para: outside enteron: intestine (i.e. beside the intestine) These are the preparations which are given other than oral routes. These are  Sterile,  Pyrogen free preparations intended to be administered parenterally (outside alimentary tract). PARENTERALS

Advantages: o Quick onset of action o Suitable for the drugs which are not administered by oral route o Useful for unconscious or vomiting patients. o Duration of action can be prolonged by modifying formulation. o Suitable for nutritive like glucose & electrolyte. o Suitable for the drugs which are inactivated in GIT or HCl (GI fluid)

Disadvantages: Once injected cannot be controlled (retreat) Injections may cause pain at the site of injection Only trained person is required If given by wrong route, difficult to control adverse effect Difficult to save patient if overdose Sensitivity or allergic reaction at the site of injection Requires strict control of sterility & non pyrogenicity than other formulation.

Necessities of Parenteral preparations:  Sterility  Pyrogen  Free from particulate matter  Clarity  Stability  Isotonicity  Solvents or vehicles used must meet special purity and other standards.  Restrictions on buffers, stabilizers, antimicrobial preservative. Do not use coloring agents.  Must be prepared under aseptic conditions.  Specific and high quality packaging.

Parental Routes of Administration: Most Common: 1. Subcutaneous ( SC) 2. Intramuscular (IM) 3. Intravenous (IV) Others: 4. Intra-arterial (IA) 5. Intrathecal 6. Intraarticular 7. Intrapleural 8. Intracardial 9. Intradermal (Diagnostic)

Subcutaneous (SC;) The injection is given under the skin Need to be isotonic Upto 2 ml is given Using ½ to 1 inch 23 gauge needle or smaller needle Given: Vaccines Insulin Scopolamine Epinephrine

Intramuscular (IM): Striated muscle fibre 0.5 to 2 ml sometimes upto 4 ml 1 to 1.5 inch & 19 to 22 gauge needle is used Preferably isotonic Principle sites: Gluteal (buttocks) Deltoid (upper arms) Vastus lateralis (lateral thigh) Given: Solutions Emulsions Oils Suspension

Intravenous (IV):  Into the vein  1 to 1000 ml  1 inch,19 to 20 gauge needle with injection rate 1ml/ 10 sec. for volume upto 5 ml & 1 ml/ 20 sec. for volume more than 5 ml. Given:  Aqueous solutions  Hydro alcoholic solutions  Emulsions  Liposome

 IV infusion of large volume fluids ( ml) has become increasingly popular. This technique is called as Venoclysis.  This is used to supply electrolytes & nutrients to restore blood volume & to prevent tissue dehydration.  Combination of parenteral dosage forms for administration as a unit product is known as an IV admixture.  Lactated Ringer Injection USP  NaCl Injection USP (0.9 %)– (replenish fluid & electrolyte)  Dextrose Injection USP (fluid & electrolyte)

Intra-arterial (IA): Direct into the artery 2 to 20 ml 20 to 22 gauge Solutions & emulsions can be administered Given: Radio opaque media Antineoplastic Antibiotics

Intrathecal: Also called intra-spinal Directly given into the spinal cord 1 to 4 ml 24 to 28 gauge Must be isotonic Given: LA Analgesics Neuroleptics

 Intraarticular:  Given directly into the joints  2 to 20 ml  5 inch 22 gauge  Must be isotonic  Given:  Morphine  LA  Steroids  NSAID’s  Antibiotics

 Intrapleural:  Given directly into the pleural cavity or lung  Used for fluid withdrawal  2 to 30 ml  2 to 5 inch, 16 to 22 gauge needle  Given:  LA  Narcotics  Chemotherapeutic agents

Intracardial:  Directly given into the heart  0.2 to 1 ml  5 inch, 22 gauge needle Given:  Cadiotonics  Calcium salts as a calcium channel blockers

 Intradermal:  Also called as diagnostic testing  0.05 ml  ½ inch, 25 to 26 gauge needle  Should be isotonic  Given:  Diagnostic agents

Official Types of Injections: 1. Solutions of Medicinal Example: Codeine Phosphate Injection Insulin Injection 2. Dry solids or liquid concentrate does not contain diluents etc. Example: Sterile Ampicillin Sodium 3. If diluents present, referred to as.....for injection Example: Methicillin Sodium for injection

4. Suspensions "Sterile....Suspension" Example: Sterile Dexamethasone Acetate Suspension 5.Dry solids, which upon the addition of suitable vehicles yield preparations containing in all respects to the requirements for sterile suspensions. Title: Sterile....for Suspension Example: Sterile Ampicillin for Suspension 6. Injectable Emulsions: Example: Propofol injection

