CHAPTER III MATERIALS AND METHODS 3.0...

CHAPTER III MATERIALS AND METHODS

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3.0 MATERIALS & METHOD

Drug profile

3.1: Zaltoprofen (ZLT)

Zaltoprofen is effective NSAIDs, which is manufactured by Japan. ZLT, 2-(10,

11-dihydro-10-oxodibenzo [b, f] thiepin-2-yl) propionic acid is a potent non-steroidal

anti-inflammatory drug (NSAID). Propionic acid derivatives are widely regarded as the

drugs of first choice in the management of patients with inflammatory joint disease

because they are the class of NSAIDs with the lowest incidence of side effects. Ibuprofen

was “the first of the propionic” launched in 1969. Since then, Ibuprofen proved to be

effective as a prescription drug in a range of painful non rheumatic condition and on the

basis of its good safety. It has been used clinically for treatment of post-operative pain

and record was approved as an OTC analgesic in 1983 in the United Kingdom and in

1984 in the USA. In India, the drug is widely used in clinical practice. ZLT belongs to

this class of NSAIDs. It has been used clinically for treatment of post-operative pain and

low back pain for more than ten years. Zaltoprofen is a preferential COX-2 inhibitor3 and

selectively inhibits prostaglandin E2 (PGE2) production at inflammatory sites.2 and to

induce apoptosis in a variety of cell lines. Zaltoprofen is a unique compound that also has

anti-bradykinin activity. Its analgesic effects may be a result of inhibition of bradykinin

B2 receptor-mediated bradykinin responses not only of cyclooxygenases but also of

bradykinin-induced 12-lipoxygenase inhibitors.

Table no. 3.1 : Detail drug profile of Zaltoprofen

Appearance Zaltoprofen occurs as white to light yellow, Crystals or

crystalline powder

Structure

Taste & odor Tasteless & odorless

IUPAC name (2RS)-2-(10-Oxo-10,11-dihydrodibenzo[b,f] thiepin-2-

S

OCH

CH3

O

OH


29

yl)propanoic acid

Molecular formula C17H14O3S

Molecular weight 298.36

Dose 80 mg, TID

Category Anti-inflammatory

Melting point 135 – 139oC

Loss on drying Not more than 0.5% (1 g, 105oC, 4hours)

Solubility Freely sol in acetone, chloroform; sol in methanol;

slightly sol in ethanol, benzene. Practically insoluble in

water, cyclohexane

Containers and storage

Tight containers and store in cool, dry, dark place.

Usage Anti-inflammatory activity

Mechanism of action:

Zaltoprofen is a nonsteroidal anti-inflammatory drug that exhibits anti-

inflammatory, analgesic and antipyretic activities. It is a COX-2 preferential inhibitor.

The main mechanism of zaltoprofen is prostaglandin biosynthesis inhibitory action due to

the COX inhibition in the arachidonic acid metabolism system. Besides this, membrane

stabilizing action such as leukocyte migration inhibitory action and lysosomal enzyme

inhibitory action are also observed with zaltoprofen. Experimental studies have shown

that Prostaglandin biosynthesis inhibitory action in the stomach tissue is weaker with

ZLT than in case of indomethacin. ZLT was shown to have more powerful inhibitory

effect to bradykinin-nociceptor than other NSAIDs.

Dosage: 80mg, thrice a day, oral administration

Adverse effects:

Gastrointestinal- Nausea, vomiting, constipation, dry mouth, loss of apatite. Neurologic-

Drowsiness, dizziness, numbness. Hypersensitivity-Photosensitivity, itching.

Hematologic- erythrocytopenia, thrombocytosis. Renal-Blood in urine rises in creatinine.


30

3.2: Ketoprofen

Ketoprofen,(RS)2-(3-benzoylphenyl)-propionic acid (chemical formula C16H14O3)

is one of the propionic acid class of nonsteroidal anti-inflammatory drugs (NSAID) with

analgesic and antipyretic effects. Ketoprofen is generally prescribed for arthritis-related

inflammatory pains or severe toothaches that result in the inflammation of the gums.

Ketoprofen topical plasters are being extensively used for treatment of musculoskeletal

pain.

Table No. 3.2: Drug Profile of Ketoprofen

Appearance A white or almost white crystalline powder

Structure

IUPAC name : 2-(3-benzoylphenyl) propanoic acid

Molecular formula: C16H14O3

Molecular mass: 254.28 g/mol

Melting point: 940C

Half life: Conventional capsules: 1.1-4 hours

Extended release capsules: 5.4 hours

Therapeutic category Non steroidal anti inflammatory drug

(NSAID)

Route Oral, Topical, Intravenous

Solubility: Sparingly soluble in water

Soluble in methanol

O

CH3

OH O


31


The anti-inflammatory effects of ketoprofen are believed to be due to inhibition

cylooxygenase-2 (COX-2), an enzyme involved in prostaglandin synthesis via the

arachidonic acid pathway. This results in decreased levels of prostaglandins that mediate

pain, fever and inflammation. Ketoprofen is a non-specific cyclooxygenase inhibitor and

inhibition of COX-1 is thought to confer some of its side effects, such as GI upset and

ulceration. Ketoprofen is thought to have anti-bradykinin activity, as well as lysosomal

membrane-stabilizing action. Antipyretic effects may be due to action on the

hypothalamus, resulting in an increased peripheral blood flow, vasodilation, and

subsequent heat dissipation.

