1. Physiological Norms

2. 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

3. Physiological Norms Pyrogenicity Tonicity Particulate matter Stability
Storage

4. 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

5. Tonicity
Tonicity
IV 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

6. Pyrogenicity 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).
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.

7. Since endotoxins are water soluble and the monomer unit of LPS can be 10,000 Daltons, endotoxins can pass through 0.22µm filters.
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.

8. The intensity of the pyrogenic response.
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.

9. Freedom from pyrogenic contamination characterizes parenteral products in the same manner as sterility and freedom from particulate matter.
Preventing the presence of pyrogens is much preferred over removing pyrogens in parenteral products.
Preventing pyrogenic contamination primarily involves the use of ingredients, solvents, packaging materials, and processing equipment that have been depyrogenated.
Initially, then using correct and force procedures during the entire manufacturing process  minimize the possibility of pyrogen development.

10. 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.

11. Tests for pyrogenic activity
Test for pyrogens = Rabbit test
Bacterial endotoxins

12. 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

13. 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)

14. 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.

15. 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

16. Rabbit test
Interpretation of the results:
The 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.
Intervals of passing or failing of products are on the basis of summed temperature response.

17. Individual Tempt. rise (°c) Tempt. Rise in group (°c) Test
No. of Rabbits
Individual Tempt. rise (°c)
Tempt. Rise in group (°c)
Test
3 rabbits
0.5
1.4
Pass
If above not passes
3+5 = 8 rabbits
3.3
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.

18. 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

19. 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

20. 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.

21. 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.

22. The limit test for particulate matter is prescribed in I. P
The limit test for particulate matter is prescribed in I.P (A-125)
Biological risk:
-Inflammatory response
-Antigenic response
-Occlusion of blood vessels
Sources of particles
–Excipients
–Processes
–Packaging materials

23. 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…

24. Methods of monitoring particulate matter contamination
Visual method
Coulter counter method
Filtration method
Light blockage method

25. Visual method:
Simple method
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:
It is used for detection of particles less than 0.1 micrometer in diameter.
Based on electrode resistance.
Sample is evaluated between two electrode & if particle found the resistance of electrode is increased.

26. 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

27. Significance of Particulate Matter monitoring
Its presence may causes:
Septicemia
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.

28. 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

29. 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

30. 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

31. 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

32. Storage
Recommended storage conditions are usually stated on a product label and may include>
A specific temp. range
Designed 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