Transcript UF-3

Microscopic evaluation of novel topical formulation
for treatment of Arthritis
Mohit
CHITKARA COLLEGE OF PHARMACY
CHITKARA UNIVERSITY, PUNJAB - 140401
INTRODUCTION
ARTHRITIS
Psoriatic Arthritis
Osteoarthritis
ARTHRITIS
Psoriasis of Skin
Rheumatoid Arthritis
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
Immune System
Gender
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
Infection
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
Genetics
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.
DIAGNOSIS OF RHEUMATOID ARTHRITIS
PHYSICAL EXAMINATION
MEDICAL HISTORY
Joint swelling &tenderness
DIAGNOSIS
Malignancy
Loss of motion in joints
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 Xrays, Joint ultrasound and bone densitometry -Measuring bone density used primarily to
detect osteoporosis
CONVENTIONAL THERAPY
DMARD’S
Methotrexate (MTX) , Gold
Hydroxychloroquine
Sulfasalazine
Cyclosporine ,Azathioprine
NSAID’S
(Used in early weeks)
Conventional
therapy
SYNOVIAL
REPLISHNERS
BIOLOGIC RESPONSE
MODIFIERS
(Glucosamine )
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
LIMITATIONS OF ARTHRITIS THERAPY
NSAID’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
Short biological half
life
Also affect normal
cells of body
Decrease efficacy
of dugs during
chronic use
Conventional
therapy
Low oral
bioavailability
Cost of treatment
is high
Several dose
dependent toxic effect
Minimum patient
compliance
Can’t maintain a constant
plasma level
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
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
PROBLEM IN PRESENT ANTI-ARTHRITIC THERAPY AND PROPOSED
STRATEGY FOR SITE-SPECIFIC DRUG DELIVERY
Carrier
ELASTIC LIPOSOMES/ FATTY ACIDS AS CARRIER SYSTEM
Modified lipid carriers that enable drug to reach
deeper skin layers.
Colloidal particles, typically consisting
phospholipids and surfactant molecules.
Lipid Bilayers
of
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
biocompatible.
Prolong the drug release.
are
biodegradable
and
Aqueous
Cavity
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
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
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 hydrosolubility 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 D EV E LO P NO V EL D RU G D ELIV E RY SYST E M, W HICH
P ROVI D E S US TAI NED AN D TA RG E TE D D E LIV E RY OF
D MR’ D TO T H E I R TA RG ET S I TE ( JO I NT S).
 TO
P REPA RE ,
CH A RA CT ERI ZE
AND
OP TIMI Z E
DI FF E RENT V ESI CUL A R FO RMU LATIONS ( ,FATTY A CI D
V ES I C LES , N I O SOMES , ELA S TI C LI P O S OMES )
 TO CA RRY O U T STA BI LI TY A ND SK IN P ERM EATIO N
S T U D I E S O F O P T I MIZE D V E S I CU L AR F O RMU L ATIO N .
 TO COMPA RE IN VIV O A N TI -A RTH RI TIC AC TI VITY OF
D EVELOP ED
V ESI CU LA R
F O RMULATION S
W ITH
MA RK E T E D F O RMU L ATIO N .
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)
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
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 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).
PREFORMULATION STUDIES
SOLUBILITY STUDIES
S. NO. SOLVENTS
1.
Distilled Water
2.
Phosphate Buffer Saline
(PBS) pH 7.4
3.
Methanol
PARTITION COEFFICIENT
SOLUBILITY
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
25 mg /mL
24.2 mg /ml
65mg/ml
Table :- Solubility Profile of MTX in Different Solvents
Table :- Partition coefficient data of MTX
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
255°C
253-255°C
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
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
2.
4
0.1608
0.157
Y= 0.0739 x – 0.1339
R2 =0.9993
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
STANDARD CURVE OF MTX IN PBS (pH 7.4) BY UV
SPECTROPHOTOMETRIC METHOD
S. No.
Concentration
(g/ml)
Absorbance
Regressed
Absorbance
1.
2
0.0796
0.0709
2.
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
Statistical Parameter
Y=0.0343 X + 0.0017
R2 = 0.996
Table :- Standard Curve of MTX in PBS (pH 7.4) at Max 302 nm
0.8
0.7
Absorbance
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
10
12
14
16
Concentration (g/ml)
Fig.:- Standard Curve of MTX in PBS (pH 7.4) at Max 302 nm
18
20
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
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
700000
y = 148370x + 288443
2
600000
R = 0.9904
Peak area
500000
400000
300000
200000
100000
0
0
0.5
1
1.5
2
Concentration (g/ml)
Fig.:- Standard Curve of MTX In Distilled Water by HPLC Method
2.5
Table1 Size and entrapment efficiency of the prepared oleic acid vesicles
UF-1,2,3-ufasomes with different molar ratio of drug
DEVELOPMENT OF VESICULAR CARRIERS BASIC PRINCIPLE
Fatty acid vesicles
PC + FATTY ACID
Transfersomes
PC + SURFACTANT
Liposomes
PC + CHOLESTEROL
Partially dehrdrate
Skin surface
Stratum corneum
(15% Water)
PC + SURFACTANT
“Transdermal osmotic gradient”
Dermal layer
(75% Water)
Applied always
nonocclusively
Transfersomes prevent
complete dehydration
Liposomes are less deformable therefore
they dehydrate completely and fuse
Due to deformability transfersomes pass
through narrow pores in the skin
Resulting in better
skin permeation
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
Size and entrapment efficiency of the prepared oleic acid vesiclesUF1,2,3-ufasomes with different molar ratio of drug
Formulatio Oleic acid: 5n code
MTX (Molar
ratio)
UF-1
9:1
Entrapment
efficiency
Particle
Size(nm)
PDI
(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 vesiclesUF1,2,3-ufasomes with different molar ratio of drug
Formulatio Oleic acid:
n code
Glucosamine
(Molar ratio)
UF-1
9:1
Entrapment
efficiency
Particle
Size(nm)
PDI
(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
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
B
Figure MTX UF-3 with different concentration of oleic acid
D
C
Optimized with span 20,conc of oleic acid 80%,ph 7.4,80mM
A
figure –( A)-Optimized MTX, (B) Glucosamine ufasomal formulation
B
At different pH,conc of oleic acid 90%
pH 5.5
pH 7.4
pH 8.5
pH8.5
pH 6.5
pH7.4
pH 5.5
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