Dosage: Greater than 25mg is commonly prescribed for pain relief.

Side effects:

Side effects are usually mild and mainly involved the GI tract. Most common adverse

GI effect is dyspepsia (11% of patients). May cause nausea, diarrhea, abdominal pain,

constipation and flatulence.

3.3: Lornoxicam

Lornoxicam is a non-steroidal anti-inflammatory drug of the oxicam class with

analgesic, anti-inflammatory and antipyretic properties. It is available in oral and

Parenteral formulations. Lornoxicam differs from other oxicam compounds in its potent

inhibition of prostaglandin biosynthesis, a property that particularly explains the

pronounced efficacy of the drug. Lornoxicam is used for the treatment of various types of

pain, especially resulting from inflammatory diseases of the joints, osteoarthritis, surgery,

sciatica, and other inflammations.

Table no: 3.3 Drug profile of Lornoxicam

Appearance

Structure

IUAC name (3E)-6-chloro-3-[hydroxy(pyridin-2-

ylamino)methylene]-2-methyl-2,3-dihydro-4H

N

N

O

NS

OH

S

Cl

O O

H


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thieno[2,3-e][1,2]thiazin-4-one 1,1-dioxide

Molecular formula C13H10ClN3O4S2

Molecular mass 371.81 g/mol

Half life: 3 – 4 hrs

Therapeutic category: Non steroidal anti inflammatory drug (NSAID)

Route: Oral, Parenteral

Solubility: Poorly soluble in water, Soluble in 0.1N NaOH

Solution


Lornoxicam's anti-inflammatory and analgesic activity is related to its inhibitory

action on prostaglandin and thromboxane synthesis through the inhibition of both COX-1

and COX-2. This leads to the reduction of inflammation, pain, fever, and swelling, which

are mediated by prostaglandins. However, the exact mechanism of lornoxicam, like that

of the other Non steroidal anti-inflammatory drugs (NSAIDs) has not been fully

determined.

Dosage

The adult dosage of Lornoxicam for pain relief is 8-16mg daily and maximum of

24mg/day. The daily dosage for Osteoarthritis is 12 mg daily in 2-3 divided doses, up to

16 mg daily.

Adverse effects:

The most common side effects reported with the regular use of the tablet form of

Lornoxicam include dizziness, headache, stomach pain, upset stomach, diarrhea, nausea,

vomiting and indigestion. As an injection, users most commonly report headache,

flushing, insomnia and redness and irritation at the injection spot.

3.4 Meloxicam

Meloxicam is a nonsteroidal anti-inflammatory drug (NSAID) used to relieve the

symptoms of arthritis, primary dysmenorrhea, fever; and as an analgesic, especially

where there is an inflammatory component. It is closely related to piroxicam. In Europe it

is marketed under the brand names Movalis, Melox, and Recoxa. In North America it is


33

generally marketed under the brand name Mobic. In Latin America, the drug is marketed

as Tenaron.

Table no. 3.4: Drug profile of Meloxicam

Appearance A Pale Yellow Powder

Structure

IUPAC name : 4-hydroxy-2-methyl-N-(5-methyl-1,3-

thiazol-2-yl)-1,1-dioxo-2H-1$l^{6},2-

benzothiazine-3-carboxamide

Molecular formula: C14H13N3O4S2

Molecular mass: 351.4 g/mol

Melting point: 2540C

Half life: 15-20 hours

Therapeutic category Non steroidal anti inflammatory drug

(NSAID)

Route Oral

Solubility: Sparingly soluble in water


Anti-inflammatory effects of meloxicam are believed to be due to inhibition of

prostaglandin synthetase (cylooxygenase), leading to the inhibition of prostaglandin

synthesis. As prostaglandins sensitize pain receptors, inhibition of their synthesis may be

associated with the analgesic and antipyretic effects of meloxicam.

Dosage: For treatment of osteoarthritis, rheumatoid arthritis and other pain conditions

(Adults):

Consider administration of 80 mg of Zaltoprofen thrice daily.

SN

O

N

CH3

OH

N

H

O O

S

CH3


34

Adverse effects:

Gastrointestinal discomfort, ulcer, Nausea, vomiting etc.

3.5: Method

3.5.1: Procurement of drug and excipients:

The drug, excipients, chemicals/ reagents and equipments used for various experiments are

enlisted as follows:

Zaltoprofen was gifted by ZCL chemicals Ltd. Mumbai, Maharashtra, India.

Poloxamber407, Eudragit RS 100, Eudragit RL 100 were purchased from Yarrow

chemicals Mumbai, Maharashtra and are of AR grade.

3.5.2: Materials:

Table No: 3.5: Shows the list of drug, excipients and chemicals used during the

study.

