5. materials and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/35967/10/10_chapter...

Materials and Methods

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5. MATERIALS AND METHODS

5.1 Materials Table 3. List of materials

S. No. Materials/Chemicals Manufacturer/Supplier

1 Artemether IPCA Laboratories, Mumbai, India

2 Curcumin Sami Laboratories, Bangalore, India

3 Simvastatin Ind Swift Pvt. Ltd, Chandigarh, India

4 Acetonitrile( HPLC grade) Sigma chemical Ltd., USA

5 0.5% Triethylamine Sigma chemical Ltd., USA

6 Ammonium acetate Rankem Ltd., Delhi, India

7 Isopropyl alcohol Rankem Ltd., Delhi, India

8 Capmul MCM Abitec, USA

9 Captex 8000 Abitec, USA



12 Glyceryl monostearate (Capmul GMS-50K) Abitec, USA

13 Plurol oleique CC 497 Gattefosse, Mumbai, India

14 Labrafac lipophile WL 1349 Gattefosse, Mumbai, India

15 Labrasol Gattefosse, Mumbai, India

16 Compritol 888 ATO (Glyceryl dibehenate) Gattefosse, Mumbai, India

17 Precirol ATO 5 (Glyceryle distearate) Gattefosse, Mumbai, India

18 Cremophor EL BASF, Mumbai, India

19 Medium chain triglyceride Lipoid GmbH, Ludwigshafen, Germany

20 Stearic acid S.D Fine chemicals Ltd. Mumbai, India

21 Tween 80 LR S.D Fine chemicals Ltd. Mumbai, India

22 Tween 20 LR S.D Fine chemicals Ltd., Mumbai, India

23 Ethanol S.D Fine chemicals Ltd., Mumbai, India

24 Potassium bromide (IR Grade) S.D Fine Chemicals Ltd., Mumbai, India

25 Lactose AR S.D Fine Chemicals Ltd., Mumbai, India

26 Sucrose AR S.D Fine Chemicals Ltd., Mumbai, India

27 Dextrose AR S.D Fine Chemicals Ltd., Mumbai, India

28 Orthophosphoric acid LR S.D Fine Chemicals Ltd., Mumbai, India

29 Sodium chloride AR S.D Fine Chemicals Ltd., Mumbai, India

30 Potassium chloride AR S.D Fine Chemicals Ltd., Mumbai, India

31 Polyethylene glycol 400 LR S.D Fine Chemicals Ltd., Mumbai, India

32 Polyethylene glycol 200 LR S.D Fine Chemicals Ltd., Mumbai, India

33 Calcium chloride AR Merck Pvt Ltd., Mumbai, India

34 Trimyristin Sigma Aldrich, Bangalore, India

35 Pluronic F68 (Polaxamer 188) Sigma Aldrich, Bangalore, India

36 Triton X 100 Sigma Aldrich, St Louis, USA

37 Dialysis bag (MWCO-12,000-14,000 g/mL) Himedia labs, Mumbai, India

38 Hydrochloric acid Fischer Chemicals Ltd., Mumbai, India

39 Dimethyl sulfoxide Qualigens, Fine Chemicals, Mumbai, India

40 Ethylene diamine tetraacetic acid Loba Chemie Pvt Ltd., Mumbai, India

41 Sodium lauryl sulphate Loba Chemie Pvt Ltd., Mumbai, India

42 Monosodium phosphate anhydrous Loba Chemie Pvt Ltd., Mumbai, India

43 Disodium phosphate anhydrous Loba Chemie Pvt Ltd., Mumbai, India


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5.2 Equipments Table 4. List of equipments

5.3 Selection of drugs

The present parenteral therapy for management of CM consists of either quinine,

quinidine, ARM or ARS. Among these drugs, ARM was selected as a potent lipophilic

anti-malarial drug. Based on the literature survey, CUR was selected as an

immunomodulatory and anti-malarial agent for an adjunctive therapy. ARM is rapidly

acting drug whereas CUR has been found to exert sustained ant-malarial action in

animal studies. This is a prerequisite for the selection of drugs for an adjunctive therapy.

S. No. Equipment Manufacturer/Supplier

1 UV/Visible spectrophotometer UV-1700 series, Shimadzu, Japan

2 High performance liquid chromatography Shimadzu LC 2010A HT, Japan

3 Fourier transform infrared spectrophotometer Shimadzu, Japan

4 Differential Scanning Calorimeter DSC Q 200 TA Instruments, USA

5 Powder X-Ray Diffractometer Bruker AXS D8, USA

6 Zetasizer Malvern ZS 90, UK

7 Transmission electron microscope Topcon 002B, USA

8 Scanning electron microscope FEI Quanta 200F SEM/EDAX, UK

9 Franz diffusion cells Kovai Glass Works, Coimbatore, India

10 Dissolution apparatus Labindia, Mumbai, India

11 Deep freezer Labline Instruments, Kochi, India

12 Freeze dryer Christ alpha 2-4 LD plus, Germany

13 Brookfield DV II Ultra+Viscometer Brookfield Engineering Laboratories, Inc., USA

14 Electroconductometer (Conductivity meter 305) Systronic, Mumbai, India

15 Centrifuge Remi Instruments, Mumbai, India

16 Digital pH meter Eutech Instruments, Mumbai, India

17 Digital electronic balance Sartorius, Bangalore, India

18 Magnetic stirrer Remi Equipments, Mumbai

19 Blade stirrer with speed regulator Remi Instruments, Mumbai, India

20 Vortex mixer Yorco Instruments, Delhi, India

21 Sonicator Bandelin RK 100 H, Germany

22 Isothermal shaker IKA® KS 400I, Germany

23 Dissolution apparatus Electro lab, Mumbai, India

24 Water bath Scientec, Mumbai, India

25 Abbe’s Refractometer Bausch and Lomb Optical Company, NY, USA

26 Light microscope Olympus BX-51, Thornwood, USA

27 Laser scanning confocal microscope Carl Zeiss, NY, USA


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5.4 Drug Profile

5.4.1 Artemether

Source and chemical nature: It is a methyl ether derivative of artemisinin isolated from

the Chinese anti-malarial plant, Artemisia annua; family: Asteraceae. Structurally, it is a

sesquiterpene lactone with an inner peroxide bridge which is responsible for its anti-

malarial activity. It is also known as DHA methyl ether and is relatively lipophilic drug.

Physico-chemical Properties

Molecular formula: C16H26O5

Molecular weight: 298.37g/mol

Melting point: 86-90°C

Occurrence: white crystalline powder

Log P: 3.02-3.48

pKa (strongest basic): -3.9

Solubility: ARM is practically insoluble in water; very soluble in dichloromethane,

dimethylsulfoxide and dimethyl formamide; freely soluble in ethyl acetate, methanol and

ethanol.

Pharmacokinetics: Although ARM is a potent antimalarial, poor bioavailability and

rapid clearance are observed with it in both human and animal models due to extensive

first pass metabolism. The absorption of ARM is increased when taken with food. In

healthy, normal volunteers, orally administered ARM is metabolized in the liver to its

active metabolite, DHA by cytochrome P450 enzymes (3A4, 3A5, 2C19 and 2B6) having

half life of ~1 h.

Half life: 3-5h

Protein binding: 95.4%

Bioavailability: low oral bioavailability (~40%)

Pharmacological properties: It possesses anti-malarial, anti-protozoal, anti-helminthic

and anti-cancer activities.

Toxicity: Animal studies on acute toxicity show that the LD50 of ARM in mice is

300mg/kg/day for oral and 50mg/kg/day for IM administration; in rats, the LD50 is a

single IM injection of 597mg/kg dose.

Stability: Due to the effect of endoperoxide linkage, ARM is quite unstable under heat

and it easily decomposes, most probably by the opening of the lactone ring. In addition,

it undergoes degradation in acidic conditions and is sensitive to light.


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5.4.2 Curcumin

Source and chemical nature: It is a hydrophobic polyphenol isolated from the rhizomes

of turmeric (Curcuma longa L.) and related species (family: Zingiberaceae), has been used

traditionally as an Indian spice. It is a bis-α, β-unsaturated β-diketone.

Physico-chemical Properties

Molecular formula: C21H20O6

Molecular weight: 368.38g/mol

Melting point: 179-183°C

Occurrence: bright yellow crystalline solid

Log P: 2.85

pKa: 7.8, 8.5 and 9.0

Solubility: Sparingly soluble in water and ether; readily soluble in dimethylsulfoxide,

dimethyl formamide, glacial acetic acid, ethanol or acetone. The solubility of drug in

these solvents is ~1mg/mL, and in acetone it is ~20mg/mL.

Pharmacokinetics: CUR shows low systemic bioavailability after oral dosing, probably

due to rapid first-pass metabolism and some degree of intestinal pre-metabolism. Its

metabolism on the one hand involves, successive reductions, which transform CUR to

hexahydrocurcuminol and hexahydrocurcumin (probably through the intermediates

dihydrocurcumin and tetrahydrocurcumin), and, on the other hand, rapid molecular

modification by conjugation, mostly in the liver, to glucuronide, sulfate and glucuronide-

sulfate forms. Moreover, when given orally, 40% of the drug is excreted unchanged in the

feces. It also undergoes extensive enterohepatic recirculation, resulting in its rapid

elimination in bile and urine.

