Microscopic evaluation of novel topical formulation for treatment of Arthritis

CHITKARA COLLEGE OF PHARMACY CHITKARA UNIVERSITY, PUNJAB - 140401

Mohit

INTRODUCTION

ARTHRITIS

Osteoarthritis

Rheumatoid Arthritis

Psoriatic Arthritis

ARTHRITIS

Psoriasis of Skin Joint Affected by Osteoarthritis

ARTHRITIS(Cont…….)

Factors Involved in Autoimmune Disorders

Genetic disposition,

Environmental factors,

Endocrinological factors

Immune dysfunction

In indvidual with a susceptible genotype,

exposure to above factors initiate an

autoimmune response to self and foreign

antigen through modulation of cytokines

production and effector cell function

ARTHRITIS(Cont…….)

ETIOLOGY

Gender

Genetics

Immune System

ETIOLOGY

Unknown antigen initiates the immune response resulting in rheumatoid arthritis

Women get arthritis 2-3 times more often than men Remission when they get pregnant.Women have higher absolute number of CD4 lymphocytes relative to men,.

Found in 20% of general population Not a diagnostic tool, many people who have the marker either do not have or will never get rheumatoid arthritis.

Infection

Bacterial infections are most important eg. septic arthritis, reactive arthritis, osteomyletis and osteitisViral for eg. rubella virus, human parvovirus, hepatitis B virusFungal eg. Candida

DIAGNOSIS OF RHEUMATOID ARTHRITIS  

LAB TEST

Complete blood Count- Low WBC count suggests felty’s syndrome

Platelet count is elevated in severe inflammation

Erythrocyte sedimentation rate (ESR) 60% of people have an elevated ESR

C-reactive protein

Rheumatoid factor (RF) +ive seropositive & -ive seronegative

Imaging studies Swelling of soft tissues and loss of bone density around the joints (X-ray) , MRI - Detect early inflammation before it is visible on X-rays, Joint ultrasound and bone densitometry -Measuring bone density used primarily to detect osteoporosis

DIAGNOSIS

PHYSICAL EXAMINATION

Joint swelling &tenderness

Malignancy

Loss of motion in joints

MEDICAL HISTORY

CONVENTIONAL THERAPY 

CORTICOSTERIODS

Conventional therapy

NSAID’S

(Used in early weeks)

BIOLOGIC RESPONSE MODIFIERS

Remicade

DMARD’SMethotrexate (MTX) , GoldHydroxychloroquineSulfasalazineCyclosporine ,Azathioprine

Approved in 1996, Enbrel (etanercept) is the first biologic response modifier to receive FDA approval for patients with moderate to severe rheumatoid arthritis

SYNOVIAL REPLISHNERS

(Glucosamine )

LIMITATIONS OF ARTHRITIS THERAPYNSAID’S

About 80% of patient experience gastrointestinal side effect including gastric ulcer, perforation and

hemorrhage etc

Colchicine

Poor solubility leads to high variability in oral bioavailability (e.g. Celecoxib, Colchicine have variable

oral bioavailability from 24 to 88%) Short biological half life (e.g. Colchicine has only 20 min.)

Systemic side effects

High systemic side effects (e.g. Rofecoxib showed cardiotoxic and renal side effects leading to its

withdrawal from market, Methotrexate has shown prominant hepatotoxic and bone marrow depression

Cost

High cost of treatment (e.g. Methotrexate and TNF-α)

Every year 1.5% of patient with rheumatoid arthritis are hospitalized with gastrointestinal problems

PROBLEMS IN PRESENT CONVENTIONAL THERAPY

Several dose dependent toxic effect

Can’t maintain a constant plasma level

Conventional therapy

Short biological half life

Low oral bioavailability

Minimum patient compliance

Cost of treatment is high

Decrease efficacy of dugs during

chronic use

Also affect normal cells of body

Deliver a steady state infusion for prolong period of time

Reduce the adverse effect and toxicity

Improve the therapeutic utility of drug by reducing the problems like

First pass metabolism

GI irritation

GI decomposition

Low absorption

Increase the half life of drug

Reduce the frequency of administration

Improved patient compliance

Self administration is possible

Drug input can be terminated at any time

ADVANTAGES OF TOPICAL DRUG DELIVERY

STRUCTURE AND FUNCTION OF HUMAN SKIN

Most extensive organ of body covering area of 2 m2 . Receive approximately one third of blood supply