Sr.No Particular Gifted from

1 Zaltoprofen ZCL Laboratory,Mumbai

2 Meloxicam Yarrow chem Product, Mumbai

3 Ketoprofen Yarrow chem Product, Mumbai

4 Lornoxicam Yarrow chem Product, Mumbai

5 Eudragit RS100 Yarrow chem Product, Mumbai

6 Poloxamber 407 Yarrow chem Product, Mumbai

3.5.3. Instruments and equipments:

The different instruments used for experiments are enlisted in Table.No.1.6

Table No. 3.6 : Details of Equipments/Instrument used.

Sr.

No

Name of the Equipment /

Instrument Make Model

1 Digital analytical balance Shimadzu AY120

(0.1 to 120 gms)

2 UV-Visible double beam

spectrophotometer shimadzu 3650

3 FTIR spectrophotometer Shimazdu Spectrophotometer-430


35

8400s,Japan

4 Magnetic stirrer Remi SPEED 1500

5 Programmable melting point

apparatus (Digital)

Digital Apparatus

VEEGO VMP-D

6 Differential scanning

Calorimetry

Mettler Toledo

Switzerland

MettlerDSC1Star

System

7 Centrifuge Machine Remi Motar RQ-126/D

8 Homogenizer Remi-Elektrotechnik

RQT-127A

(8000 rpm with 100ml

capacity)

9 Distillation Water Assembly Distillation Unit

Mono Quartz Cap Capacity 2-5 Litres

10 pH meter Ri Digital pH Model 152 R

11 Scanning electron

microscope JOEL JSM-6390lv

12 Hot Air Oven Universal Hot air

Oven -----

13 Stability Chamber Remi-Instruments Compressor model

372LAG

14 Sonicator LABMAN Scientific

Instruments LMUC-4

15 Zeta sizer Malvern Instrument

UK NanoZS90

3.6: Characterization of Drugs:

Characterization of all drug (Zaltoprofen, Ketoprofen, Lornoxicam, Meloxicam )

& polymer were carried out with following test:-

I. Description: Appearance & color

II. Determination of melting point of zaltoprofen

III. Spectrophotometric characterization using,

A) UV- Visible spectrophotometer


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B) FT-IR spectrophotometer

IV. Determination of thermal behavior by: Differential Scanning Calorimetry (DSC)

V. Compatibility of drug with polymer by FTIR & Differential Scanning Calorimetry

(DSC)

3.6.1: Characterization of Zaltoprofen

I) Description: The powder was examined for appearance (colour) and nature.

II) Melting point determination:

The melting point of drug Zaltoprofen was determined by using open capillary

method melting point apparatus . The melting point was determined by introducing small

amount of substance in the capillary which is attached to graduated thermometer and then

constant heat was applied with the assembly suspended in the paraffin bath. The drug

sample was tested in temperature range of 140-147o

C and the temperature at which it

melt is noted.

III) Spectrophotometric Characterization of Zaltoprofen :

A) UV- Visible spectrophotometric characterization:

a) Determination of λ max:

UV spectrum of Zaltoprofen was carried out in phosphate buffer pH 7.4.

Zaltopofen was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH in a

100 ml volumetric flask. The solution of 20µg/ml was kept in a fused silica cuvette. The

UV spectrum was recorded in the range of 200-800 nm by shimadzu double beam UV-

visible spectrophotometer against blank buffer solution pH 7.4. Wavelength for

maximum absorbance was recorded. The λ max of Zaltoprofen in pH 7.4 buffer solution

was 338 nm.

b) Calibration curve by UV- Visible spectrophotometer:

Preparation of standard calibration curve of Zaltoprofen:

UV spectrum of Zaltoprofen was carried out in phosphate buffer pH7.4

Zaltoprofen was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH in

a 100 ml volumetric flask. The solution of 20µg/ml was kept in a fused silica cuvette.

The UV spectrum was recorded in the range of 200-800 nm by shimadzu double beam

UV-visible spectrophotometer 3650 against blank buffer solution pH 7.4. Wavelength for


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maximum absorbance was recorded. The λ max of Zaltoprofen in Ph 7.4 buffer solution

was 338 nm. And linearity for calibration curve is 5-30 µg/ml.

B) Determination of FTIR spectrum:

FTIR spectra of Zaltoprofen was studied. Above samples were mixed with KBr of

IR grade in the ratio of 1:100 and compressed using motorized pellet press (Kimaya

Engineers, India) at 10-12 tones pressure. The pellets were then scanned using FTIR

spectrophotometer (Shimadzu 8400S, Japan). The FTIR spectrum of zaltoprofen drug

were compared with that of the FTIR Spectrum of pure drug zaltoprofen , to confirm any

changes occur or not in the principle peaks of spectra of plain drug Zaltoprofen. spectra

was obtained by using a FTIR spectrometer-430 (Shimadzu 8400S, Japan)

IV) Determination of thermal behavior by Differential scanning calorimeter (DSC)

Zaltoprofen was assessed by carrying out thermal analysis. The inert atmosphere

was maintained by purging nitrogen gas throughout the experiment at the rate of 40

ml/min. The samples (1-2mg) were carefully transferred and heated in a crimped

aluminum pan for accurate results. The samples were heated from 30°C– 300°C at the

rate of 10°C/min.

