CONTENTS

1
Microscopic evaluation of novel topical
formulation for treatment of Arthritis
Mohit
CHITKARA COLLEGE OF PHARMACY CHITKARA
UNIVERSITY, PUNJAB - 140401
2
INTRODUCTION
3
ARTHRITIS
Osteoarthritis
Psoriatic Arthritis
ARTHRITIS
Rheumatoid Arthritis
Psoriasis of Skin
Joint Affected by Osteoarthritis
4
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

5
ARTHRITIS(Cont.)
6
ETIOLOGY
Gender
Immune System

• 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,.

ETIOLOGY
Genetics
Infection

• 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.

• Bacterial infections are most important eg.
  septic arthritis, reactive arthritis,
  osteomyletis and osteitis
• Viral for eg. rubella virus, human parvovirus,
  hepatitis B virus
• Fungal eg. Candida

7
DIAGNOSIS OF RHEUMATOID ARTHRITIS  

• PHYSICAL EXAMINATION
• Joint swelling tenderness
• Malignancy
• Loss of motion in joints

MEDICAL HISTORY
DIAGNOSIS

• LAB TEST
• Complete blood Count-
  Low WBC count suggests feltys 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

8
CONVENTIONAL THERAPY 

• DMARDS
• Methotrexate (MTX) , Gold
• Hydroxychloroquine
• Sulfasalazine
• Cyclosporine ,Azathioprine

NSAIDS (Used in early weeks)
Conventional therapy
SYNOVIAL REPLISHNERS (Glucosamine )
BIOLOGIC RESPONSE MODIFIERS Remicade
CORTICOSTERIODS
Approved in 1996, Enbrel (etanercept) is the
first biologic response modifier to receive FDA
approval for patients with moderate to severe
rheumatoid arthritis
9
LIMITATIONS OF ARTHRITIS THERAPY

• NSAIDS
• 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-a)
• Every year 1.5 of patient with rheumatoid
  arthritis are hospitalized with gastrointestinal
  problems

10
PROBLEMS IN PRESENT CONVENTIONAL THERAPY 
Short biological half life
Also affect normal cells of body
Low oral bioavailability
Decrease efficacy of dugs during chronic use
Conventional therapy
Cost of treatment is high
Minimum patient compliance
11
ADVANTAGES OF TOPICAL DRUG DELIVERY

• 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

12
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)
• Epidermis
• Dermis
• Hypodermis

13
PROBLEM IN PRESENT ANTI-ARTHRITIC THERAPY AND
PROPOSED STRATEGY FOR SITE-SPECIFIC DRUG DELIVERY
Carrier
14
ELASTIC LIPOSOMES/ FATTY ACIDS AS CARRIER SYSTEM
Lipid Bilayers

• 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
15
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
Acute arthritis
Interaction with relevant cells

• FCA enhances antibody production primarily
  because of the depot effect
• Nonspecific 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
16
DRUG INTRODUCTION
Chemical structure of methotrexate

• 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

17
DRUG INTRODUCTION
Chemical structure of glucosamine

• The daily oral dose requirement of glucosamine is
  1500mg/day
• Available in oral tablet and injectable form
• Oral bioavailability of drug molecule is just
  26(subjected to uptake and degradation by the
  liver )

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.
18
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
19
THE OBJECTIVES OF THE PROPOSED RESEARCH WORK

• To develop novel drug delivery system, which
  provide sustained and targeted delivery of DMRD
  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.

20
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
• Phase contrast microscopy
• Transmission electron microscopy
• 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)

21
METHODLOGY(cont.)

• 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 study
• Fluorescence 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

22
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

23
EXPERIMENTAL WORK DONE

• IDENTIFICATION (Drug selected for study)
• Ultraviolet Absorption Maxima (?Max)
• Methotrexate and glucosamine - 100µg /ml stock
  solution in distilled water
• Scanned- Between 200-400nm
  exhibits maxima at 267 nm
• Results are concordant with the value given in
  the official books (Merck Index, 1996).
• Infrared Spectral Assignment
• The IR spectra of MTX was recorded using
  (Perkin Elmer, IR Spectrophotometer).
• Nuclear Magnetic Resonance
• The NMR spectra of MTX was recorded using
  (Bruker, NMR).

