Physiological Norms

Physiological Norm

When inject able solutions are formulated , effort should be made to mimic the body's

normal serum values for: PH , Isotonocity , to create a pyrogen free

preparation ,stable and free from any particulate matter

Physiological NormsPyrogenicityTonicityParticulate matterStabilityStorage

pH

pH is used to describe the degree of acidity of the solution . pH range from O-14.

Values below7 representing greater acidity , above 7 represent less acidity or greater alkalinity

A solution having pH about 7 neutral .Plasma pH is 7.4 and parenteral solution

should try to stay around that number.pH cannot seen , but tested after preparation

Tonicity

TonicityIV solution should be as close to istonic as

possible.A good reference point to remember is that :

0.9 % Na cl and 5% dextrose injections are both approximately isotonic

Pyrogens are products of metabolism of microorganisms.

The most potent pyrogenic substances (endotoxins) are

constituents (lipopolysaccharides, LPS) of the cell wall of

gram-negative bacteria (e.g., Pseudomonas sp, Salmonella

sp, Escherichia coli).

o Gram-positive bacteria and fungi produce pyrogens

but of lower potency and of different chemical

nature. Gram-positive bacteria produce peptidoglycans

while fungi produce glucans, both of which can cause

nonendotoxin pyrogenic responses.

1. Pyrogenicity

Since endotoxins are the most potent pyrogens.

Gram-negative bacteria are global in the environment,

especially water and the risk of their presence as

contaminants in sterile products

Side effects of Pyrogens,

if present in parenteral drug products and injected into

patients, fever, chills, pain in the back and

legs, and malaise.

While pyrogenic reactions are rarely fatal, they can cause

serious discomfort and, in the seriously ill patient, shock-like

symptoms that can be fatal.

Since endotoxins are water soluble and the monomer unit of

LPS can be 10,000 Daltons, endotoxins can pass through

0.22µm filters.

The intensity of the pyrogenic response.

o The degree of hazard will be affected by:-

the medical condition of the patient,

the potency of the pyrogen,

the amount of the pyrogen,

the route of administration (intrathecal >intravenous >,

intramuscular > subcutaneous).

When injected into humans in sufficient amounts, pyrogens will cause

a variety of adverse physiological responses.

The most common or recognizable response is an increase in body

temperature, from which the name “pyrogen” is derived (Greek “pyro” =

fire; “gen” = beginning).

Pyrogenic responses rarely are fatal unless the patient is very sick and

the dose is very large.

o Freedom from pyrogenic contamination characterizes parenteral

products in the same manner as sterility and freedom from

particulate matter.

o Preventing the presence of pyrogens is much preferred over removing

pyrogens in parenteral products.

o Preventing pyrogenic contamination primarily involves the use of

ingredients, solvents, packaging materials, and processing equipment

that have been depyrogenated.

o Initially, then using correct and force procedures during the

entire manufacturing process minimize the possibility of

pyrogen development.

Pyrogens contaminate biological materials because of their ability to

resist autoclaving as well as to pass through many filters.

Several techniques are used to remove them from

injectable products.

Strict control of the cleanliness of equipment and Containers,

distillation of water, and limited processing times.

Pyrogens may destroyed by being subjected to dry heat at

175°C for 3 hr.

Other pyrogens removal techniques (less effective) include:

filtration, absorption or ad- sorption, chemical oxidation

or combination of these.

Tests for pyrogenic activity

Test for pyrogens = Rabbit test

Bacterial endotoxins

Test for pyrogens = Rabbit test

The development of the test for pyrogens reach in 1920

A pyrogen test was introduced into the USPXII (1942)

The test consists of measuring the rise in body temperature in healthy

rabbits by the intravenous injection of a sterile solution of the substance

under the test.

Why the Rabbit?

Reproducible pyrogenic response

Other species not predictable

Similar threshold pyrogenic response to humans

Rabbit Pyrogen Test

Rabbits must be healthy and mature

New Zealand or Belgian Whites used

Either sex may be used

Must be individually housed between 20 and 23°C

Rabbit test

Selection of animals (healthy, adult, not less than 1,5 kg,…)

Housing of animals (environmental problems: presence of

strangers (unknown place), noise, T, …)

Equipment and material used in test (glass ware, syringes,

needles)

Retaining boxes (comfortable for rabbits as possible)

Thermometers (standardized

position in rectum, precision of

0.1°C)

Rabbit test

Preliminary test (Sham Test)

Intravenous injection of sterile pyrogen-free saline solution

To exclude any animal showing an unusual response to

the trauma (shock) of injection

Any animal showing a temperature variation greater than

0.5 oC is not used in the main test

All glass ware, syringes and needles must be pyrogen free

by heating at 250 oC for not less than 30 min.

Rabbit test -

Main test:

–Group of 3 rabbits

–Preparation and injection of the product:

Warming the product to 37±2 °C

Dissolving or dilution

Injection site: ear vein

The injected volume: about 10ml per kg of body

weight over 10min. duration

Record temperature at 30-min intervals for 3hours

Rabbit test

Interpretation of the results:

oThe test is carried out on the first group of 3 rabbits; if

necessary on further groups of 3 rabbits to a total of 4 groups,

depending on the results obtained.

oIntervals of passing or failing of products are on the basis of

summed temperature response.