V) Compatibility of ZLT with polymer by FTIR & Differential Scanning

Calorimetry (DSC):

a) Fourier transformer infrared Spectroscopy (FTIR):

The infrared spectrum of physical mixture of Eudragit RS100: Zaltoprofen (1:1),

Eudragit RL100: Zaltoprofen (1:1), Eudragit RS100: Poloxamer F128 (407): Zaltoprofen

(1:1:1) and Eudragit RL100: Poloxamer F127 (407): Zaltoprofen (1:1:1) were recorded

by potassium bromide dispersion technique in which mixture of polymer: Zaltoprofen

and potassium bromide was placed in sample holder and an infrared spectrum was

recorded using FTIR Spectrophotometer-430 ( Shimadzu 8400S, Japan ). The identified

peaks were compared with the principle peaks of reported IR spectrum of Zaltoprofen,

Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407) and the interaction between

the meloxicam & polymer as well as Stabilizer was studied

b) Zaltoprofen- polymers compatibility study by DSC:

Physical mixture of Zaltoprofen with polymers (Eudragit RS100, Eudragit RL100

and Poloxamer 407) in the ratio of 1:1 were assessed by carrying out thermal analysis. .


38

Thermogram for drug zaltoprofen & polymer and stabilizer in 1:1:1 ratio was taken for

obtained using DSC (Mettler DSC 1 star system, Mettler-Toledo, Switzerland). The drug

was sealed in perforated aluminum pan and heated at constant rate of 10°C/min over the

temperature ranges of 30-300°C.

3.6.2: Characterization of ketoprofen:

I) Description: The powder was examined for appearance (colour, odour) and nature.


The melting point of ketoprofen was determined by using open capillary method

melting point apparatus . The melting point was determined by introducing small amount

of substance in the capillary which is attached to graduated thermometer and then


sample was tested in temperature range of 95-100o C and the temperature at which it

melt is noted.

III) Spectrophotometric characterization of ketoprofen :



UV spectrum of ketoprofen was carried out in phosphate buffer pH 7.4.

ketoprofen was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH in a



visible spectrophotometer against blank buffer solution pH 7.4. Wavelength for

maximum absorbance was recorded. The λ max of Ketoprofen was determined in pH 7.4

buffer solution and was found to be 260 nm.


Preparation of standard calibration curve of ketoprofen:

UV spectrum of ketoprofen was carried out in phosphate buffer pH 7.4.

ketoprofen was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH in a



visible spectrophotometer against blank buffer solution pH 7.4. And linearity calibration


39

curve was carried out by using standard Ketoprofen solutions in the range 4-14µg/ml and

the absorbance with respect to 4,6,8,10,12,14µg/ml were determine for linearity.


FTIR spectra of ketoprofen was studied. Above samples were mixed with KBr of



spectrophotometer (Shimadzu 8400S, Japan). The FTIR spectrum of Ketoprofen drug

were compared with that of the FTIR Spectrum of pure drug Ketoprofen , to confirm any

changes occur or not in the principle peaks of spectra of plain drug Ketoprofen spectra


IV) Determination of thermal behaviour by Differential scanning calorimetry (DSC)

Melting point of drug was determined by using differential scanning calorimetry.

Thermogram for Ketoprofen was obtained using DSC (Mettler DSC 1 star system,

Mettler-Toledo, Switzerland). The drug was sealed in perforated aluminum pan and

heated at constant rate of 10°C/min over the temperature ranges of 30-300°C.

V) Compatibility of ketoprofen with polymer by FTIR & Differential Scanning

Calorimetry (DSC):

a) Fourier transform infrared Spectroscopy (FTIR):

The infrared spectrum of physical mixture of Eudragit RS100: ketoprofen (1:1), Eudragit

RL100: ketoprofen (1:1), Eudragit RS100: Poloxamer F128 (407): ketoprofen(1:1:1) and

Eudragit RL100: Poloxamer F128 (407): ketoprofen (1:1:1) were recorded by potassium

bromide dispersion technique in which mixture of polymer: ketoprofen and potassium

bromide was placed in sample holder and an infrared spectrum was recorded using FTIR

Spectrophotometer-430 ( Shimadzu 8400S, Japan ). The identified peaks from these

were compared with the principle peaks of reported IR spectrum of Ketoprofen, Eudragit

RS100, Eudragit RL100 and Poloxamer F128 (407).and the interaction between the

meloxicam & polymer as well as Stabilizer was studied.

b) Ketoprofen- polymers compatibility study by DSC:

Physical mixture of ketoprofen with polymers (Eudragit RS100, Eudragit RL100 and

Poloxamer 407) in the ratio of 1:1 were assessed by carrying out thermal analysis. .