Half life: 1.39h

Protein binding: 60%

Bioavailability: low oral bioavailability (<0.1%)

Pharmacological properties: A large number of in vitro and in vivo studies in both

animals and humans have indicated that CUR exhibits promising pharmacological

activities including anti-oxidant, anti-inflammatory, anti-angiogenic, anti-spasmodic,

anti-microbial and anti-plasmodial activities.

Toxicity: Even at 8g/day, no toxicity is reported from humans and animals.

Stability: Under physiological conditions, CUR can exist in both an enol and a bis-keto

form, which coexist in equilibrium. In acidic and neutral solutions as well as in the solid

state, keto form predominates, and CUR acts as a potent donor of H-atoms. In contrast,


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under alkaline conditions (≥pH 8), the enolic form predominates, and the phenolic part

of the molecule plays the principal role as an electron donor. In solution, it has been

demonstrated that 90% of CUR degrades to trans-6-(4’-hydroxy-3’-methoxyphenyl)-2,4-

dioxo-5-hexanal,vanillin, feruloylmethane, and ferulic acid within 30min. CUR is

similarly unstable at basic pH. Yellow CUR changes to dark red colour at alkaline pH

and under physiological conditions the λ max for CUR is observed at 420nm. Aqueous

solution of CUR should not be stored for more than 12h. It should be kept in a tightly

closed container, protected from light and stored in a cool place.

5.5 Analytical method development

Qualitative and quantitative analysis of ARM and CUR were developed using reverse

phase-high performance liquid chromatography (RP-HPLC).

5.5.1 Selection of detection wavelength

100µg/mL concentration (conc.) of ARM and CUR solutions was prepared separately in

acetonitrile (ACN). The solutions were scanned in the UV-Visible region of 200-800nm

and the spectrum was recorded using photodiode array (PDA).

5.5.2 Preparation of standard stock solutions

10mg of ARM and CUR working standards were accurately weighed and transferred

into a 10mL volumetric flask separately and dissolved in ACN and made up to the

volume with the same solvent to produce 1mg/mL (1000µg/mL) of ARM and CUR

stock solutions, respectively. Stock solutions were further diluted to 100µg/mL by taking

10mL of respective drug solution (1mg/mL) and diluting upto 100mL in volumetric

flask with ACN. These solutions were then stored in the refrigerator at 5oC±3oC until

further analysis.

5.5.3 Linearity and range of artemether

Linearity and range were analyzed by preparing calibration curves using different conc.

of the standard solution. Calibration curve was plotted using mean peak area (x) versus

the respective conc. (y) of standard drug solutions. Linearity for ARM was established

over the range of 2-25µg/mL using the weighted least square regression analysis. From

the stock solution (100µg/mL), aliquots of 0.2, 0.4, 0.5, 1.0, 1.5, 2.0 and 2.5mL were

pipetted out in 10mL volumetric flasks and made up to the volume with ACN to obtain

the conc. of 2-25µg/mL and analyzed at 209nm by RP-HPLC. Calibration curve data

was subjected to linear regression analysis to obtain intercept, slope and regression

equation. All measurements were made in triplicate and mean±S.D. was recorded.


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5.5.4 Linearity and range of curcumin

Linearity and range were analyzed by preparing calibration curves using different conc.

of the standard solution. Calibration curve was plotted using mean peak area (x) versus

the respective conc. (y) of standard drug solutions. Linearity for CUR was established

over the range of 1-50µg/mL using the weighted least square regression analysis. From

the stock solution (100µg/mL), aliquots of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mL were

pipetted out in 10mL volumetric flasks and made up to the volume with ACN to obtain

the conc. of 1-50µg/mL and analyzed at 418nm by RP-HPLC. Calibration curve data

was subjected to linear regression analysis to obtain intercept, slope and regression

equation. All measurements were made in triplicate and mean±S.D. was recorded.

5.5.5 Mobile phase preparation

A weighed quantity (0.9635gm) of ammonium acetate was dissolved in 500mL of milli Q

water and pH was adjusted to 3.0 with ortho-phosphoric acid. The resultant buffer

solution was filtered through 0.45μm nylon membrane filter and degassed. Mobile phase

used was ACN and 25mM ammonium acetate buffer (pH 3.0) in ratio of 70:30 v/v.

5.5.6 Optimized Chromatographic Conditions of artemether and curcumin

5.5.7 Analytical method validation

The RP-HPLC method was validated according to the ICH guidelines, Q2 (R1)

(ICH, 2005). Validation of optimized HPLC method was done with respect to the

following parameters:

Stationary phase Hibar C18 (250 x 4.6mm i.d., 5m)

Mobile Phase ACN: Ammonium acetate buffer

Mobile phase ratio 70:30

Flow rate 1.0mL/min

Sample volume 20ml using Rheodyne 7725i injector

Detection λ 209nm (ARM) and 418nm (CUR)

pH 3.0

Buffer strength 25mM

Data station LC-20AD (PDA)

Retention time 10.7±0.1min (ARM) and 4.3±0.1min (CUR)

Run time 12.0 min (ARM) and 5.0min (CUR)


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Specificity

Specificity is the ability of a method to discriminate between the intended analyte and

other components in the sample. A method is said to be specific when it produces a

response only for a single analyte in the presence of other interferences. The specificity of

the method is to analyze unequivocally the analyte in the presence of other endogenous

compounds (degradants, excipients, impurities). It was carried out by comparing the

standard retention time spectra and the sample retention time spectra.

Accuracy

Accuracy is expressed as the closeness of agreement of trueness. It was determined by

standard addition method. For this purpose, known quantities of ARM and CUR were

supplemented to the sample solution previously analyzed. The results of this solution

were compared with the true results. This experiment was carried out by analyzing

replicates (n=6) at 3 quality control (QC) levels. The mean, standard deviation (S.D.) and

percentage relative standard deviation (% R.S.D.) was calculated. Accuracy was

calculated by comparing the averaged measured conc. to the actual conc., and was

expressed in percentage nominal.

%Nominal = (Measured conc./Actual conc.) x 100

Precision

Precision was measured by interday (day to day precision, on 3 different days) and

intraday (repeatability on the same day) variations by analyzing 6 replicates over 3

different conc. of ARM (5.0, 10.0, 15.0µg/mL) and CUR (7.5, 15.0, 22.5µg/mL) at same

optimized chromatographic conditions. Precision was evaluated by calculating the

relative standard deviation (R.S.D.) of measured conc. at each sample based on linearity

plots. In all situations, R.S.D. values were <5%, which was considered to be acceptable.

%R.S.D. = (S.D./Mean) x 100

Limit of detection and quantitation

Limit of detection (LOD) and quantitation (LOQ) of the method were estimated by

injecting a series of dilute solutions with known conc. by visual observation and

signal-to-noise ratio.

LOD = 3.3 σ/s;

LOQ = 10 σ/s,

where, σ is the standard deviation of response and s is slope of the calibration curve


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Robustness

Robustness of the method was studied by injecting the standard solutions with slight

variations in the optimized conditions ±4% in the ratio of ACN in mobile phase, ±0.1

mL of the flow rate and ±0.1 in the pH value.

5.6 Bioanalytical method development

Bioanalytical methods were developed for estimation of ARM and CUR in rat plasma by

RP-HPLC.

5.6.1 Preparation of standard stock solutions

10mg of ARM and CUR working standards were accurately weighed and transferred

into a 10mL volumetric flask separately and dissolved in ACN and made up to the

volume with the same solvent to produce a 1mg/mL (1000µg/mL) of ARM and CUR

stock solutions, respectively. Stock solutions were further diluted to 100µg/mL by taking

10mL of respective drug solution (1mg/mL) and diluting upto 100mL in volumetric flask

with ACN. These solutions were then stored in the refrigerator at -200C ± 20C until

further analysis.

5.6.2 Linearity and range for artemether

Seven point calibration curve was prepared by serial dilution of ARM stock solution

(100µg/mL) in the range of 1-15µg/mL. The concentrations were corrected for potency

and amount weighed. Calibration standards were prepared daily by spiking 0.2mL of

blank plasma with 200µL of the appropriate working solution resulting in conc. of 1, 2, 4,

5, 10, 12 and 15µg of ARM per mL plasma. 200µL of CUR was added as an internal

standard (IS) at conc. of 50ng/mL. To the resulting solution, 200µL of precipitating

agent (10% perchloric acid) was added. The mixture was vortexed for 5min and

centrifuged at 4000rpm for 10min. The supernatant layer was separated and analyzed. A

plot with the resulting peak area ratios of ARM to IS (response factor) was obtained

against the conc. QC samples viz. low quality control (LQC) 2µg/mL, medium quality

control (MQC) 10µg/mL and high quality control (HQC) 15µg/mL were prepared by

spiking 200µL aliquot of blank plasma with 200µL of spiking solution of drug as well as

the IS. All solutions were stored in the refrigerator at 5.0±3.0oC. The bulk spiked

calibration and QC samples were stored at -20oC until further analysis.

