S U B M I T T E D T OD r S I J U E . N

H O DD E P T O F P H A R M A C O L O G Y

P R E S E N T E D B Y

S H I J I N A N

1 S T Y R M . P H A R MP H A R M A C O L O G Y

Screening Methods for Arthritis

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Contents……

Introduction

Classification

Methods

Reference

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Arthritis is the inflammation of one or more joints, which results in soreness, swelling, stiffness, discomfort, and restricted movement.

It involves the breakdown of cartilage.

Cartilage normally protects the joint, allowing for even movement.

Cartilage also absorbs shock when pressure is placed on the joint.

Without the usual amount of cartilage, the bones rub together, causing soreness, swelling and stiffness.

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Causes of Rheumatoid Arthritis

RA is caused by a combination of genetic and environmental factors that trigger an abnormal immune response.

Possible causes: Genetic factors-Certain genes that play a role in the immune

system are associated with RA development. Defects in the immune system can cause ongoing

inflammation. Environmental factors-Certain infectious agents, such as

some viruses or bacteria, may increase susceptibility to RA. Other factors-Some evidence suggests that hormonal factors

may promote RA development in combination with genetic factors and environmental exposure.

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Types of arthritis

There are over 100 types of arthritis

Osteoarthritis - cartilage loses its elasticity. If the cartilage is stiff it becomes damaged more easily. The cartilage, which acts as a shock absorber, will gradually wear away in some areas

Rheumatoid arthritis - this is an inflammatory form of arthritis. The synovial membrane (synovium) is attacked, resulting in swelling and pain

Infectious arthritis (septic arthritic) - an infection in the synovial fluid and tissues of a joint. It is usually caused by bacteria, but could also be caused by fungi or viruses. Bacteria, fungi or viruses may spread through the bloodstream from infected tissue nearby, and infect a joint

Juvenile rheumatoid arthritis (JRA) - means arthritis that affects a person aged 16 or less. JRA can be various forms of arthritis; it basically means that a child has it

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antirheumatic drugs

1. Immunosuppressants:Methotrexate,Azathioprine, Cyclosporine2. Sulfasalazine3. Anti-malarials : Chloroquine or Hydroxychloroquine4. Leflunomide5. Gold, Auranofin6. d-Penicillamine

Screening Methods…….

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In vitro8

Modulation of cellular proteoglycan metabolism

Cellular chondrocytic chondrolysis

Cartilage explant chondrolysis

In vivo9

Canine anterior cruciate ligament (ACL) transection model

Chymopapain-induced cartilage degeneration in the rabbit

Spontaneous OA model in STR/1N mice

Modulation of cellular proteoglycan metabolism

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Tissue and cell preparation

Fetlocks of freshly slaughtered steers (age 18 to20 months) are skinned and the metacarpo-phalangeal joint opened under semi-sterile conditions.

Articular cartilage is then carefully removed

The tissue is washed with Ham’s F12 to remove adherent synovial fluid.

The pieces are then transferred into a 150 ml trypsinizing flask, containing serum

Incubated with gentle stirring for 1 h at 37 °C and 95%humidity

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Filtration –Centrifugation(800 rpm)

Resuspension and washing is performed with HBSS and cells are counted and checked for vitality under the microscope using the Eosin staining

Assay

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The assay starts 5 days after cell preparation. Compounds are added to the medium in a final concentration

The concentration can be varied according to the expected potency of the drug studied.

An untreated control group as well as standard compound

groups are included.

As standard compound,pentosane polysulfate to check for matrix increase,or retinoic acid to cause matrix decrease

At the end of treatment, the medium is removed,

wells washed with 500 μl of medium without supplements,and 1 ml/well of the staining solution is added

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After removal of the supernatant,

washing steps are performed for 10 min each:

• 3 × 500 μl/well 3% acetic acid,

• 1 × 500 μl/well3% acetic acid in 25% ethanol,

• 2 × 500 μl/well 50% ethanol,

• 1 × 500 μl/well70% ethanol.