Thermogram for drug Ketoprofen & polymer and stabilizer in 1:1:1 ratio was taken for


40




3.6.3: Characterization of Lornoxicam:



The melting point of Lornoxicam was determined by using open capillary method




sample was tested in temperature range of 100-110 o C and the temperature at which it

melt is noted.

III) Spectrophotometric characterization of Lornoxicam :



UV spectrum of Lornoxicam was carried out in phosphate buffer pH 7.4.

Lornoxicam was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH in

a 100 ml volumetric flask. The solution of 20µg/ml was kept in a fused silica cuvette.


UV-visible spectrophotometer against blank buffer solution pH 7.4. Wavelength for

maximum absorbance was recorded. The λ max of Lornoxicam in pH 7.4 buffer solution

was found to be 377 nm.


Preparation of standard calibration curve of Lornoxicam:

UV spectrum of Lornoxicam was carried out in phosphate buffer pH7.4.

Lornoxicam drug was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4

pH in a 100 ml volumetric flask. The solution of 10µg/ml was kept in a fused silica

cuvette. The UV spectrum was recorded in the range of 200-800 nm by shimadzu double

beam UV-visible spectrophotometer against blank buffer solution pH 7.4. The λ max of

Lornoxicam in Ph 7.4 buffer solution was 377 nm. And linearity calibration curve was


41

carried out by using standard Lornoxicam solutions in the range 5,10,15,20,25 µg/ml and

the absorbance were determined for linearity.


FTIR spectra of Lornoxicam was studied. Above samples were mixed with KBr

of IR grade in the ratio of 1:100 and compressed using motorized pellet press (Kimaya


spectrophotometer (Shimadzu 8400S, Japan). The FTIR spectrum of Lornoxicam drug

were compared with that of the FTIR Spectrum of pure drug Lornoxicam , to confirm any

changes occur or not in the principle peaks of spectra of plain drug lornoxicam spectra




Thermogram for Lornoxicam was obtained using DSC (Mettler DSC 1 star system,



V) Compatibility of Lornoxicam with polymer by FTIR & Differential Scanning

Calorimetry (DSC):


The infrared spectrum of physical mixture of Eudragit RS100: Lornoxicam(1:1),

Eudragit RL100: Lornoxicam (1:1), Eudragit RS100: Poloxamer F128 (407): Lornoxicam

(1:1:1) and Eudragit RL100: Poloxamer F128 (407): Zaltoprofen (1:1:1) were recorded

by potassium bromide dispersion technique in which mixture of polymer: Lornoxicam

and potassium bromide was placed in sample holder and an infrared spectrum was

recorded using FTIR Spectrophotometer-430 ( Shimadzu 8400S, Japan ). The identified

peaks were compared with the principle peaks of reported IR spectrum of Lornoxicam,

Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407) and the interaction between

the Lornoxicam & polymer as well as Stabilizer was studied.

b) Lornoxicam- polymers compatibility study by DSC:

Physical mixture of Lornoxicam with polymers (Eudragit RS100, Eudragit RL100

and Poloxamer 407) in the ratio of 1:1 were assessed by carrying out thermal analysis.

Thermogram for drug Lornoxicam & polymer and stabilizer in 1:1:1 ratio was taken


42

for obtained using DSC (Mettler DSC 1 star system, Mettler-Toledo, Switzerland). The

drug was sealed in perforated aluminum pan and heated at constant rate of 10°C/min over

the temperature ranges of 30-300°C.

3..6.4 :Characterization of Meloxicam:



The melting point of Meloxicam was determined by using open capillary method




sample was tested in temperature range of 260-265 0C and the temperature at which it

melt is noted.

III) Spectrophotometric characterization of Meloxicam:



UV spectrum of Meloxicam was carried out in phosphate buffer pH7.4. Meloxicam drug

was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH in a 100 ml

volumetric flask. The solution of 20µg/ml was kept in a fused silica cuvette. The UV

spectrum was recorded in the range of 200-800 nm by shimadzu double beam UV-visible

spectrophotometer against blank buffer solution pH 7.4. Wavelength for maximum

absorbance was recorded. The λ max of Meloxicam in Ph 7.4 buffer solution was found

to be 362 nm.


Preparation of standard calibration curve of Meloxicam:

UV spectrum of Meloxicam was carried out in phosphate buffer pH 7.4.

Meloxicam drug was weighed 0.01 gm accurately and transfer in phosphate buffer 7.4 pH

in a 100 ml volumetric flask. The solution of 10µg/ml was kept in a fused silica cuvette.


UV-visible spectrophotometer against blank buffer solution pH 7.4. The λ max of

Meloxicam in PH 7.4 buffer solution was 362 nm. And linearity calibration curve was


43

carried out by using standard Meloxicam solutions in the range 5-30µg/ml

(5,10,15,20,25,30 µg/ml ).


