leading to lower entrapment efficiency. Moreover the optimum ratio of PC : CHOL
results a narrow size distribution with an average vesicle size of 523.6, 376.6 and 404.3nrn
for formulation A2, A5 and A8 respectively, may featured a distinctive speculation of higher
entrapment efficiency of both hydrophilic and lipophilic dugs Dutta et a1.,(2000). The drug
molecules (hydrophilic nature) may also influence liposome formation by an ability to bind
water molecules and form liquid crystal structures in aqueous solutions. Drug release studies
of plain liposomal formulations were observed for 48 hrs. A2, A5 and A8 formulations
demonstrated 72.3* 1.56%, 76.5 * 1.24%, 71.5 * 1.44% cumulative release for rifampicin,
isoniazid and pyrazinamide respectively.
When the conventional liposomes were compared with pH sensitive and gel cored liposomes
for their encapsulation efficiency, there are no significant changes observed between plain
and pH sensitive liposomal formulations. However gel cored liposomes showed a statically
higher encapsulation than plain liposomes for both isoniazid and pyrazinamide (P < 0.005)
suggesting that the poly acrylic acid (PAA) undergoes ionisation; this makes an abrupt
change in apparent dissociation constant of the surrounding with respect to bulk, facilitate the
ionisation of hydrophilic drugs and thereby improve the encapsulation efficacy Thomas et
a1.,(1994). Further the change along the polymer backbone of PAA increases the
hydrodynamic volume of the liposomal core, which may further contribute to the higher
encapsulation of both isoniazid and pyrazinamide in gel cored liposomes Cevc,(1987).
Further a higher encapsulation efficiency of rifampicin was observed in gel cored liposomes
that may be associated to smaller size distribution of gel cored liposomes, which may implies
the change in pH induced by PAA, facilitate the liposome formation, doing so narrowed the
velocity distribution of the molecules potentially narrowing the size distribution of the
liposomes as well, clearly characterized by size and PDI of the gel cored formulations, which
is found to be a size range of 465.9 k 2.4, 339.0 2.6 nm and 341.0 k 1.6 and PDI 0.16 k
0.08,O.ll k 0.10 and 0.17 * 0.09 for formulations A20, A23 and A 26 respectively.
The polydispersity index is an important parameter for ensuring predictable drug release
prompted by a uniform surface area available for diffusion. Result indicated that all liposome
suspension, ensuring homogenous particle size distribution.
In vitro drug release studies were carried out in dialysis bag. The release pattern of the drugs
is shown in Figure 2. The liposomal dispersion of rifampicin was found to release about 72.3
* 1.5 % of the drug after 48hrs Similarly, in case of isoniazid and pyrazinamide 76.5 & 1.24
% and 71.5 k 1.44 % of released was observed indicated a slow and sustained release of
drugs from conventional liposomal formulations. When in vitro release profiles of plain
liposomes were compared with pH sensitive liposomes, pH-sensitive liposomes demonstrated
a significant decrease in the cumulative release of both hydrophilic and lipophilic drugs,
suggesting following speculations. Firstly the combination CHEMIDOPE is the crucial factor
determining the ability of such liposomes to undergo destabilization upon acidification.
Secondly pH sensitive liposome undergoes protonation at acidic pH, which is rate limiting
factor in case of pH sensitive liposome which will govern the drug release mechanism. To
establish the above hypothesis, quenching assay of pH sensitive liposomes was performed
using carboxyfluorescein Ropert et a1.,(1995).
Table 6: Evaluation parameters of plain, pH sensitive and gel cored liposome.
Formulation
type
v) ii
.8a d -
.C- cd CI a
w>
2 E
2
$4 % o 5 .-S
Code PS in (nm)
RIFAMPICIN
ZP in (mV)
A1
A2
A3
PDI
605.7 3.4
523.6 2.7
661.8 5.3
ISONIAZID
EE (%)
-22.0 k 1.8
-27.3 h 2.1
-14.5 * 5.2
A4
A5
A6
Percentage
cumulative
release after
48hrs
0.341* 0.01
0.299k 0.01
0.310* 0.01
568.2 * 3.3
376.6*5.7
590.0 * 2.0
PYRAZINAMIDE
44.40 * 7.9
56.08 * 4.6
39.60 * 1.9
-24.3 0.3
-31.2k4.1
-19.4 3.2
A7
A8
A9
83.1 1.83
72.3 * 1.56
89.4 * 2.87
0.298* 0.03
0.288* 0.01
0.289* 0.02
494.9 * 4.4
404.3 * 6.5
553.4 3.5
RIFAMPICIN
56.16 * 1.6
68.67 * 1.5
45.33 * 9.3
-24.5 -+ 5.3
-27.6 k 2.9
-17.7 * 1.9
A10
Al l
A 12
93.2 2.21
76.5 * 1.24
95.2 * 2.27
0.274* 0.05
0.210* 0.04
0.270* 0.02
825.63 * 8.9
526.86
10.14
604.66
28.83 * 1.1
42.33 zt 8.1
22.67 * 7.9
-12.65 * 1.8
-18.94 *
06.3 1
-14.12 iz
85.7 * 2.34
71.5 * 1.44
89.3 2.59
0.201 * 0.01
0.207 * 0.08
0.297 * 0.01
52.00 * 2.1
62.60 * 00.72
56.00 * 01.57
69.58 *
0.56
62.65 *
1.12
67.80