No. of Rabbits

Individual Tempt. rise (°c)

Tempt. Rise in group (°c)

Test

3 rabbits0.51.4Pass

If above not passes

3+5 = 8 rabbits

0.53.3Pass

If above test not passes perform the test again

If above test not passes, the sample is said to be pyrogenic or go thr’ the sources of contamination of pyrogen.

Disadvantages of rabbit test

High variability in response.

Difficulty in controlling all factors.

Antipyretic drugs such asaspirin, acetaminophen and

morphine mask pyrogenic effect (i.e., misleading in

results).

Some other drugs have their inherent pyrogenic

Bacterial endotoxins

To detect or quantify endotoxins of gram-negative

bacterial origin

Reagent: Amoebocyte lysate from horseshoe crab

(Limulus polyphemu sorTachypleus tridentatus).

The name of the test is also Limulus amebocyte lysate

(LAL ) test

Limulus polyphemus = horseshoe

Mechanism of LAL

The test is based on the primitive blood-clotting mechanism of

the horseshoe crab

enzymes located with the crab's amebocyte

) from the blood)

Samples

(endotoxins)

initiation of an enzymatic coagulation cascade

proteinaceous gel

This method showed to be more sensitive, more rapid and easier to perform.

2. Particulate Matter

Unwanted mobile insoluble matter other than gas bubbles

present in the given product.

It may be dangerous when the particle size is larger than

R.B.C.& may block the blood vessel.

This type of products are immediately rejected from the

batch.

The limit test for particulate matter is prescribed in

I.P. 1996 (A-125)

Biological risk:

-Inflammatory response

-Antigenic response

-Occlusion of blood vessels

Sources of particles

–Excipients

–Processes

–Packaging materials

Sources of particulate matter

Intrinsic contamination:

–Originally present in products

e.g. Barium ions may react or leach with Sulphur ion which

are already present in formulation may produce barium

sulphate crystals.

Extrinsic contamination:

–Material comes from outside or environment

e.g. coming off the material from body & cloths of person

Entry of particle from ceiling, walls & furniture

May be in the form of cotton, glass rubber, plastics, tissues,

insect fragments, bacterial contamination, dust, papers etc…

Methods of monitoring particulate matter contamination

Visual methodCoulter counter methodFiltration methodLight blockage method

Visual method:

o Simple method

o Filled container are examined against

strong illuminated screen by holding neck

& rotating it slowly or inverted it to keep

out the foreign matter.

Coulter counter method:

o It is used for detection of particles less than 0.1 micrometer

in diameter.

o Based on electrode resistance.

o Sample is evaluated between two electrode & if particle

found the resistance of electrode is increased.

Filtration method:

–It is used for counting the particles in hydraulic fluids.

–Sample passed through filter

–Material is collected on filter

–Evaluated under microscope.

–Disadvantage:

Skilled & trained person is required

Significance of Particulate Matter monitoring

Its presence may causes:

o Septicemia

o Fever &blockage of blood vessels

Quality of product may affect

As per USP

–LVP (large volume products): Not more than 50

particles/ml (size10 or more than 10 micrometer) & 5

particles/ml (size more than 25 micrometer)

–SVP: 10,000 particles/container of size 10 micrometer or

greater & not more than 1000 particles/container greater

than 25 micrometer.

Stability

Stability of parenteral preparations must be assured so that patient receive the intended dose.

Hydrolytic and oxidation degradations are the most common forms of instability, but rarely shows as cloudiness or color changes

Rate of hydrolysis may be affected by: change in temp, pH of the solution .

Oxidation is affected by : temp. pH, light, oxygen and impurities( heavy metals )

Choice of packaging is also important for product stability

Storage

Monitoring the storage condition in the pharmacy is necessary to ensure that sterile products retain their respective quality attributes

Controlled Temp . storage areas such as , refrigerators, freezer, should be monitored at

least once daily with results documented

storage

1-Iv solutions, sterile commercial product, and suppliers should be stored according to manufacturer labeling or USP product monographs to preserve stability of ingredients

2 -Most sterile products are aqueous solutions for which hydrolysis is the most common chemical degradation reaction

storage

In general rate of chemical reaction increases exponentially for each 10 degree increase in temp.

Example: storage of B lactam antibiotic solution for one day at room temp. will have an equivalent hydrolytic effect of approximately 3-5 days at cold tem.

Cold also may cause harm as well Example refrigeration may cause precipitation , and

freezing may break an emulsion or denature proteins

Storage

Recommended storage conditions are usually stated on a product label and may include>

A specific temp. rangeDesigned place ( refrigerate )

Instruction as ( protect from light )These instructions should be followed carefully

In absence of specific instructions, a sterile product should be stored at controlled room temp. away from excessive or variable heat , cold and light