FTIR spectra of Meloxicam was studied. Above samples were mixed with KBr of



spectrophotometer (Shimadzu 8400S, Japan). The FTIR spectrum of Meloxicam drug

were compared with that of the FTIR Spectrum of pure drug Meloxicam , to confirm any

changes occur or not in the principle peaks of spectra of plain drug Meloxicam spectra




Thermogram for Meloxicam was obtained using DSC (Mettler DSC 1 star system,



V) : Compatibility of Meloxicam with polymer by FTIR & Differential Scanning

Calorimetry (DSC)


The infrared spectrum of physical mixture of Eudragit RS100: Meloxicam (1:1), Eudragit

RL100:Meloxicam (1:1), Eudragit RS100: Poloxamer F128 (407): Meloxicam (1:1:1)

and Eudragit RL100: Poloxamer F128 (407): Meloxicam(1:1:1) were recorded by

potassium bromide dispersion technique in which mixture of polymer: Meloxicam and

potassium bromide was placed in sample holder and an infrared spectrum was recorded

using FTIR Spectrophotometer-430 (Shimadzu 8400S, Japan ). The identified peaks

from these were compared with the principle peaks of reported IR spectrum of

Meloxicam, Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407) and the

interaction between the meloxicam & polymer as well as Stabilizer was studied.

b) Meloxicam- polymers compatibility study by DSC:

Physical mixture of Meloxicam with polymers (Eudragit RS100, Eudragit RL100 and

Poloxamer 407) in the ratio of 1:1 were assessed by carrying out thermal analysis..

Thermogram for drug meloxicam & polymer and stabilizer in 1:1:1 ratio was taken for


44




3.7: Characterization of Polymer

The polymers Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407) used

for the formulation were characterized for following parameters,

i. Determination of melting point

ii. Spectrophotometric characterization using, IR spectrometry

iii. Determination of thermal behaviour by: Differential scanning calorimetry .

i. Melting point:

The melting point of Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407)

open capillary method. The melting point were determined by introducing small amount

of substance in the capillary attached to graduated thermometer and constant heat was

applied with the assembly suspended in the paraffin bath. The polymer samples were

tested in temperature range 30oC -300

oC and point at which polymer melts was noted.

ii. Determination of FTIR spectrum:

The FTIR spectrum of Eudragit RS100, Eudragit RL100 and Poloxamer F128

(407) were recorded by potassium bromide dispersion technique in which mixture of

polymer and potassium bromide was placed in sample holder and an infrared spectrum

were recorded using FTIR spectrophotometer-430(Shimadzu 8400S, Japan). The

identified peaks were compared with the principle peaks of reported IR spectrum of

Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407) and it was found that the

polymer sample was suitable .

iii) Determination of thermal behaviour by Differential Scanning Calorimetry

(DSC):

Eudragit RS100, Eudragit RL100 and Poloxamer F128 (407) were assessed by

carrying out thermal analysis. Melting point of drug was determined by using differential

scanning calorimetry. Thermogram for polymer was obtained using DSC ( Mettler DSC 1

star system, Mettler-Toledo, Switzerland). The drug was sealed in perforated aluminum

pan and heated at constant rate of 10°C/min over the temperature ranges of 30-300°C.


45

3.8: Formulation of polymeric nanosuspension:

3.8.1) Preparation of Zaltoprofen nanosuspensions

Zaltoprofen nanosuspensions were prepared by the Quassi emulsification solvent

diffusion method.[128] The ZLT(80 mg) and Eudragit RS100/RL100 (160 mg ) were co-

dissolved in 5 ml of methanol. The solution was to be slowly injected with a syringe

containing thin Teflon tube into 20 ml water containing stabilizer poloxamber 407 and it

was maintained at low temperature in ice bath protected from sun light. During injection

the mixture was stirred well by a high speed homogenizer at 5500 rpm speed. The

solution immediately turned into pseudo emulsion of the drug and polymer methanol

solution in the external aqueous phase. The counter diffusion of methanol and water out

of and into the emulsion micro droplets respectively results into the formation of

nanosuspension. Formulation were prepared with varying polymer & stabilizer ratio

overall 8 formulation of drug zaltoprofen were prepared with two different polymer

Eudragit RS100 & Eudragit RL100 with a stabilizer such as poloxamber 407(Pluronic

F127)and the formulation were code as ZRS-F1,ZRS-F2,ZRS-F3,ZRS-F4,ZRL-F5,ZRL-

F6,ZRL-F7,ZRL-F8.

Table no. 3.7 : Details about formulation contents of Zaltoprofen polymeric

nanosuspension batches.