5.6.3 Linearity and range of curcumin

Seven point calibration curve was prepared by serial dilution of CUR stock solution

(100µg/mL) in the range of 50-2000ng/mL. The concentrations were corrected for

potency and amount weighed. Calibration standards were prepared daily by spiking

0.2mL of blank plasma with 200µL of the appropriate working solution resulting in conc.


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of 50, 100, 125, 250, 500, 1000 and 2000ng of CUR per mL plasma. 200µL of simvastatin

was added as an internal standard (IS) at conc. of 500ng/mL. To the resulting solution,

200µL of precipitating agent (10% perchloric acid) was added. The mixture was vortexed

for 5min and centrifuged at 4000rpm for 10min. A plot with the resulting peak area ratios

of CUR to IS (response factor) was obtained against the conc. QC samples viz. LQC

75ng/mL, MQC 750ng/mL and HQC 1250ng/mL were prepared by spiking 200µL

aliquot of blank plasma with 200µL of spiking solution of drug as well as the IS. All

solutions were stored in the refrigerator at 5.0±3.0oC. The bulk spiked calibration and

QC samples were stored at -20oC until further analysis.

5.6.4 Preparation of blank plasma

Blank plasma (200µL) was transferred into 2.0mL centrifuge tube and 200µL of

precipitating agent (10% perchloric acid) were added. The resulting solution was

vortexed for 5min and centrifuged at 4000rpm for 10min. The supernatant layer was

separated and analyzed.

5.6.5 Mobile phase preparation

A weighed quantity (0.9635gm) of ammonium acetate was dissolved in 500mL of milli Q

water and the pH was adjusted to 3.0 with ortho-phosphoric acid. The resultant buffer

solution was filtered through 0.45μm nylon membrane filter and degassed. Mobile phase

used was ACN and 25mM ammonium acetate buffer (pH 3.0) in the ratio of 70:30 v/v.

5.6.6 Optimized Chromatographic Conditions for artemether and curcumin

Stationary phase Hibar C18 (250 x 4.6mm i.d., 5m)

Mobile Phase ACN: Ammonium acetate buffer

Mobile phase ratio 70:30

Flow rate 1.0mL/min

Sample volume 20ml using Rheodyne 7725 i injector

Detection λ 209nm (ARM) and 232nm (CUR)

Ph 3.0

Buffer strength 25mM

Data station LC-20AD (PDA)

Retention time of ARM and IS 14.43±0.1min and 4.88±0.1min

Retention time of CUR and IS 4.93±0.1min and 14.34±0.1min

Run time 16.0 min


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5.6.7 Bioanalytical method validation

RP-HPLC method was validated according to the ICH guidelines, Q2 (R1) (ICH, 2005).

Validation of optimized HPLC method was done with respect to following parameters:

Specificity

Specificity is the ability of a method to discriminate between the intended analyte and

other components in the sample. A method is said to be specific when it produces a

response only for a single analyte in the presence of other interferences. The specificity of

the method is to analyze unequivocally the analyte in the presence of other endogenous

compounds (degradants, excipients, impurities). It was carried out by comparing the

standard retention time spectra and the sample retention time spectra.

Accuracy

It was determined by analyzing the percentage recovery of ARM and CUR in plasma

samples. For this purpose, known quantities of ARM and CUR were supplemented to

the blank plasma samples. This experiment was carried out by analyzing replicates (n=6)

at three QC levels (LQC, MQC and HQC). The mean, S.D. and % R.S.D. were

calculated. Accuracy was calculated by comparing the averaged measured conc. to the

actual conc., and was expressed in percentage recovery.

% Recovery = (Measured conc./Actual conc.) x 100

Precision

Precision was measured by inter-day (day to day precision, on 3 different days) and

intra-day (repeatability on the same day) variations by analyzing six replicates over three

QC levels of ARM (2, 10, 15µg/mL) and CUR (75, 750, 1250ng/mL) at same optimized

chromatographic conditions. Precision was evaluated by calculating R.S.D. of measured

conc. at each sample based on linearity plots. In all situations, R.S.D. values were <5%,

which was considered to be acceptable.

%R.S.D. = (S.D./Mean) x 100

Limit of detection and quantitation

LOD and LOQ of the method were estimated by injecting a series of dilute solutions

with known conc. by visual observation and signal-to-noise ratio.

LOD = 3.3 σ/s

LOQ = 10 σ/s

where, σ is the standard deviation of response and s is slope of the calibration curve


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Stability in rat plasma

The stability of spiked samples of ARM and CUR was assessed at different storage

conditions viz. short-term (at room temperature over 4h), long-term (at -20oC for 30

days), and 2 freeze-thaw cycles by analyzing the replicates (n=6) at 3 QC levels.

5.7 Solubility studies

The solubility of drugs were determined in different oils, oil mixtures, surfactants and

co-surfactants separately by adding an excess amount of drugs (usually in increments of

1mg till the drug saturation was achieved) to 2mL of each excipients (Table 5) in 5mL

capacity stoppered vials, and mixed using a vortex mixer (Yorco Instruments, Delhi,

India) (Jain et al., 2013b). These vials were kept at 25±0.5oC in an isothermal shaker

(IKA® KS 4000i, Germany) for 72h to reach equilibrium. The equilibrated vials were

removed from the shaker and centrifuged at 4000rpm for 15min using centrifuge (Remi

Instruments, Mumbai, India). The supernatant was taken and filtered through a 0.45µ

membrane filter (Sartorius, Germany). The conc. of drugs was determined after suitable

dilution using HPLC (n=3).

Table 5. List of excipients used for solubility studies

Oils

Capmul MCM (MCM), Captex 8000 (CTX 8000), Captex 500 (CTX 500),

Captex 300 (CTX 300), Labrafac lipophile WL 1349 (Labrafac), Medium chain triglyceride (MCT)

Oil mixtures CTX 500+MCT(1:1), CTX 500+MCM(1:1), CTX 500+Labrafac (1:1)

Surfactants Cremophor EL (Crem EL), Labrasol, Tween 80 (T 80), Tween 20 (T 20)

Co-surfactants Polyethylene glycol 400 (PEG 400), Polyethylene glycol 200 (PEG 200), Ethanol, Plurol oleique CC 497 (PQ)

5.8 Partition coefficient studies

The partition coefficient (PC) of drugs was determined in various solid lipids viz. stearic

acid (SA), glyceryl monostearte (GMS), compritol 888 ATO (compritol), precirol ATO 5

(precirol) and trimyristin (TM) by isothermal shaker method as reported previously

(Sood et al., 2013). Ten mg of drug was dispersed in a blend of melted lipid (1g) and hot

phosphate buffer (PB) (1mL) pH 7.4, after which it was shaken for 30min over a hot

water bath shaker maintained at 70oC. Aqueous phase was then separated from lipid

after cooling by centrifugation at a speed of 10,000rpm for 20min. The clear supernatant

obtained was suitably diluted with ACN and drug content was estimated using HPLC

(n=3). PC was calculated as:

PC = (Ci - C)/C

where, Ci = initial amount of drug added (10mg)

C = conc. of drug in pH 7.4 PB


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5.9 Solubility and solution state stability studies

Saturation solubility of both the drugs was performed in simulated nasal fluid (SNF) pH

6.4 containing 1% w/v sodium lauryl sulphate (SLS) at 25±1.0oC in isothermal shaker.

SNF consisted of monosodium phosphate anhydrous (447mg), disodium phosphate

anhydrous (210mg), sodium chloride (4.4g), potassium chloride (1.5g) and calcium

chloride dehydrate (368mg) dissolved and made upto 500mL with distilled water having

final pH of 6.4. The drug was added in increments of 1mg till the saturation was

achieved. Drug solubility was determined by HPLC at the end of 72h (n=3). For stability

studies, standard solutions of drug (5-25µg/mL) were prepared in SNF pH 6.4 containing

1% w/v SLS and stored at 37±0.5oC for 72h. The samples were assayed for drug content

using HPLC method at 0, 24, 48 and 72h (n=3).

5.10 Compatibility studies

Compatibility of drug and lipid was studied using Fourier transform infrared

spectroscopy (FTIR) and differential scanning calorimetry (DSC).

5.10.1 Procedure for FTIR

A physical mixture of drug and lipid (either alone or in combination) was prepared and

mixed with anhydrous potassium bromide (KBr) in 1:4 ratio. About 100mg of this

mixture was ground into fine powder using mortar and pestle followed by compression to

form a transparent KBr pellet using a Beckmann hydraulic press (Beckman Instruments

Inc., Fullerton, USA) set at 15 tons pressure. Each KBr pellet was scanned at 4mm/s at a

resolution of 2cm over a wave number region from 4000 to 400 cm-1 in a FTIR

spectrophotometer (Shimadzu, Japan). The FTIR spectrum of the physical mixture (1:1)

was compared with those of pure drug and lipid and infra red peak matching method was

done to detect any appearance or disappearance of peaks.