For the purpose of transdermal/topical drug delivery, we can examine the structure and function of human skin categorized into four main layers:Stratum corneum (Stratum corneum is the rate limiting barrier)EpidermisDermisHypodermis

PROBLEM IN PRESENT ANTI-ARTHRITIC THERAPY AND PROPOSED STRATEGY FOR SITE-SPECIFIC DRUG DELIVERY

0

0.2

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0.60.8

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1.2

1.4

1.6

0 2 4 6 8 10 12 14

Time (hr)

Conc

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(g/

m

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Carrier

ELASTIC LIPOSOMES/ FATTY ACIDS AS CARRIER SYSTEM

Modified lipid carriers that enable drug to reach deeper skin layers.

Colloidal particles, typically consisting of phospholipids and surfactant molecules.

Pass through skin pores of size less than their own diameter.

Serve as rate limiting membrane barrier for systemic absorption of drugs.

Accommodate both hydrophilic and lipophilic drugs.

Liposomal surfactants are biodegradable and biocompatible.

Prolong the drug release.

Aqueous Cavity

Lipid Bilayers

IN VIVO MODELS FOR ARTHRITIS

Antigen-Adjuvant induced model for arthritis

Intraarticular injections of soluble antigen (same mice previously immunized to the same antigen )

Water in oil emulsion by combining one volume of FCA with one volume of aqueous antigen solution

Interaction with relevant cells

Acute arthritis

FCA enhances antibody production primarily because of the depot effectNonspecific immunopotentiation of macrophages by surfactant and the mycobacterium

The adjuvant is a mixture of non-metabolizable oil (mineral oil), a surfactant (Aracel.A) and mycobacterium (M.tuberculosis or M.butyricum) is considered to be one of the most effective adjuvant

DRUG INTRODUCTION

Methotrexate (MTX) is a folic acid antagonist preferably used for long-term therapy of rheumatoid arthritis

Available in oral tablet and injectable form

Poor bioavailability &systemic use of this drug may provoke any of a number of side effects mainly hepatotoxicity and bone marrow suppression agranulocytosis and thrombocytopenia

Chemical structure of methotrexate

DRUG INTRODUCTION

The daily oral dose requirement of glucosamine is 1500mg/dayAvailable in oral tablet and injectable form

Oral bioavailability of drug molecule is just 26%(subjected to uptake and degradation by the liver )

Chemical structure of glucosamine

A major problem in topical administration of these proposed drugs is its hydro-solubility and dissociation at physiological pH so its capacity for passive diffusion is thus limited.

ARTHRITIC DRUGS MARKET

The major players in the arthritis drug market include

Abbott Laboratories

Johnson & Johnson

Amgen

Roche

Pfizer

As of 2008, Abbott Laboratories' Humira, which was approved by the FDA in 2003, is the

highest selling drug in the arthritis market, with sales growing 50% from 2007 to 2010 to $8.5

billion

THE OBJECTIVES OF THE PROPOSED RESEARCH WORK

TO DEVELOP NOVEL DRUG DELIVERY SYSTEM, WHICH PROVIDE SUSTAINED AND TARGETED DELIVERY OF DMR’D TO THEIR TARGET SITE (JOINTS).

TO PREPARE, CHARACTERIZE AND OPTIMIZE DIFFERENT VESICULAR FORMULATIONS(,FATTY ACID VESICLES, NIOSOMES, ELASTIC LIPOSOMES)

TO CARRY OUT STABILITY AND SKIN PERMEATION STUDIES OF OPTIMIZED VESICULAR FORMULATION.

TO COMPARE IN VIVO ANTI-ARTHRITIC ACTIVITY OF DEVELOPED VESICULAR FORMULATIONS WITH MARKETED FORMULATION.