After shaking the plates gently for 10 min

100 μl/well of each supernatant is then transferred to round-bottom microtiter-plates

The extinction photometrically assessed in the plate-reader at a

wavelength of 610 nm.

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EVALUATION

The extinction is expressed in percentage as staining

density with the control values defined as 100%. Values ≥ 110% are interpreted as stimulation of matrix formation,

values lower than 80% as inhibition of matrix formation. Experiments with 8 wells/treatment usually exhibit a standard deviation below 7%.

Cellular chondrocytic chondrolysis

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Assay The assay is started 6 days after tissue preparation Except for a control group, interleukin-1α is added in a

concentration of 8 U/well, Except for an IL-1-control group, compounds are added to the

medium in a final concentration The concentration can be varied according to the expected

potency of the drug studied. At the end of an 8 day treatment, the medium is replaced by a

medium and incubated for 24 h. The supernatant is removed Mixed 1 : 1 with 8 M GuHCl, and separated with a PD10-

Sephadex column .

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The samples containing the incorporated sulfate derived from medium as well as explant extraction are then mixed with scintillating fluid and assessed in a β-scintillation counter

EVALUATION

Counts per minute (cpm) from medium and gel fraction are calculated and related to the total well content.

The data are converted into percent incorporation, with the values of the untreated control group or those of the IL-1 control group serving as 100%.

Chymopapain-induced cartilage degenerationin the rabbit

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Intraarticular injection of chymopapain into the rabbit knee joint results in cartilage degradation with rapid loss of proteoglycans

Procedure

Animal:New Zealand White rabbits

18 mg chymopapain (Sigma) are dissolved in 1 ml 0.9% NaCl,and 50 mg l-cysteine-HCl is added for activation

The preparation is then passed under sterile conditions through a 0.22 μm Millipore filter, and tested for activity prior to each operation

One injection of 1.8 mg chymopapain in 0.1 ml per joint usually results in a proteoglycan loss/cartilage dry weight of ca 40% after 10 to 12 days.

2 to 4 animals per experiment receive chymopapain in 0.9% NaCl in one joint, and 0.9% NaCl into the contralateral knee to assess the proteoglycan loss caused by the enzyme.

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Care is taken with all injections to apply the same volume to both joints, as the contralateral serves as internal control.

If the drug is given orally, only one knee receives chymopapaintreatment, whereas if the drug is applied intraarticularly into one knee

both knees are treated with chymopapain.

The animals are sacrificed after 10 to 14 days,

And from defined regions of weight-bearing areas in the joint full thickness samples are obtained for histology and assessment of proteoglycan content

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EVALUATION

The histological appearance of the articular cartilage is assessed with a modified Mankin score, emphasizing more earlier changes, and separating degradative aspects, (e.g. loss of safranin staining),

The proteoglycan content is expressed as % change to the contralateral knee, or, comparing groups, to chymopapain-treated or untreated control groups.

To relate the proteoglycan content to cartilage dry weight has been found to be more reliable than choosing wet weight as baseline, as the cartilage water content is known to change in this model according to matrix composition.

Spontaneous OA model in STR/1N mice

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Beginning at the age of 10 weeks, male mice are trained to walk on a slowly rotating cylinder

recording the mean walking time of each mouse. Mice with a moderate activity (neither dropping off too soon,

nor staying on for hours) are then selected to enter the experiment.

In groups of 8 to 10 animals the drug is applied systemically for 8 weeks,

The mobility of each animal is recorded once or twice a week on the rotating cylinder.

The body weight is recorded regularly as well At the end of the experiment, the animals are sacrificed, Both knee joints dissected, fixed, decalcified, and embedded

in defined orientation for histology.

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EVALUATIONThe mean walking time decreases with age and disease progression, and is

recorded in a time-dependent graph over the treatment period.

Reference22

Drug Discovery and Evaluation by H.Gerhard Vogel

Pg no:775-787