5.10.2 Procedure for DSC

DSC analysis was performed using DSC Q200 (TA Instruments, USA). The instrument

was calibrated for temperature and heat flow using high purity indium standard. Briefly,

about 5mg of samples were accurately weighed in non-hermetically sealed aluminum

pans and crimped. DSC thermograms covered the range from 20-100oC and 20-200oC for

ARM and CUR respectively, at a heating rate of 10oC/min under constant purging of

nitrogen at a flow rate of 50mL/min. An empty pan, sealed in the same way as that of

the sample, was used as a reference. DSC thermograms were analyzed for pure drug,

lipid and its physical mixture (1:1) using TA universal analysis software.


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5.11 Purity analysis

The purity of ARM and CUR was analysed using DSC as per procedure described

earlier. The thermograms of the drugs were subjected to purity analysis using Vant Hoff’s

equation that measures the fraction melted as a function of temperature.

where, Ts = Sample temperature

To = Theoretical melting point for 100% pure crystalline compound

R = Gas constant

X = Total mole fraction impurity

F = Fraction melted at Ts

5.12 Formulation of NLCs

Drug (ARM and CUR) loaded NLC formulations were prepared by a microemulsion

method as reported earlier (Sood et al., 2013). The chosen solid lipid and liquid lipid

were melted at 70oC, to which drug was added under continuous stirring for 5min. Ten

mL of hydrophilic surfactant solution heated at same temperature was added to the

melted lipid as a continuous phase, with mechanical stirring for 15min. A clear warm

o/w ME was formed under stirring at a temperature above the melting point of the lipid

used. NLC dispersions were obtained by dispersing the warm o/w ME dropwise into an

ice cold distilled water (3-4oC) in a beaker under continuous stirring (triple blade stirrer)

for 3h at a ratio of 1:5 (ME:water, v/v). The dispersion was centrifuged at 10,000rpm for

20min and supernatant was discarded. The resulting NLC pellets were redispersed using

millipore water and centrifuged again. The procedure was repeated twice to remove both

free surfactant and free drug molecules and the resultant dispersion was finally

lyophilized. In a similar manner, blank NLC formulation was also prepared without

incorporating the drug to lipid matrix. Preparation of NLC using the aforementioned

method is illustrated in Fig.7.

F

1 )

H

T X R( - T T

2o

os

fD=


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Fig.7. Representation of preparation of NLC using microemulsion technique

5.12.1 Study on the effect of formulation/ process variables

The effect of formulation/process variables such as stirring time, stirring speed, lipid

conc., type of surfactant, conc. of surfactant and ratio of internal phase to external phase

on the particle size (PS) and polydispersity index (PDI), several batches of blank lipid

nanoparticles (LNP) consisting of only solid lipid were prepared and evaluated. To

investigate the effect of formulation/process variables, each time one parameter was

varied, keeping the others as constant. From the results obtained, optimum level of

variables were selected and kept constant in the subsequent evaluations.

Effect of stirring time

Four different batches of LNP were prepared corresponding to 1, 2, 3 and 4h stirring

time, keeping the following parameters as constant,

Lipid conc. : 1% w/v

Stirring speed : 2000rpm

Surfactant conc. (Pluronic F 68) : 2% w/v

Internal:External phase ratio : 1:10

Effect of stirring speed

Four different batches of LNP were prepared corresponding to 1000, 1500, 2000 and

2500rpm stirring speed, keeping the following parameters as constant,

Lipid conc. : 1% w/v

Stirring time : 3h




61 | P a g e

Effect of lipid concentration

Four different batches of LNP were prepared corresponding to 0.5%, 1%, 1.5% and 2%

w/v lipid conc., keeping the following parameters as constant,


Stirring time : 3h



Effect of surfactant concentration

Four different batches of LNP were prepared corresponding to 1%, 2%, 3% and 4% w/v

surfactant conc., keeping the following parameters as constant,


Stirring time : 3h

Lipid conc. : 1.5% w/v


Effect of surfactant type

Four different batches of LNP were prepared using different type of surfactants viz. T 20,

T 80, Crem EL and Pluronic F68, keeping the following parameters as constant,

Stirring time : 3h


Lipid conc. : 1.5% w/v

Surfactant conc. : 3% w/v


Effect of ratio of internal phase to external aqueous phase

Four different batches of LNP were prepared and corresponding to different ratios of

internal to external aqueous phase (1:1, 1:5, 1:10, 1:20), keeping the following parameters

as constant,


Stirring time : 3h

Lipid conc. : 1.5 % w/v

Surfactant conc. : 3 % w/v


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5.12.2 Optimization of NLCs by central composite design

Based on number of factors and their level, CCD was used for formulation optimization

of ARM-NLC and CUR-NLC. CCD is one of the techniques of RSM for optimization of

pharmaceutical dosage forms and its rotatable characteristic enables it to identify

optimum responses around its center point without changing the predicting variance

(Zhang et al., 2010). The effect of 4 independent variables viz. conc. of lipid, liquid lipid

to total lipid ratio, drug to lipid ratio and surfactant conc. on dependent variable viz. PS,

drug loading (DL) and entrapment efficiency (EE) were studied at 5 different levels,

coded as –α, -1, 0, 1 and +α. The value for alpha (2) was intended to fulfil the rotatability

in the design. The coded and uncoded independent variables for both ARM-NLC and

CUR-NLC are given in Table 6. A total of 30 experiments with 6 centre points for

statistical assessment of the pure error sum of squares were carried out using Design

expert® software (Version 8.0.7.1, M/s Stat-Ease, Minneapolis, USA).

Table 6. Variables for central composite design

Independent factors Levels

Coded Uncoded -2 -1 0 1 2

A Lipid Conc. (%) 0.3 0.6 0.9 1.2 1.5

B Liquid lipid to total lipid ratio 0.05 0.13 0.20 0.28 0.35

C Drug to lipid ratio 0.05 0.08 0.10 0.13 0.15

D Surfactant Conc. (%) 1.0 1.5 2.0 2.5 3.0

5.12.3 Optimization of cryoprotectant concentration

Lyophilization of the NLC dispersions was carried out by using 2%, 3% and 5% (w/v) of

sucrose, lactose and dextrose as cryoprotectants. The NLC dispersions (10mL) were

frozen in aqueous cryoprotectant solution at -20oC for about 24h and then the samples

were transferred to the freeze-dryer (Christ, Alpha 2-4 LD plus, Germany) operated at -

40oC and pressure of 0.001bar for 72h to obtained the finely dispersed NLC powders for

further experiments. Lyophilized NLC were characterized for redispersibility, mean PS

and mean PDI by dynamic light scattering experiment before and after freeze-drying

procedure. The measurements were used to determine the Sf/Si ratio of lyophilized

NLC, where Sf and Si indicates mean PS after and before freeze-drying respectively. To

assess the redispersibility of lyophilized NLC, 50mg of product was redispersed in 1mL

of millipore water and visually assessed using following grading system:

Grade A: Readily redispersible (<15 sec, clear solution)

Grade B: Moderately redispersible (>15 sec, clear to translucent solution)

Grade C: Poorly redispersible ( does not redisperse, presence of large particles)


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5.13 Formulation of NEs

5.13.1 Construction of pseudoternary phase diagrams

NEs were prepared using spontaneous nano-emulsification method and phase behaviour

was studied using pseudoternary phase diagrams. The phase diagrams containing oil,

surfactant, co-surfactant and double distilled water were developed using the aqueous

titration method (Fig. 8). (Shafiq et al., 2007). The selected oil phase was heated gently at

45-50oC for 5min. Surfactant and co-surfactant (smix) were mixed together in different

volume ratios (1:1, 1:2, 2:1, 3:1) and heated at same temperature. These smix ratios were

chosen to reflect the increasing conc. of co-surfactant with respect to surfactant and

increasing conc. of surfactant with respect to co-surfactant for the detailed study of the

phase diagrams for the formulation of NE. Mixture of oil and smix were prepared in

different volume ratios (1:1, 1:2, 1:3, 1:4, 2:1) in screw-cap glass tubes and were vortexed

(Yorco Instruments, Delhi) to form homogenous isotropic mixtures. Each mixture were

then slowly titrated with aqueous phase (double distilled water) and stirred at room

temperature to attain equilibrium. Aqueous phase was added with increment of 5µL

using micropipette at each interval to each oil-smix mixture under vortex mixing. The

calculation for the addition of aqueous phase was done by calculating the percentage of

each component of the NE present at each 5µL addition. After equilibrium, the samples

were visually observed for the following categories:

Fig.8. (a) Preparation of NE by aqueous titration method (b) Visual observation of clear

nanoemulsion (NE) and turbid/milky emulsion (E)

Transparent and easily flowable : o/w nanoemulsion (NE)

Milky or cloudy/phase separation : Emulsion (E)

Milky gel : Emulsion gel (EG)

Transparent gel : Nanoemulsion gel (NG)


64 | P a g e

Phase diagrams were constructed using Chemix Software Ver.3.50 (MN, USA). Oil,

surfactants and co-surfactant were grouped in 2 different combinations for phase studies

(Table 7). The percentage calculation for different oil: smix ratios are given in Table 8-12.