Batch Drug

(mg)

Polymer (mg) Surfactant

Poloxamer 407 (%)

Distilled water

(mL) Eudragit

RS100

Eudragit

RL100

F1 80 80 - 0.5 20

F2 80 160 - 0.5 20

F3 80 80 - 1 20

F4 80 160 1 20

F5 80 80 0.5 20

F6 80 - 160 0.5 20

F7 80 80 1 20

F8 80 160 1 20


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3.8.2) Preparation of Meloxicam Nanosuspensions

Meloxicam nanosuspensions were prepared by the Quassi emulsification solvent

diffusion method.[128] The Meloxicam (15mg) and Eudragit RS100/RL100 (30 mg)

were co-dissolved in 6 to 7 ml of methanol it was stirred continuous for 3 Hrs. The

solution was to be slowly injected with a syringe containing thin Teflon tube into 40 ml

water containing stabilizer poloxamber 407 and it was maintained at low temperature in

ice bath protected from sun light. During injection the mixture was stirred well by a high

speed homogenizer at 6000-6500 rpm speed for 5 Hrs. The solution immediately

turned into pseudo emulsion of the drug and polymer methanol solution in the external

aqueous phase. The counter diffusion of methanol and water out of and into the emulsion

micro droplets respectively results into the formation of nanosuspension. Formulation

were prepared with varying polymer & stabilizer ratio overall 8 formulation of drug

meloxicam were prepared with two different polymer Eudragit RS100 & Eudragit

RL100 with a stabilizer such as poloxamber 407(Pluronic F127)and the formulation were

code as (MRS-F1,MRS-F2,MRS-F3,MRS-F4,MRL-F5,MRL-F6,MRL-F7,MRL-F8)

[128-131]

Table no. 3.8: Details about formulation contents of Meloxicam polymeric


Batch Drug

(mg)


Poloxamer 407

(%)

Distilled

water (mL) Eudragit

RS100

Eudragit

RL100

MRS-F1 15 15 - 0.5 20

MRS-F2 15 30 - 0.5 20

MRS-F3 15 15 - 1 20

MRS-F4 15 30 1 20

MRL-F5 15 15 0.5 20

MRL-F6 15 - 30 O.5 20

MRL-F7 15 15 1 20

MRL-F8 15 30 1 20


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3.8.3) Preparation of Ketoprofen Nanosuspensions

Ketoprofen nanosuspensions were prepared by the Quassi emulsification solvent

diffusion method.[128] The drug (75 mg) and Eudragit RS100/RL100 (150 mg ) were

co-dissolved in 5 ml of methanol. The solution was to be slowly injected with a syringe



the mixture was stirred well by a high speed homogenizer 5500 rpm agitation speeds for

5 Hrs. The solution immediately turned into pseudo emulsion of the drug and polymer

methanol solution in the external aqueous phase. The counter diffusion of methanol and

water out of and into the emulsion micro droplets respectively results into the formation

of nanosuspension. Formulation were prepared with varying polymer & stabilizer ratio

overall 8 formulation of drug ketoprofen were prepared with two different polymer


F127)and the formulation were code as (KRS-F1,KRS-F2,KRS-F3,KRS-F4,KRL-

F5,KRL-F6,KRL-F7,KRL-F8.[128-131]

Table no.3.9 : Details about formulation contents of Ketoprofen polymeric


Batch Drug

(mg)


Poloxamer 407

(%)

Distilled

water (mL) Eudragit

RS100

Eudragit

RL100

KRS-F1 75 80 - 0.5 20

KRS-F2 75 160 - 0.5 20

KRS-F3 75 80 - 1 20

KRS-F4 75 160 1 20

KRL-F5 75 80 0.5 20

KRL-F6 75 - 160 O.5 20

KRL-F7 75 80 1 20

KRL-F8 75 160 1 20


48

3.8.4) Preparation of Lornoxicam Nanosuspensions

Lornoxicam nanosuspensions were prepared by the Quassi emulsification solvent

diffusion method.The Lornoxicam (8 mg) and EudragitRS100/RL100 (16/32 mg) were

co-dissolved in 5 ml of methanol. The solution was to be slowly injected with a syringe



the mixture was stirred well by a high speed homogenizer at 5500 rpm speeds for 5 hrs.

The solution immediately turned into pseudo emulsion of the drug and polymer methanol

solution in the external aqueous phase. The counter diffusion of methanol and water out

of and into the emulsion micro droplets respectively results into the formation of

nanosuspension. Formulation were prepared with varying polymer & stabilizer ratio

overall 8 formulation of drug Lornoxicam were prepared with two different polymer


F127)and the formulation were code as (LRS-F1,LRS-F2,LRS-F3,LRS-F4,LRS-

F4,LRL-F5,LRL-F6,LRL-F7,LRL-F8). [128-131]

Table no. 3.10 : Details about formulation contents of polymeric nanosuspension

batches.( Lornoxicam )

Batch Drug

(mg)


Poloxamer 407

(%)

Distilled

water (mL) Eudragit

RS100

Eudragit

RL100

LRS-F1 8 8 - 0.5 20

LRS-F2 8 16 - 0.5 20

LRS-F3 8 8 1 20

LRS-F4 8 16 1 20

LRL-F5 8 8 0.5 20

LRL-F6 8 16 O.5 20

LRL-F7 8 8 1 20


49

LRL-F8 8 16 1 20

3.9) EVALUATION PARAMETERS OF NANOSUSPENSIONS

3.9.1) Particle size analysis:

Scanning electron microscopy (SEM) is a method for high resolution surface

imaging. The SEM uses an electron beam for surface imaging. The advantages of SEM

over light microscopy are greater magnification and much larger depth of field. Different

elements and surface topographies emit different quantity of electrons, due to which the

contrast in a SEM micrograph (picture) is representative of the surface topography and

distribution of elemental composition on the surface. [131,132 ]

The Particle size was performed at STIC Cochin, the evaluation was carried out with

SEM Make: JEOL Model JSM-6390lv.