METHODLOGY

Identification and characterization of drug

Estimation of drugs in buffers and biological fluids by spectroscopy

Preparation of proposed vesicular system

Microscopic studies and characterization of proposed system

I.Phase contrast microscopy

II.Transmission electron microscopy

III.Scanning electron microscopy

In vitro characterization of vesicular system

Shape

Size and size distribution studies ( Dynamic light scattering methods)

Entrapment efficiency (Minicolumn centrifugation methods)

Degree of deformability (Extrusion method)

Zeta potential ( Zeta meter)

Turbidity measurement (Nephalometer)

No. of vesicles per cubic mm (Hemocytometer)

Phospholipid-ethanol interaction study (Differential Scanning Calorimetry)

In vitro skin permeation and deposition study (Using Diffusion Cell)

Stability study of the optimized formulation

In vivo studyFluorescence microscopy of rat viable skin to determine the extend of penetration of

vesicular formulation (Qualitative)

Confocal laser scanning Microscopy (CLSM) of rat viable skin to determine the rate and

extend of penetration (Quantitative)

Histopathological study of inflamed joint

METHODLOGY(cont….)

IMPORTANCE OF PROPOSED RESEARCH INVESTIGATION (National &International market status…)

Prevalence of arthritis in India increased drastically for last one decade

Dramatic increase in the demand of anti-arthritis drug

The market for rheumatoid arthritis therapeutics is estimated to reach over $20B in

2011

Proposed novel formulations will selectively deliver the drug to the targeted

inflamed joint

Naturally taken up by cells of mononuclear phagocytic system (MPS)

Biocompatible and biodegradable as they are made from natural phospholipid

Reducing the dose of the drug by minimizing the systemic exposure of drug and

increasing the deposition in deeper layer of skin

Easy to scale up, as procedure is simple, do not involve lengthy procedure and

unnecessary use of pharmaceutically unacceptable additives

EXPERIMENTAL WORKEXPERIMENTAL WORK DONE DONE IDENTIFICATION (Drug selected for study)IDENTIFICATION (Drug selected for study) Ultraviolet Absorption Maxima (Ultraviolet Absorption Maxima (Max)Max)

Methotrexate and glucosamine :- 100µg /ml stock solution in distilled water Methotrexate and glucosamine :- 100µg /ml stock solution in distilled water Scanned:- Between 200-400nm exhibits maxima at 267 nmScanned:- Between 200-400nm exhibits maxima at 267 nm

Results are concordant with the value given in the official books (Merck Index, 1996).Results are concordant with the value given in the official books (Merck Index, 1996).

Infrared Spectral AssignmentInfrared Spectral Assignment The IR spectra of MTX was recorded using (Perkin Elmer, IR Spectrophotometer).The IR spectra of MTX was recorded using (Perkin Elmer, IR Spectrophotometer).

Nuclear Magnetic ResonanceNuclear Magnetic Resonance The NMR The NMR spectra of MTX was recorded using (Bruker, NMR). of MTX was recorded using (Bruker, NMR).

S. NO. SOLVENTS SOLUBILITY

1. Distilled Water 25 mg /mL

2. Phosphate Buffer Saline (PBS) pH 7.4

24.2 mg /ml

3. Methanol 65mg/ml

PREFORMULATION STUDIESPREFORMULATION STUDIES

Table :- Solubility Profile of MTX in Different Solvents

S. No. SOLVENTSYSTEM

PARTITIONCOEFFICIENT

1. n-octanol : Distilled Water

1.2220.13

2. n-octanol: PBS (pH 74)

1.1270.15

Table :- Partition coefficient data of MTX

PARTITION COEFFICIENTPARTITION COEFFICIENT SOLUBILITY STUDIESSOLUBILITY STUDIES

PREFORMULATION STUDIESPREFORMULATION STUDIES S. NoS. No.. ParameterParameter StandardStandard ObservatioObservatio

nn

11 I.R. spectrumI.R. spectrum

22 0.002% w/v solution in HPLC 0.002% w/v solution in HPLC grade methanol observed grade methanol observed spectrophotometrically.spectrophotometrically.

Exhibit maxima at 302 Exhibit maxima at 302 nm. nm.