Physical state was plotted on a pseudo-three-component phase diagram with 3 axis

representing aqueous, oil and smix at a fixed volume ratio. For each smix ratio, a

separate phase diagram was constructed and only NE points were plotted (shaded area),

as for formulation development, only the NE area is of interest. Clear transparent and

isotropic samples were deemed to be within the NE region. From each phase diagram

constructed, optimized range of oil, surfactant and co-surfactant concentrations were

selected from the NE region to prepare the NE drug delivery system using DoE.

Table 7. Excipients grouped in different combinations

Group Oil Surfactant Co-surfactant

I MCM+CTX 500 Crem EL PEG 400

II MCM+CTX 500 T 20+ Crem EL PEG 400

Table 8. Percentage calculation of oil, smix and water for phase diagram (oil and smix ratio 1:1)

Oil (µL) Smix (µL) Water

(µL)

Water added at each interval

(µL) Total volume (µL) Oil (%) Smix (%) Water (%)

10 10 5 0 25 40.00 40.00 20.00

10 10 10 5 30 33.33 33.33 33.33

10 10 15 5 35 28.57 28.57 42.85

10 10 20 5 40 25.00 25.00 50.00

10 10 25 5 45 22.22 22.22 55.55

10 10 30 5 50 20.00 20.00 60.00

10 10 35 5 55 18.18 18.18 63.63

10 10 40 5 60 16.66 16.66 66.66

10 10 45 5 65 15.38 15.38 69.23

10 10 50 5 70 14.28 14.28 71.42

10 10 55 5 75 13.33 13.33 73.33

10 10 60 5 80 12.50 12.50 75.00

10 10 65 5 85 11.76 11.76 76.47

10 10 70 5 90 11.11 11.11 77.77

10 10 75 5 95 10.52 10.52 78.94

10 10 80 5 100 10.00 10.00 80.00

10 10 85 5 105 9.52 9.52 80.95

10 10 90 5 110 9.09 9.09 81.81

10 10 95 5 115 8.69 8.69 82.60



(µL)



10 20 5 0 35 28.57 57.14 14.28

10 20 10 5 40 25.00 50.00 25.00

10 20 15 5 45 22.22 44.44 33.33

10 20 20 5 50 20.00 40.00 40.00

10 20 25 5 55 18.18 36.36 45.45

10 20 30 5 60 16.66 33.33 50.00

10 20 35 5 65 15.38 30.76 53.84

10 20 40 5 70 14.28 28.57 57.14

10 20 45 5 75 13.33 26.66 60.00

10 20 50 5 80 12.50 25.00 62.50

10 20 55 5 85 11.76 23.52 64.70

10 20 60 5 90 11.11 22.22 66.66

10 20 65 5 95 10.52 21.05 68.42

10 20 70 5 100 10.00 20.00 70.00

10 20 75 5 105 9.52 19.04 71.42

10 20 80 5 110 9.09 18.18 72.72

10 20 85 5 115 8.69 17.39 73.91

10 20 90 5 120 8.33 16.66 75.00

10 20 95 5 125 8.00 16.00 76.00


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(µL)



10 30 5 0 45 22.22 66.66 11.11

10 30 10 5 50 20.00 60.00 20.00

10 30 15 5 55 18.18 54.54 27.27

10 30 20 5 60 16.66 50.00 33.33

10 30 25 5 65 15.38 46.15 38.46

10 30 30 5 70 14.28 42.85 42.85

10 30 35 5 75 13.33 40.00 46.66

10 30 40 5 80 12.50 37.50 50.00

10 30 45 5 85 11.76 35.29 52.94

10 30 50 5 90 11.11 33.33 55.55

10 30 55 5 95 10.52 31.57 57.89

10 30 60 5 100 10.00 30.00 60.00

10 30 65 5 105 9.52 28.57 61.90

10 30 70 5 110 9.09 27.27 63.63

10 30 75 5 115 8.69 26.08 65.21

10 30 80 5 120 8.33 25.00 66.66

10 30 85 5 125 8.00 24.00 68.00

10 30 90 5 130 7.69 23.07 69.23

10 30 95 5 135 7.40 22.22 70.37



(µL)



10 40 5 0 55 18.18 72.72 9.09

10 40 10 5 60 16.66 66.66 16.66

10 40 15 5 65 15.38 61.53 23.07

10 40 20 5 70 14.28 57.14 28.57

10 40 25 5 75 13.33 53.33 33.33

10 40 30 5 80 12.50 50.00 37.50

10 40 35 5 85 11.76 47.05 41.17

10 40 40 5 90 11.11 44.44 44.44

10 40 45 5 95 10.52 42.10 47.36

10 40 50 5 100 10.00 40.00 50.00

10 40 55 5 105 9.52 38.09 52.38

10 40 60 5 110 9.09 36.36 54.54

10 40 65 5 115 8.69 34.78 56.52

10 40 70 5 120 8.33 33.33 58.33

10 40 75 5 125 8.00 32.00 60.00

10 40 80 5 130 7.69 30.76 61.53

10 40 85 5 135 7.40 29.62 62.96

10 40 90 5 140 7.14 28.57 64.28

10 40 95 5 145 6.89 27.58 65.51



(µL)



20 10 5 0 35 57.14 28.57 14.28

20 10 10 5 40 50.00 25.00 25.00

20 10 15 5 45 44.44 22.22 33.33

20 10 20 5 50 40.00 20.00 40.00

20 10 25 5 55 36.36 18.18 45.45

20 10 30 5 60 33.33 16.66 50.00

20 10 35 5 65 30.76 15.38 53.84

20 10 40 5 70 28.57 14.28 57.14

20 10 45 5 75 26.66 13.33 60.00

20 10 50 5 80 25.00 12.50 62.50

20 10 55 5 85 23.52 11.76 64.70

20 10 60 5 90 22.22 11.11 66.66

20 10 65 5 95 21.05 10.52 68.42

20 10 70 5 100 20.00 10.00 70.00

20 10 75 5 105 19.04 9.52 71.42

20 10 80 5 110 18.18 9.09 72.72

20 10 85 5 115 17.39 8.69 73.91

20 10 90 5 120 16.66 8.33 75.00

20 10 95 5 125 16.00 8.00 76.00


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5.13.2 Optimization of NEs using Box-Behnken design

Based on initial screening, conc. of oil, surfactant and co-surfactant were optimized from

the NE region of pseudoternary phase diagrams. BBD was used statistically to optimize

the effect of 3 independent variables namely conc. of oil (%), surfactant (%) and

co-surfactant (%) and evaluate the main effects, interaction effects and quadratic effects

of these formulation ingredients on the dependent variables namely globule size (GS)

(nm) and zeta potential (ZP) (mV). Design-Expert® software (Version 8.0.7.1, M/s Stat-

Ease, Minneapolis, USA) was used to conduct the study. A total of 17 experiments with

5 centre points were designed by the software (in order to allow the estimation of pure

error) and experiments were run in random order. Table 13 shows the coded and

uncoded independent variables.

Table 13. Variables for Box-Behnken design

Independent factors Design level

Coded Uncoded -1 0 +1

A Conc. of Oil (%) 12.00 16.00 20.00

B Conc. of surfactant (%) 24.00 29.50 35.00

C Conc. of co-surfactant (%) 12.00 16.00 20.00

5.14 Evaluation

5.14.1 Particle size and zeta potential analysis

The mean PS/GS and ZP of NLCs and NEs were determined using a zetasizer ZS 90

(Malvern Instruments, UK). The mean PS/GS was measured based on photon

correlation spectroscopy technique that analyzes the fluctuations in dynamic light

scattering due to brownian motion of the particles. The mean diameter was obtained at

an angle of 90° in 10 mm diameter cells at 25°C. The ZP, reflecting the electric charge on

the particle surface, is a very useful way of evaluating the physical stability of any

colloidal system. It was determined based on an electrophoretic light scattering technique

(Jain et al., 2013a). All PS/GS and ZP measurements were carried out at 25oC using

disposable polystyrene cells and disposable plain folded capillary zeta cells, respectively,

after appropriate dilution of all samples with original dispersion medium. Three replicate

analyses was performed for each formulation, and data presented as mean±S.D.

5.14.2 Determination of entrapment efficiency and drug loading percentage

EE and DL percentage of lyophilized NLC were determined according to the procedure

described earlier (Sood et al., 2013). Weighed quantity of lyophilized drug loaded NLC

(10mg) were suspended in hydroalcholic solution (ethanol and water in 50:50) under

water bath 70oC for 30min. This ensures melting of NLC and release of entrapped drug


67 | P a g e

in media. The solution is allowed to cool at room temperature to preferentially

precipitate the lipid. The amount of drug in the supernatant after centrifugation

(10,000rpm for 30min) was determined by HPLC (n=3).

Drug entrapment efficiency (%) = Wdrug/ Wtotal x 100

Drug loading (%) = Wdrug/ Wlipid x 100

Wdrug; analyzed amount of drug in the supernatant,

Wtotal; total amount of drug used in formulation,

Wlipid; weight of lyophilized NLC formulation

5.14.3 Scanning electron microscopy

External surface morphology of lyophilized drug loaded NLC was recorded using

scanning electron microscopy (SEM) (FEI QUANTA 200 SEM/EDAX, UK) at 20kV as

an accelerating voltage (Sood et al., 2013). Weighed amount of samples (5-7mg) were

mounted on an aluminium stub with double sided adhesive tape. The tape was firmly

attached to the stub and lyophilized sample was scattered carefully over its surface. The

stub with the sample was then sputter coated with a thin layer of gold to make the sample

conductive. Processed sample was subjected to SEM analysis. The images were captured

under magnification of 10,000-15,000x and recorded.