All the formulation of drug Zaltoprofen ,Meloxicam, Ketoprofen ,Lornoxicam

with EudragitRS100 & Eudragit RL100 with a Stabilizer Poloxamber 407( Pluronic

F127) were subjected to the particle size determination and the particle size was

determined and recorded.

3.9.2) Zeta potential of the Drug:

Zeta potential measurements were run at 250C with electric field strength of 23

V/m, using Zetasizer (Nano ZS 90, Malvern Instruments, UK). To determine the zeta

potential, samples of drug nanosuspension were diluted and placed in electrophoretic

cell. The zeta potential was calculated as described by Helmholtz–Smoluchowski

equation. All the formulation of drug Zaltoprofen ,Meloxicam, Ketoprofen ,Lornoxicam

with EudragitRS100 & Eudragit RL100 with a stabilizer Poloxamber 407( Pluronic

F127) after formulating the suspension or nanosuspension the Zeta Potential was

determined to know the stability of the nanosuspension it is very important parameter

because the biphasic dosage form i.e suspension stability depend upon the charge or the

total electrical double layer formed around the disperse nanoparticle and the Zeta

potential give stability measure so all formulation was determined with zeta potential and

recorded.[131,133]


50

3.9.3) Percentage of drug entrapment in the polymeric nanosuspension:

Percentage Entrapment efficiency:

In order to determine the % entrapment around 2 ml of formulation were taken in

the Nessler’s cylinder tube (10 ml) the solution was centrifuge in the centrifuge machine

at 2000-3000 rpm for 4 hrs. The supernatant layer was filter through whatmann filter

paper number 41and diluted with phosphate buffer 7.4 pH up to 10 ml and the resultant

solution were analyse at particular wavelength of drug in nm using UV Double beam

Spectrophotometer-3650 [134]

These was carried out for three time and the result were calculated .The

Percentage entrapment efficiency was calculated according to the equation or formula:

% EE = Total dug content –Free dissolve drug × 100 ------------------equation (1)

Dug amount used

These procedure is followed to all the formulation of drug Zaltorofen, Meloxicam

,Ketoprofen ,Lornoxicam

3.9.4) Saturation Solubility

Saturation solubility is an important physical properties which depend on the temperature

and responsible for the dissolution medium. However, below a size of approximately 1–

2μm, the saturation solubility is also a function of the particle size. Saturation solubiity of

plain drug and dry nanosuspension formulations were carried out in distilled water for

which 5 mg of drug and dry nanosuspensions (weigh equivalent to 5 mg of drug) in 2 ml

distilled water were taken separately and were allowed to be stirred in an isothermal

mechanical shaker (37.0 ± 1.0°C) for 24h. The stirred samples were further taken in test

tubes and centrifuged (Remi) at 10000 rpm for 15 minutes. The supernants were collected

and filtered through 0.22μm nylon membrane filter (Gelman Laboratory, Mumbai, India),

diluted with Phosphate buffer and absorbance was measured depending the drug

respective nm using UV-Visible spectrophotometer. The solubility was measured at

25°C. Every sample was analyzed in triplicate and the mean values and standard

deviations were reported. By these procedure the saturation solubility of the drug as well

as dry powder of nanosuspension i.e nano-size determined for the saturation solubility

and perform for drug zaltoprofen, Ketoprofen, Meloxicam, Lornoxicam.[ 135-136]


51

3.9.5) In-vitro drug Release studies

In vitro drug release of the nanosuspension was carried out by using USP Dissolution

apparatus type2 (paddle type). 5ml of nanosuspension was taken in a dialysis membrane

consisting of a spectrap or membrane (cut-off: 1200Da). This dialysis system was tied to

the paddle and the dissolution medium was Phosphate buffer pH 7.4. Dissolution was

carried in triplicate for 10 hrs at 37±10C temperature and 50rpm speed. At regular

intervals of time 1ml of sample from the external medium was taken and replaced with

fresh phosphate buffer and all the samples were analysed at nm of respective drug using

U.V spectrophotometer.

By using these procedure all drug Zaltoprofen ,Meloxicam, Ketoprofen

,Lornoxicam nanosuspension with Eudragit RS100 & Eudragit RL100 with stabilizer

poloxamber 407(Pluronic F127) were analyse for in vitro drug dissolution . [137-138]

3.9.6) Mathematical Dissolution Model for the optimize formulation:

The formulation which show maximum drug entrap as well as saturation solubility &

drug release, optimum zeta potential which give the measure of stability such formulation

were consider as optimize formulation mathematical model like Ist order plot, Higuchi

Plot ,Korseymer peppas Plot and by applying these equation the release pattern were

determined .These study give the pattern of the drug release from the polymeric

nanosuspension .[ 138 ]

CHAPTER III MATERIALS AND METHODS 3.0...

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