Exhibit maxima at Exhibit maxima at 302 nm 302 nm

33 Retention time Retention time methanol/acetonitrile/pH 5.4 methanol/acetonitrile/pH 5.4

buffer solution ) as mobile phase buffer solution ) as mobile phase using C 18 column at the flow rate using C 18 column at the flow rate

of 1 ml/min of 1 ml/min

RT for MTX was RT for MTX was reported as 9.5 min. reported as 9.5 min. (Pereira (Pereira et. al.et. al., 2000; , 2000;

Seki Seki et al.,et al., 1991) 1991)

RT for MTX was RT for MTX was found 9.8 min. found 9.8 min.

44 Melting range Melting range 255°C 255°C 253-255°C 253-255°C

55 Solubility Solubility Sparingly soluble in Sparingly soluble in water, slightly soluble in water, slightly soluble in

chloroform and have chloroform and have good solubility in good solubility in

methanol and practically methanol and practically insoluble in ether. insoluble in ether.

Sparingly soluble Sparingly soluble in water, slightly in water, slightly

soluble in soluble in chloroform, chloroform, soluble in soluble in

methanol and methanol and practically practically

insoluble in ether insoluble in ether

Table:- PREFORMULATION STUDIES

STANDARD CURVE OF MTX IN DISTILLED STANDARD CURVE OF MTX IN DISTILLED WATER BY UV SPECTROPHOTOMETRIC WATER BY UV SPECTROPHOTOMETRIC

METHODMETHOD

S . No. Concentration (g/ml)

Absorbance RegressedAbsorbance

Statistical Parameter

1. 2 0.0839 0.0839 Y= 0.0739 x – 0.1339 R2 =0.9993

2. 4 0.1608 0.157

3. 6 0.2339 0.2324

4. 8 0.3098 0.3078

5. 10 0.3879 0.3832

6. 12 0.4579 0.4586

7. 14 0.5294 0.5341

8. 16 0.6074 0.6094

9. 18 0.6852 0.6848

10. 20 0.7637 0.7602

Table :- Standard Curve of MTX In Distilled Water at Max 302 nm

S. No. Concentration (g/ml)

Absorbance Regressed Absorbance

Statistical Parameter

1. 2 0.0796 0.0709 Y=0.0343 X + 0.0017R2 = 0.9962. 4 0.1410 0.1393

3. 6 0.2076 0.2077

4. 8 0.2758 0.2761

5. 10 0.3428 0.3445

6. 12 0.4082 0.4129

7. 14 0.4747 0.4813

8. 16 0.5499 0.5497

9. 18 0.6317 0.6181

10. 20 0.6886 0.6865

Table :- Standard Curve of MTX in PBS (pH 7.4) at Max 302 nm

STANDARD CURVE OF MTX IN PBS (pH 7.4) STANDARD CURVE OF MTX IN PBS (pH 7.4) BY UV SPECTROPHOTOMETRIC METHODBY UV SPECTROPHOTOMETRIC METHOD

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 2 4 6 8 10 12 14 16 18 20

Concentration (g/ml)

Ab

so

rba

nc

e

Fig.:- Standard Curve of MTX in PBS (pH 7.4) at Max 302 nm

HPLC ASSAY OF MTXHPLC ASSAY OF MTX

S. No. Concentration(g/ml)

Peak area StatisticalParameter

1 0.0 0.0 y = 148370x + 288443

R2 = 0.99042 0.2 305375

3 0.4 360342

4 0.8 403575

5 1.2 475400

6 2.0 580025

Table :- Standard Curve of MTX in Distilled Water by HPLC Method

y = 148370x + 288443

R2 = 0.9904

0

100000

200000

300000

400000

500000

600000

700000

0 0.5 1 1.5 2 2.5

Concentration (g/ml)

Pe

ak

are

a

Fig.:- Standard Curve of MTX In Distilled Water by HPLC Method

DEVELOPMENT OF VESICULAR CARRIERS -BASIC PRINCIPLE

PC + SURFACTANT

Applied always nonocclusively

Transfersomes

PC + SURFACTANT PC + CHOLESTEROL

Partially dehrdrate Skin surface

“Transdermal osmotic gradient”

Stratum corneum (15% Water)