5.14.4 Transmission electron microscopy

The shape and morphology of drug loaded NLC dispersion and drug loaded NE were

analyzed using transmission electron microscopy (TEM) (TOPCON 002B, USA) at an

accelerating voltage of 200kV (Jain et al., 2013b). Prior to the analysis, the samples were

diluted 100 times with double distilled water and a drop (5-10µL) was placed onto

carbon-coated 200-mesh copper grids to create a thin film. Before the film dried on the

grid, the samples were negatively stained with 2% w/v phosphotungstic acid by adding a

drop of the staining solution to the film for 30s; any excess droplets were drained off with

a filter paper. The grid was allowed to air-dry under room temperature. Digital

micrograph and soft imaging viewer software were used to capture the image of samples.

5.14.5 Differential Scanning Calorimetry

Crystalline behaviour of both drug and lipid was studied by DSC. Thermograms were

recorded for pure drug, lipid and lyophilized drug loaded NLC. DSC was carried out as

per the procedure described earlier. Degree of crystallinity of lyophilized drug loaded

NLC was calculated by comparing the enthalpy of NLC with enthalpy of bulk lipid

(Freitas and Müller, 1999). The melting enthalpy of bulk lipid was used as a reference

(100%) to calculate the percentage of crystallinity of NLC (Varshosaz et al., 2012).


68 | P a g e

where, ∆H freeze-dried NLC indicates enthalpy of freeze dried NLC

∆H bulk indicates enthalpy of bulk lipid.

5.14.6 Powder X-ray diffraction

Powder X-ray diffraction (PXRD) patterns were recorded for pure drug, lipid, physical

mixture (drug and lipid in 1:1) and lyophilized drug loaded NLC. PXRD pattern of

samples were collected using a Bruker AXS D8 Advance powder diffractometer, USA.

The samples were exposed to Cu Kα radiation generated at 40kV, 35mA and scanned

from 3o to 80o, 2θ at a step size of 0.020o and step time of 31.2s.

5.14.7 Fourier transform infrared spectroscopy

Solid state characterization of lyophilized drug loaded NLC formulation along with plain

drug and lipid were done using FTIR as per the procedure described earlier.

5.14.8 Thermodynamic stability tests

Optimized drug loaded NE formulation was subjected to different thermodynamic

stability tests (n=3) (Shafiq-un-nabi et al., 2007).

Centrifugation test: The samples were centrifuged at 3,500rpm for 30min.

Heating cooling cycle test: The samples were subjected to 6 cycles between refrigerator

temperature 4oC and 45oC with storage at each temperature for 48h.

Freeze thaw cycle test: The samples were subjected to 3 cycles between -21oC and +25oC

with storage at each temperature for 48h.

5.14.9 Drug content

The formulation was diluted to required conc. using ACN as solvent and drug content of

NE was estimated using HPLC method (Jain et al., 2013b). The drug content (n=3) was

calculated as:

Drug content (%) = Analyzed content/Theoretical content x 100

5.14.10 Refractive index and percent transmittance

The refractive index of the system was measured by an Abbe’s refractometer (Bausch and

Lomb optical company, NY, USA) by placing a drop of NE formulation onto the slide

(n=3) and it was compared to refractive index of distilled water. The percent

transmittance of the system was measured at 650nm using UV spectrophotometer

(Shimadzu, Japan) keeping distilled water as blank (n=3) (Ghosh et al., 2006).


69 | P a g e

5.14.11 Viscosity determination

The viscosity of the NE formulation (0.5g) was determined as such without dilution

using Brookfield DV-II ultra+viscometer (Brookfield Engineering Laboratories, Inc.,

Middleboro, MA, USA) with spindle # CPE 40 at 25±0.5ºC (n=3). The software used

for the calculations was Rheocalc V2.6. In the small volume adapter, the NE was filled

and the angular velocity was increased gradually from 10, 20, 50 and 100rpm. The

hierarchy of the angular velocity was reversed (Jain et al., 2013b).

5.14.12 Electroconductivity study

To determine the nature of the continuous phase and to detect the phase inversion

phenomenon, the electrical conductivity measurement of the NE formulation was carried

out by an electroconductometer (Conductivity meter 305, Systronic) by inserting the

probe in NE taken in a beaker (n=3) (Ghosh et al., 2006). The tested NE was prepared

with a 0.01N aqueous solution of sodium chloride (NaCl) instead of distilled water.

5.14.13 Determination of pH

The pH of the formulation was measured using digital pH meter (n=3) (Eutech

instruments, Mumbai).

5.14.14 In vitro Release Studies

The release of drug from developed formulations (NLC and NE) and solution was

performed in SNF pH 6.4 containing 1% SLS using the dialysis bag method (Sood et al.,

2013). For both the drugs, solution was prepared by dissolving 27.6mg/mL of ARM

(ARM-SOL) and 11.7mg/mL of CUR (CUR-SOL) in a mixture of 1mL ethanol and

2mL propylene glycol and finally volume was made to 10mL with distilled water

separately. Dialysis membrane having pore size of 2.4nm and molecular weight cut off

12,000-14,000 (Dialysis membrane-150, HiMedia, Mumbai, India) was used. The bags

were soaked in distilled water for 24h before use. Drug solution, lyophilized drug loaded

NLC and drug loaded NE were placed in dialysis bags separately and sealed at both the

ends. The bags were placed in baskets (USP Dissolution apparatus Type-I, Lab India,

Mumbai) and immersed in dissolution medium (SNF) maintained at 37±0.5oC and

stirred at 100rpm. The volume of dissolution medium for NLC and NE was 200mL and

500mL respectively. Aliquots of the samples were withdrawn from dissolution medium

at regular time intervals and same volume of fresh dissolution medium was replaced to

maintain a constant volume. The samples were analyzed for drug content by HPLC

(n=3). The drug release profile was constructed by plotting the cumulative percent drug

release versus time (h).


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5.14.15 Ex vivo Permeation Studies on Nasal Mucosa

To investigate the permeation efficacy of drug from lyophilized NLC, NE and solution

across the freshly excised sheep nasal mucosa, ex vivo permeation studies were performed

using the Franz diffusion cell with surface area of 1.79cm2 and volume of 25mL (Kovai

Glass Works, Coimbatore, India) (Seju et al., 2011). The freshly excised sheep nasal

mucosa was collected from the slaughter house in PBS, pH 6.4. Excised superior nasal

membrane was cut to an appropriate size and thickness (0.2mm), made free from

adhered tissues and mounted between the donor and receptor compartment of the Franz

diffusion cell, with mucosal side facing the donor compartment. The mounted tissue was

allowed to stabilize and stirred under SNF pH 6.4 containing 1% SLS for 15min on a

magnetic stirrer. The diffusion cell was thermostated at 37±0.5oC. Solution from both the

compartments was removed after 15min, and the receptor compartment was freshly filled

with SNF. The mounting of nasal membrane was done on the rim of the receptor

compartment; the donor compartment of diffusion cell was placed over it and secured

with a clamp to avoid the leakage of diffusion media. Permeation studies of pure drug

solution, lyophilized drug loaded NLC reconstituted with SNF and drug loaded NE were

carried out by placing 1mL onto stabilized sheep nasal membrane on donor

compartment and continuously magnetic stirred at 600rpm. Aliquot (0.5mL) of media

were withdrawn from the receptor compartment at predetermined time intervals, filtered

through 0.45µm nylon filter paper and analyzed for drug content using HPLC. Each

removed sample was replaced immediately by an equal volume of fresh diffusion media

maintained at 37±0.5oC to maintain the constant volume at each time interval. Each

study was carried out for a period of 6h, during which the amount of drug permeated

across the sheep nasal mucosal membrane was determined at each sampling point using

HPLC (n=3). The permeation profile was constructed by plotting the amount of drug

permeated per unit skin surface area (µg/cm2) versus time (h). The steady state flux (Jss,

µg /cm2.h) was calculated from slope of the plot using linear regression analysis.

5.14.16 Release Kinetics

In vitro dissolution has been recognized as an important element in drug development.

Under certain conditions, it can be used as a surrogate for the assessment of

bioequivalence. Several theories/kinetic models describe the drug dissolution from

immediate and modified release dosage forms. There are several models to represent the

drug dissolution profiles where ft is the function of t (time) related to the amount of drug

dissolved from the pharmaceutical dosage system. To compare dissolution profiles


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between two drug products, model dependent (curve fitting), statistic analysis and model

independent methods can be used (Costa and Lobo, 2001).

In order to elucidate mode and mechanism of drug release, the in vitro data was

transformed and interpreted at graphical interface constructed using various kinetic

models. Zero order release describes the drug dissolution of several types of modified

release pharmaceutical dosage forms, as in the case of transdermal systems, matrix

tablets with low soluble drugs, coated forms, osmotic systems etc., where the drug release

is independent of conc.