1 12.37 1 00.24 1 1.17
ISONIAZID
A13 635.35 -3 1.32 k 0.235 k 0.02 61.88 * 02.32 73.54
08.34 03.65 2.34
A14 467.45 -36.65 i 0.217 k 0.01 68.45 k 03.56 ' 64.32 *
12.45 08.73 1.28
A15 525.65 * -33.45 * 0.3 12 * 0.02 63.62 * 03.34 68.74 *
1 08.64 1 04.69 1 2.12
PYRAZINAMIDE
A16 1 589.54 k 1 -28.45 * 1 0.198 * 0.03 1 33.73 h 02.31 1 64.67 *
1 09.48 1 03.78 1 1.78
RIFAMPICIN
A19 566.3 l 2.4 -30.4 * 1.10 0.12 0.02 47.60 3.0 78.1 k 1.83
A20 465.9 2.4 -3 1.6 * 0.63 0.16 * 0.08 60.26 k 5.7 64.3 1.56
A2 1 634.1 h 2.3 -26.9 * 0.56 0.13 * 0.01 44.30* 0.9 73.4 k 2.87
a
ISONIAZID
8 A22 455.6 * 4.8 -29.2 * 1.80 0.16 k 0.07 71.30 k 3.5 82.3 1.18
3 A23 339.0 k 2.6 -33.2 2.10 0.1 1 * 0.10 78.60 * 2.25 68.8 h 1.34
a .C1
A24 488.0 k1.4 -22.7k1.73 O.lOhO.01 65.60h1.41 85.061.40
4
-a PYRAZINAMIDE
2 A25 381.2 * 3.7 -29.4 * 2.90 0.08 * 0.01 44.20 * 0.80 79.4 k 1.04
S
d
0
A26 341.0 * 1.6 -36.8 * 3.08 0.17 0.09 53.50 * 5.07 65.8 * 2.01
A27 41 1.4k1.2 -21.8* 1.90 0.10*0.02 40.10*1.53 82.6k2.01
Values are expressed as mean + S.D. (n=3)
The result of drug quenching efficiency of pH sensitive liposomes (table 7) indicates that pH
liposome preparations were found to have a marked effect on the rate at which
carboxyfluorescein (hydrophilic moiety) escaped from the liposomes at different pH Kirby et
a1.,(1980). At pH 5.5 the liposomes were sensitive revealing that drug is likely to be released
into the cytoplasm from the endosomes, thereby escaping the lysosomal pathway, which
normally leads to the excretion of liposomal contents from the cells. It was observed that the
release pattern of pH sensitive liposomes was much slower for all drugs when compared to
plain and gel cored liposoms. The speculation further supported by the data obtained from the
quenching assay in table 7.
Table 7: Percentage (%) calcein leakage at different pH (mean *S.D., n=3)
I S.No I pH of the incubation medium I Percentage decrease in quenching
efficiency
1 4.0 0.003* 0.89
2 4.5 05.38% 2.70
3 5 08.89* 0.92
4 5.5 39.45* 3.06
after 48 hrs in compare to plain liposomes where 72.3 k 1.56,76.5 k 1.24 and 7 1.5 * 7.44 %
released was observed for rifampicin, isoniazid and pyrazinamide respectively and 64.3
1.56, 68.8 k 1.34 and 65.8 * 2.01 % in case of gel cored liposomes.
When the in vitro release profile of plain liposomes were compared with gel cored liposomes,
there was no significant changes found in the percentage cumulative release at pH 7.4.
However the gelling study conformed that PAA undergoes transition from sol to gel at acidic
pH, leads to slow and sustained cytosolic release of hydrophilic drugs. Further the
incorporation of ionisable moiety into phospholipids backbone disappear the electrostatic
repulsive forces within the phospholipids network, thereby the additive effect of hydrophobic
interaction and Vandenvall force overcome the net repulsive forces offer a more compact
conformation of the phospholiid vesicles, leads to slow release of lipophilic drug (RIF). The
slow release of rifampicin f'urther reinforced by the high molecular weight of rifampicin from
the rigid phospholipd membrane. These results are in line with earlier reports in which
inclusion of PAA in liposome resulted in prolonged drug retention at acidic pH. The
entrapment efficiency, molar ratio, zeta potential, PDI and % cumulative release of optimised
liposomal formulations for all the drugs are shown in Table 8.
Table 8: .In vitro characterization of optimised formulations.

We Claim
1. A pharmaceutical formulation for macrophage targeted anti-tubercular multidrug therapy
comprising ligand appended liposomal delivery systems.
2. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems comprising a gel cored liposomal formulations having PC: CHOL
in the molar ratio of 7:3 of rifampicin; 5mg, isoniazid; 2mg and pyrazinamide; 2mg
respectively.
3. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems comprising a plain liposomal formulations.
4. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems comprising a pH sensitive liposomal formulations having
D0PE:CHEM in the molar ratio of 4:Ol.
5. The pharmaceutical formulation as claimed in claim 2, wherein said gel cored formulation
having half-life ( t , ~o)f Dry Powder Inhalers(DP1) is found to be 18.12 h 1.9 h, 19.14 k 1.8 h
and 28.06 k 0.7 h for rifampicin, isoniazid and pyrazinamide respectively.
6. The pharmaceutical formulation as claimed in claim 1, wherein said ligand appended
liposomal delivery systems is coated by Mannan.
7. The pharmaceutical formulation as claimed in any one of claims 1-6, wherein said
pharmaceutical formulation is used in site specific and multidrug therapy of tuberculosis.