Dermal layer (75% Water)

Transfersomes prevent complete dehydration

Due to deformability transfersomes pass through narrow pores in the skin

Resulting in better skin permeation

Liposomes are less deformable therefore they dehydrate completely and fuse

LiposomesFatty acid vesicles

PC + FATTY ACID

Table1 Size and entrapment efficiency of the prepared oleic acid vesiclesUF-1,2,3-ufasomes with different molar ratio of drug

VISUALIZATION OF ELASTIC LIPOSOMES

TEM ( X 1,80, 000) Photomicrograph of liposomal formulation

Optical microscopy ( X 450) Photomicrograph liposomal

formulation

Figure - TEM Photomicrograph of UF-3 formulation of glucosamine

Formulation code

Oleic acid: 5-MTX (Molar ratio)

Entrapment efficiency

Particle Size(nm)

PDI

UF-1 9:1 (39.4±2.1) 505 ± 15 0.367 ± 0.037

UF-2 8:2 (45.4±2.1) 523±12 0.468 ± 0.037

UF-3 7:3 (51.0±4.2%), 632±17 0.262 ± 0.037

UF-4 6:4 (49.4±2.7) 531±16 0.489 ± 0.037

UF-5 5:5 (48.4±2.4) 404±13 0.581 ± 0.037

Size and entrapment efficiency of the prepared oleic acid vesiclesUF-1,2,3-ufasomes with different molar ratio of drug

Formulation code

Oleic acid: Glucosamine (Molar ratio)

Entrapment efficiency

Particle Size(nm)

PDI

UF-1 9:1 (37.4±1.1) 525 ± 15 0.367 ± 0.027

UF-2 8:2 (55.4±1.1) 553±12 0.368 ± 0.017

UF-3 7:3 (49.0±3.2%), 632±17 0.262 ± 0.036

UF-4 6:4 (49.4±2.7) 631±16 0.489 ± 0.033

UF-5 5:5 (48.4±2.4) 414±23 0.681 ± 0.047

Size and entrapment efficiency of the prepared oleic acid vesiclesUF-1,2,3-ufasomes with different molar ratio of drug

Fig. 2. Differential scanning calorimetry traces of oleic acid (a), oleic acid –MTX vesicles (b), oleic acid –MTX 8:2 vesicles (c) and oleic acid –MTX 9:1 vesicles (d).

A D

CB

Figure MTX UF-3 with different concentration of oleic acid

Optimized with span 20,conc of oleic acid 80%,ph 7.4,80mM

A B

figure –( A)-Optimized MTX, (B) Glucosamine ufasomal formulation

At different pH,conc of oleic acid 90%

pH 5.5pH 7.4

pH 8.5

pH8.5

pH 5.5

pH 6.5

pH7.4

Figure 4. Photomicrograph of oleic acid vesicles dispersion incubated at different pH (400× magnification).

Figure . Vesicle growth at low pH values 80mM concentration of oleic acid

Figure: Flourescence microscopy of optimized formulation

Figure 17 Confocal microscopy of different formulation

Figure 15 Histological View of Saggital Section of Rat Knee Joint Samples.

Figure : Histological View of Saggital Section of Rat Knee Joint Samples.

Conclusion • The results of the present study demonstrated that proposed vesicular formulation possess

great potential for skin accumulation, prolonging release, improving site specific delivery and reducing the skin toxicity of glucosamine and methotrexate . This formulation seems to represents an attractive strategy for site-specific sustained delivery of glucosamine and methotrexate. In addition they are cost effective and therapeutically viable. Sustained release behavior and drug retention in the deeper part of skin might be beneficial for the longterm effects of drugs. The oleic acid vesicles seemingly fuse with the skin and release the contents. They are seen to penetrate intact and to form drug depots in the skin. The fatty acid in addition may serve as a penetration enhancer, thus by circumventing the stratum corneum barrier potential they may lead to better permeation of the drug molecules.

Acknowledgement

1. Dr. Sandeep Arora, Director, Chitkara College of Pharmacy, Chitkara

University, India

2. Dr Arvind Sharma, Associate Professor, Chitkara College of Pharmacy, Chitkara

University, India