Qt = Qo + Kot

where, Qt is the amount of drug released in time t, Qo is the initial amount of the drug in

the solution and Ko is the zero order release constant

First order describes the release from system where release is conc. dependent e.g.

pharmaceutical dosage forms containing water soluble drugs in porous matrices.

log Qt = log Qo + K1 t /2.303

where Qt is the amount of drug released in time t, Q is the initial amount of drug in the

solution and K is the first s order release constant.

Higuchi described the release of drug from insoluble matrix as a square root of time

Qt = KH √t

where, Qt is the amount of drug released in time t, KH is Higuchi’s dissolution constant.

The following plots were made:

cumulative % drug release vs. time (zero order kinetic model);

log cumulative of % drug remaining vs. time (first order kinetic model) and

cumulative % drug release vs. square root of time (Higuchi model).

Mechanism of Drug release

Korsmeyer et al., (Korsmeyer et al., 1983) developed a simple, semi empirical model,

relating exponentially the drug release to the elapsed time (t)

ft = K tn

where K is a constant incorporating structural and geometrical characteristic of the drug

dosage form, n is the release exponent, ft is Mt / M∞ (fractional release of drug). Based on

‘n’ value, release can be Fickian, non-Fickian or zero order (Table 14). Depending on the

relative magnitude of the rate of polymer swelling to the rate of drug diffusion, various

release profiles may be possible. The situation where the polymer structural

rearrangement takes place rapidly in response to the swelling solvent as compared to the

rate of drug diffusion generally leads to Fickian diffusion, or the so-called first order

release, characterized by square root of time dependence in both the amount released

and the penetrating diffusion front position in slab geometry. In most systems, the


72 | P a g e

intermediate solution, which is often termed non-Fickian or anomalous diffusion, will

prevail whenever the rates of diffusion and polymer relaxation are comparable.

Table 14. Diffusional exponent and mechanism of diffusional release from various

non-swellable controlled release systems (Ritger and Peppas, 1987).

Diffusional exponent (n) Drug release mechanism

Thin film Cylindrical sample Spherical sample

0.50<n<1.0 0.45 0.43 Fickian diffusion

0.50<n<1.0 0.45<n<1.0 0.43<n<1.0 Anomalous

(Non-Fickian) transport

1.0 1.0 1.0 Zero-order release

5.15 Toxicity assessment

The toxicological assessment of the developed lipid based nano formulations (NLC and

NE) were carried out using in vitro cytotoxicity assay on SVG p12 cell line, in vitro

hemolytic toxicity on rat erythrocytes and nasal ciliotoxicity study on sheep nasal

mucosa stained with hematoxylin and eosin (H&E) stain to assess the safety of the

developed formulations to brain cells, blood and nasal epithelium respectively (n=3).

5.15.1 In vitro cytotoxicity assay on SVG p12 cell line

The toxicity studies of the blank and drug loaded formulations (NLC and NE) were

carried out in SVG p12 cells, a human brain cell line. The cells were maintained in

minimum essential medium (MEM), supplemented with 10% v/v fetal bovine serum,

penicillin (100 IU/mL), streptomycin (100μg/mL) and amphotericin B (5μg/mL) in a

humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were then seeded in

multiwall culture plates and cytotoxicity assay was carried out using cell suspension,

containing 5000 cells seeded in each well of a 96 well microtiter plate (Nunc and

Tarsons) and incubated for 24h at 37°C. Cells were treated with 250-2000μg/mL.

Control cells were incubated without the test compound and with MEM. The microtiter

plates were incubated at 37°C in a humidified incubator with 5% CO2 for a period of 72h.

Morphological changes in the cells were inspected daily and observed for microscopically

detectable alterations, i.e., loss of monolayer, granulation and vaculation in the

cytoplasm. The cytopathic effect was observed. A plot of % growth inhibition versus

conc. was plotted to determine the IC50 value (conc. of the drug that produces 50%

inhibition of the cells) by SRB (Sulphorhodamine B) assay (Skehan et al., 1990).

5.15.2 In vitro hemolytic toxicity

Fresh blood from rats was collected in a vial containing ethylene diamine tera acetic acid

(EDTA) as an anticoagulant. Blood was centrifuged at 3000rpm for 20min to remove


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white blood cells (WBC) debris and suspended RBCs were taken out. RBCs were washed

3 times with isotonic saline solution (0.15M NaCl and pH 7.4) before diluting with buffer

to prepare erythrocyte stock dispersion. RBC cell suspension was adjusted to 50%

hematocrit. Hemolysis experiments were carried out for both blank and drug loaded

formulations of NE and NLC at a conc. of 0, 5, 10, 25, 50, 100, 250 and 500μg/mL.

Incubation was carried at 37oC for a period of 1h. After incubation under shaking, debris

and intact RBCs was removed by centrifugation and 100µL of resulting supernatant was

dissolved in 2mL of an ethanol/hydrochloride and chloric acid (HCL) mixture (39:1,

99% ethanol and HCL, w/v). This mixture dissolves all components and avoids the

precipitation of hemoglobin. The absorbance of the mixture was determined at 398nm by

spectrophotometer monitoring against a blank sample. Control sample of 0% lysis (in

buffer) and 100% lysis (in Triton X 100) was employed in the haemolytic experiment

(Joshi et al., 2008). The % hemolysis caused by the test sample (n=3) was calculated by

following equation:

% Hemolysis = Absorbance of Test - Absorbance at 0% x 100

Absorbance at 100% lysis - Absorbance at 0%

5.15.3 Nasal ciliotoxicity studies on sheep nasal mucosa

Nasal ciliotoxicity studies were carried out using the freshly isolated sheep nasal mucosa

collected from a slaughter house in a phosphate buffered saline (PBS pH 6.4) (Seju et al.,

2011). Each piece was treated with drug solution in PBS pH 6.4, blank NLC, lyophilized

drug loaded NLC, blank NE, drug loaded NE, PBS pH 6.4 (as negative control) and

isopropyl alcohol (IPA) (nasal mucociliary toxicity agent used as a positive control),

respectively. After treatment for 2h, all the samples were washed properly with distilled

water and were preserved with 10% formalin until further analysis. Each sample was

sectioned and stained with H&E. The mucosa was then dissected out, and the mucocillia

was examined on an optical microscope by a pathologist.

5.16 Stability Studies

The optimized batch of lyophilized drug loaded NLC and drug loaded NE was evaluated

for stability for 6 months by storing them at 4±1oC and 25±1oC (n=3) (Hu et al., 2006).

Samples were transferred in amber coloured glass vials, sealed and were stored upright.

NLC formulations were rehydrated with millipore water and evaluated for any change in

PS (nm), ZP (mV), DL (%) and EE (%) at specified time intervals (0, 1, 3 and 6 months

of storage). Further, polymorphic transition of lipid in NLC formulations upon storage at

25±1oC was assessed using DSC thermograms and was recorded at 0 and 6 months of

storage. Similarly, NE formulations were withdrawn at specified time intervals (0, 1, 3

and 6 months of storage) and assessed for any change in GS (nm), ZP (mV), refractive


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index, drug content (%) viscosity (cP) and pH. The clarity and phase separation of the

NE formulations were determined by visual examination under light alternatively against

white and black backgrounds.

5.17 Pharmacokinetic and brain uptake studies

The animal experiments were carried out with the approval from institutional animal

ethical committee, J.S.S College of Pharmacy, Udhagamandalam, Tamil nadu, India

(Proposal number. JSSCP/IAEC/Ph.D/PH.Ceutics/04/2012-13). Pharmacokinetic and

brain uptake studies were carried out using male wistar rats as reported earlier for drug

solution and lyophilised drug loaded NLC formulation reconstituted with 1mL of

isotonic saline solution (Haque et al., 2012; Kumar et al., 2008). Rats were housed in

cages and had free access to standard laboratory diet (Lipton feed, Mumbai, India) and

water ad libitum. The animals were maintained at 22±1oC and 65±5% relative humidity

(R.H). Efficacy of IN route was compared with that of the IV route. Based on previously

published literature, the dose selected for ARM was 5mg/kg body weight (Aditya et al.,

2010) and 2.5mg/kg body weight for CUR (Chauhan et al., 2013). Grouping of animals

for ARM and CUR treated are shown in Table 15 and Table 16 respectively.

Table 15. Grouping of animals for pharmacokinetic studies for ARM formulation

Group Formulation Dose

I vehicle control

5 mg/kg

II ARM-NLC (IN) dispersed in isotonic saline solution

III ARM-SOL (IV into the tail vein)

IV ARM-SOL (IN)

Table 16. Grouping of animals for pharmacokinetic studies for CUR formulation

Group Formulation Dose

I vehicle control

2.5 mg/kg

II CUR-NLC (IN) dispersed in isotonic saline solution

III CUR-SOL (IV into the tail vein)

IV CUR-SOL (IN)

Before nasal administration, the rats were lightly anesthetized by exhaling diethyl ether.