24
PREFORMULATION STUDIES
PARTITION COEFFICIENT
SOLUBILITY STUDIES
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
S. No. SOLVENT SYSTEM PARTITION COEFFICIENT
1. n-octanol Distilled Water 1.222?0.13
2. n-octanol PBS (pH 74) 1.127?0.15
Table - Partition coefficient data of MTX
Table - Solubility Profile of MTX in Different
Solvents
25
PREFORMULATION STUDIES
S. No. Parameter Standard Observation
1 I.R. spectrum
2 0.002 w/v solution in HPLC grade methanol observed spectrophotometrically. Exhibit maxima at 302 nm. Exhibit maxima at 302 nm
3 Retention time methanol/acetonitrile/pH 5.4 buffer solution ) as mobile phase using C 18 column at the flow rate of 1 ml/min RT for MTX was reported as 9.5 min. (Pereira et. al., 2000 Seki et al., 1991) RT for MTX was found 9.8 min.
4 Melting range 255C 253-255C
5 Solubility Sparingly soluble in water, slightly soluble in chloroform and have good solubility in methanol and practically insoluble in ether. Sparingly soluble in water, slightly soluble in chloroform, soluble in methanol and practically insoluble in ether
Table- PREFORMULATION STUDIES
26
STANDARD CURVE OF MTX IN DISTILLED WATER BY UV
SPECTROPHOTOMETRIC METHOD
S . No. Concentration (?g/ml) Absorbance Regressed Absorbance Statistical Parameter
1. 2 0.0839 0.0839 Y 0.0739 x 0.1339 R2 0.9993
2. 4 0.1608 0.157 Y 0.0739 x 0.1339 R2 0.9993
3. 6 0.2339 0.2324 Y 0.0739 x 0.1339 R2 0.9993
4. 8 0.3098 0.3078 Y 0.0739 x 0.1339 R2 0.9993
5. 10 0.3879 0.3832 Y 0.0739 x 0.1339 R2 0.9993
6. 12 0.4579 0.4586 Y 0.0739 x 0.1339 R2 0.9993
7. 14 0.5294 0.5341 Y 0.0739 x 0.1339 R2 0.9993
8. 16 0.6074 0.6094 Y 0.0739 x 0.1339 R2 0.9993
9. 18 0.6852 0.6848 Y 0.0739 x 0.1339 R2 0.9993
10. 20 0.7637 0.7602 Y 0.0739 x 0.1339 R2 0.9993
Table - Standard Curve of MTX In
Distilled Water at ?Max 302 nm
27
STANDARD CURVE OF MTX IN PBS (pH 7.4) BY UV
SPECTROPHOTOMETRIC METHOD
S. No. Concentration (?g/ml) Absorbance Regressed Absorbance Statistical Parameter
1. 2 0.0796 0.0709 Y0.0343 X 0.0017 R2 0.996
2. 4 0.1410 0.1393 Y0.0343 X 0.0017 R2 0.996
3. 6 0.2076 0.2077 Y0.0343 X 0.0017 R2 0.996
4. 8 0.2758 0.2761 Y0.0343 X 0.0017 R2 0.996
5. 10 0.3428 0.3445 Y0.0343 X 0.0017 R2 0.996
6. 12 0.4082 0.4129 Y0.0343 X 0.0017 R2 0.996
7. 14 0.4747 0.4813 Y0.0343 X 0.0017 R2 0.996
8. 16 0.5499 0.5497 Y0.0343 X 0.0017 R2 0.996
9. 18 0.6317 0.6181 Y0.0343 X 0.0017 R2 0.996
10. 20 0.6886 0.6865 Y0.0343 X 0.0017 R2 0.996
Table - Standard Curve of MTX in
PBS (pH 7.4) at ?Max 302 nm
28
Fig.- Standard Curve of MTX in PBS (pH
7.4) at ?Max 302 nm
29
HPLC ASSAY OF MTX
S. No. Concentration (?g/ml) Peak area Statistical Parameter
1 0.0 0.0 y 148370x 288443 R2 0.9904
2 0.2 305375 y 148370x 288443 R2 0.9904
3 0.4 360342 y 148370x 288443 R2 0.9904
4 0.8 403575 y 148370x 288443 R2 0.9904
5 1.2 475400 y 148370x 288443 R2 0.9904
6 2.0 580025 y 148370x 288443 R2 0.9904
Table - Standard Curve of MTX in Distilled Water
by HPLC Method
30
Fig.- Standard Curve of MTX In Distilled
Water by HPLC Method
31
DEVELOPMENT OF VESICULAR CARRIERS - BASIC
PRINCIPLE
Table1 Size and entrapment efficiency of the
prepared oleic acid vesicles UF-1,2,3-ufasomes
with different molar ratio of drug
PC SURFACTANT
Fatty acid vesicles
PC FATTY ACID
32
VISUALIZATION OF ELASTIC LIPOSOMES
TEM ( X 1,80, 000) Photomicrograph of liposomal
formulation
Optical microscopy ( X 450) Photomicrograph
liposomal formulation
33
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34
Figure - TEM Photomicrograph of UF-3 formulation
of glucosamine
35
Size and entrapment efficiency of the prepared
oleic acid vesiclesUF-1,2,3-ufasomes with
different molar ratio of drug
Formulation code Oleic acid 5-MTX (Molar ratio) Entrapment efficiency Particle Size(nm) PDI
UF-1 91 (39.42.1) 505 15 0.367 0.037
UF-2 82 (45.42.1) 52312 0.468 0.037
UF-3 73 (51.04.2), 63217 0.262 0.037
UF-4 64 (49.42.7) 53116 0.489 0.037
UF-5 55 (48.42.4) 40413 0.581 0.037
36
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 91 (37.41.1) 525 15 0.367 0.027
UF-2 82 (55.41.1) 55312 0.368 0.017
UF-3 73 (49.03.2), 63217 0.262 0.036
UF-4 64 (49.42.7) 63116 0.489 0.033
UF-5 55 (48.42.4) 41423 0.681 0.047
37
Fig. 2. Differential scanning calorimetry traces
of oleic acid (a), oleic acid MTX vesicles (b),
oleic acid MTX 82 vesicles (c) and oleic acid
MTX 91 vesicles (d).
38
A
D
C
B
Figure MTX UF-3 with different concentration of
oleic acid
39
Optimized with span 20,conc of oleic acid 80,ph
7.4,80mM
B
A
figure ( A)-Optimized MTX, (B) Glucosamine
ufasomal formulation
40
At different pH,conc of oleic acid 90
pH 5.5
pH 7.4
pH 8.5
41
pH8.5
pH 6.5
pH 5.5
pH7.4
Figure 4. Photomicrograph of oleic acid vesicles
dispersion incubated at different pH (400
magnification).
42
Figure . Vesicle growth at low pH values 80mM
concentration of oleic acid
43
Figure Flourescence microscopy of optimized
formulation
44
Figure 17 Confocal microscopy of different
formulation
45
Figure 15 Histological View of Saggital Section
of Rat Knee Joint Samples.
46
Figure Histological View of Saggital Section of
Rat Knee Joint Samples.
47
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.

48
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

49
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