IN administration was carried out with help of micropipette attached to low density

polyethylene tube having 0.1mm internal diameter. Animals (n=3) were sacrificed by

cervical dislocation and blood samples of ~0.25mL were collected by cardiac puncture at

the following time points: 0.25, 0.5, 0.75, 1, 2, 4 and 6h. Blood samples were placed into

tubes containing 0.3mL of EDTA and centrifuged at 3000rpm for 15min. After

centrifugation, the plasma obtained was stored at -20oC until analysis. Brain samples

were collected by cutting open the skull, rinsed with saline solution and blotted with filter


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paper to remove the blood taint. Brain samples were homogenized in PBS (pH 7.4) to

determine the amount of drug in the brain tissue. The homogenate was centrifuged at

6000rpm for 15min at 4oC; supernatant was collected and frozen at -20oC until analysis.

Pharmacokinetic parameters like elimination rate constant (Ke), half life (t1/2) and area

under curve (AUC), Cmax (peak plasma conc.) and Tmax (time of peak plasma conc.) were

obtained directly from the plasma conc.-time profile and brain conc.-time profile. All the

data were expressed as the mean±S.E.M. (standard error mean). Further, drug targeting

efficiency (DTE%) that represents time average partitioning ratio and nose to brain direct

transport percentage (DTP%) was calculated as follows:

DTE% = (AUCbrain/AUCblood)IN / (AUCbrain/AUCblood)IV × 100

DTP% = BIN – Bx/ BIN × 100

where Bx = (BIV / PIV) x PIN, Bx is the brain AUC fraction contributed by systemic circulation through the BBB

following IN administration;

BIV is the AUC0-360 (brain) following IV administration; PIV is the AUC0-360 (blood) following IV administration; BIN is the AUC0-360 (brain) following IN administration; PIN is the AUC0-360 (blood) following IN administration

5.18 Pharmacodynamic studies

5.18.1 Animals, Parasites and Infection

The animal experiments were carried out with approval from University of Hyderabad,

Hyderabad, India. Four to five weeks old C57BL/6 female mice weighing 23-25g were

housed at University of Hyderabad animal housing facility. Mice were kept in autoclaved

plastic cages and autoclaved wheat husk was used as bedding. The animals were

maintained at a temperature of 22±1oC and 65±5% R.H, were fed a standard mouse diet

and provided with clean drinking water ad libitum throughout the studies. P. berghei

ANKA (PbA) strain was used for in vivo evaluation of anti-malarial activity. This strain

was examined and found to be free of contamination with Eperythrozoon coccoides. The

strain is known to provide the high mortality in mice, providing a good model to estimate

survival and anti-malarial efficacy in reducing parasitemia. It is sensitive to all currently

used anti-malarial drugs. PbA GFPcon 259cl2 cryovials were kept in the liquid nitrogen

at -196oC. All PRBCs were mixed with ice-cold parasite buffer (5mM Na2HPO4, 5mM

NaH2PO4, 0.9% NaCl) before giving infection. At first, the infection was given to three

‘source mice’. When any one of the source mice showed CM symptoms during the

course of infection, blood was collected from its tail vein and passaged IP to all

experimental mice so that each mouse received 104-106 parasites. Induction of infection

in experimental cerebral malaria (ECM) mice is presented in Fig.9.


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Fig.9. Induction of CM in C57BL/6 mice

5.18.2 Parasitemia

Anti-malarial efficacy was assessed by the parasitemia level and the mean survival time

for upto 4 weeks following inoculation. The thin blood smears from tail caudal vein were

fixed in methanol for 10s and stained with 10% Giemsa solution (Sigma Aldrich) for

20min. Stained slide was examined for parasitemia under a light microscope with an oil

immersion objective of 100x magnification power, (Olympus BX-51). Parasitemia was

counted as % of PRBCs in total number of normal RBCs across three fields of the

microscope (Fig.10). Parasitemia is the quantitative content of parasites in the blood and

was counted daily in all experimental cerebral malaria (ECM) mice. Survival time in

days was recorded for all the groups. Giemsa-stained blood smears, the gold standard

technique for malaria diagnosis, routinely used to detect for and quantify parasites

present in the peripheral circulation in the infected mice (Barcia, 2007).

Fig.10. Determination of parasite count by Giemsa staining


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5.18.3 Cerebral malaria prediction

Rescue treatment (drug regimen) was started only when the CM symptoms

commenced in mice. Rapid murine coma and behavioural scale (RMCBS) was

used for CM prediction as presented in Table 17. The RMCBS consists of 10

parameters, and each parameter is scored 0 to 2, with a 0 score correlating with the

lowest function and a 2 score, the highest. An animal can achieve an accumulative

score of 0 to 20. The mice that gave a score of <12 were given drugs accordingly

(Carroll et al., 2010).

Table 17. The Rapid Murine Coma and Behaviour Scale

Label Score Description

Coordination

Gait (0-2) (none-ataxic-normal)

Balance (0-2) (no body extension-extends front feet on wall- entire body lift)

Exploratory behavior

Motor performances (0-2) (none-2-3-corners explored in 90s- explores 4 corners in 15s)

Strength and Tone

Body Position (0-2) (on slide-hunched-full extension)

Limb Strength (0-2) (hypnotonic, no grasp-weak pull-back [front paw grasp only]-stong pull-back [active pull away, jerk away])

Reflexes and Self-Preservation

Touch Escape (0-2) (none-unilateral-instant and bilateral; in 3 attempts)

Pinna Reflex (0-2) (none-unilateral-instant and bilateral; in 3 attempts)

Toe Pinch (0-2) (none-unilateral-instant and bilateral; in 3 attempts)

Aggression (0-2) (none-bite attempt with tail cut-bite attempt prior to tail cut, in 5s)

Hygiene-Related Behaviour

Grooming (0-2) (ruffled, with swaths of hair out of place-dusty/piloerection-normal/clean with sheen)

5.18.4 Intraperitoneal administration

Mice showing CM symptoms were divided into 8 treatment groups of 6 mice each as

shown in Table 18. The drug doses intended were ARM 25mg/kg body weight

(Clemmer et al., 2011) and CUR 100mg/kg body weight (Reddy et al., 2005) once a day

either alone or in combination for 5 consecutive days by IP route. The therapeutic dose

for both the drugs was selected based on the previously published reports. ARM and

CUR were dissolved in 100% DMSO at a conc. of 12.5mg/mL and 50mg/mL

respectively. Nanoformulations ARM NE and CUR NE were prepared at conc.

27.6mg/mL and 11.7mg/mL respectively.


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Table 18. Grouping of animals for anti-malarial studies by IP route

Group Treatment

1 Control DMSO

2 ARM SOL

3 CUR SOL

4 ARM+CUR SOL

5 Control NE

6 ARM NE

7 CUR NE

8 ARM+CUR NE

5.18.5 Intranasal administration

Similar to IP administration, mice were divided into 8 treatment groups (n=6) as shown

in Table 19. For IN administration, the drug doses were reduced to 1/4th of that of IP

administration. ARM NE and CUR NE were given at dose of 6.125mg/kg body weight

and 25mg/kg body weight respectively once a day either alone or in combination. Drug

solutions of ARM and CUR were prepared in 100% DMSO at a conc. of 32.5mg/mL

and 125mg/mL respectively. Nanoformulations ARM NE and CUR NE was prepared at

conc. 27.6mg/mL and 11.7mg/mL respectively. Mice were anesthetized using ketamine-

xylazine cocktail at a dose of 20mg ketamine and 0.1mg xylazine per mouse (20g body

weight). Mice were laid 180 degree on their back so that head positioned itself around 45

degree from the ground. The drugs were then given into the nostril of mice through a

micropipette attached to low density polyethylene tube having 0.1mm internal diameter

(Dhuria et al., 2010).

Table 19. Grouping of animals for anti-malarial studies by IN route

Group Treatment

1 Control DMSO

2 ARM SOL

3 CUR SOL

4 ARM+CUR SOL

5 Control NE

6 ARM NE

7 CUR NE

8 ARM+CUR NE


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5.18.6 Survivability and Histopathology

Mice were checked daily for survivability under various drug regimens. Autopsy was

done in mice that died of CM. Brain, spleen and liver were first washed in PBS pH 7.4

and fixed in 4% paraformaldehyde (pH 7.4) until further histopathological analysis. Each

section were stained with H and E and studied under a light microscope (Olympus BX-

51) at 400x magnification to examine the associated histopathological changes. For

detecting degenerating neurons, other deparaffinized brain sections were stained with

Fluoro-Jade®C (FJ-C) as explained elsewhere (Schmued and Hopkins, 2000). The

degenerating neuron densities were analyzed at 400x magnification using a laser

scanning confocal microscope (Carl-Zeiss).

5.18.7 Splenomegaly

Macroscopic observation of spleens from infected mice was carried out for drug loaded

NE (IP) and control group (IP). Infected spleens were excised upon death of mice,

photographed and checked for splenomegaly.

5.18.8 Statistical analysis

Pharmacokinetic and pharmacodynamic data was analyzed by two way analysis of

variance (ANOVA) followed by Tukey post test. Values were considered as significant

when P<0.05.

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