Formulation of Parenteral: 1.Therapeutic agents 2.Vehicles i.Water ii.Water miscible vehicles iii.Non- aqueous vehicles 3.Added substances (Additives) i.Antimicrobials ii.Antioxidants iii.Buffers iv.Bulking agents v.Chelating agents vi.Protectants vii.Solubilizing agents viii.Surfactants ix.Tonicity- adjusting agents

General steps involved 1. Cleaning 2. Preparation of bulk products 3. Filtration 4. Filling of solution in or product in ampoule or vial 7. Tests for Quality control 5.Sealing 6. Sterilization

2.Solvents: o Water o Should meet compendial requirements o Water miscible vehicles o Ethyl alcohol o PEG o PG o Non aqueous vehicles o Fixed oils  Solvents used must be:  Non-irritating  Non-toxic  Non-sensitizing  No pharmacological activity of its own  Not affect activity of medicinal

Added substances (Additives) Antimicrobials: Benzyl alcohol – 10 % Benzethonium chloride % Methyl paraben – 0.18 % Propyl paraben – % Phenol – 0.5 % Antioxidants: A) Reducing agent: Ascorbic acid – 0.1 % Sodium bisulphite--0.1 – 0.15 % Sodium metabisulphite – 0.15 % Thiourea % B) Blocking agents : Ascorbic acid esters-0.01 – 0.015% BHT – 0.02 % C) Synergistic: Ascorbic acid, Citric acid, Tartaric acid D) Chelating agent: EDTA %

Buffers:  Added to maintain pH,  Change in pH may causes degradation of the products  Acetates, citrates, phosphates are generally used. Eg: Acetic Acid Adipic Acid Benzoic Acid, Citric Acid Lactic Acid Chelating agents: Eg: Disodium edetate – % Disodium calcium edetate % Tetrasodium edetate – 0.01 %

Stabilizers: Creatinine – % Glycerin – 1.5 – 2.25 % Niacinamide – % Sodium saccharin – 0.03 % Sodium caprylate – 0.4 % Solubilizing agents: Dimethylacetamide, Ethyl alcohol Glycerin Lecithin PEG – 40 castor oil PEG – 300 Polysorbate 20, 40, 80

Tonicity- adjusting agents: Glycerin Lactose Mannitol Dextrose Sodium chloride Sorbitol LABELING: Name of product Quantity of the product % of drug or amount of drug in specified volume of amount of drug and volume of liquid to be added Name and quantity of all added substances Mfg. license no. Batch no. Manufacturer/Distributor Mfg. & Expiration date Retail price (incl. of all taxes) Mfger. address Veterinary product should be so labeled

STERILITY TESTING FOR PARENTERAL PRODUCTS  Sterility testing attempts to reveal the presence or absence of viable micro-organisms in a sample number of containers taken from batch of product. Based on results obtained from testing the sample a decision is made as to the sterility of the batch.  is made after the product exposition to the one of the possible sterilization procedures  can only provide partial answers to the state of sterility of the product batch under test  is inadequate as an assurance of sterility for a terminally sterilized product  The environment in which the test is conducted  The quality of the culture conditions provided  The test method  The sample size  The sampling procedure  avoid accidental contamination of the product during the test  the test is carried out under aseptic conditions  regular microbiological monitoring should be carried out

 Appropriate conditions for the growth of any surviving organism should be provided by the culture media selection.  Factors affecting growth of bacteria  Phases of bacterial growth  Culture media for sterility testing NNutrition MMoisture AAir TTemperature ppH LLight OOsmotic pressure GGrowth inhibitors

 capable of initiating and maintaining the vigorous growth of a small number of organisms  sterile  Types of media:  Fluid thioglycollate medium  Soya-bean casein digest medium  other media  composition  specific role of some ingredients  primarily intended for the culture of anaerobic bacteria  incubation of the media:  14 days at °C

 primarily intended for the culture of both fungi and aerobic bacteria  specific role of some ingredients  incubation of the media:  14 days at °C

 Membrane filtration  Direct inoculation of the culture medium  Appropriate for : (advantage)  filterable aqueous preparations  alcoholic preparations  oily preparations  preparations miscible with or soluble in aqueous or oily (solvents with no antimicrobial effect)  solutions to be examined must be introduced and filtered under aseptic conditions  All steps of this procedure are performed aseptically in a Class 100 Laminar Flow Hood  pore size of 0.45  m  effectiveness established in the retention of micro-organisms  appropriate composition  the size of filter discs is about 50 mm in diameter

 sterilization of filtration system and membrane  filtration of examined solution under aseptic conditions (suitable volume, dissolution of solid particles with suitable solvents, dilution if necessary…)  one of two possible following procedures:  the membrane is removed, aseptically transferred to container of appropriate culture medium  passing the culture media through closed system to the membrane, incubation in situ in the filtration apparatus (sartorius, millipore ).  suitable quantity of the preparation to be examined is transferred directly into the appropriate culture medium  volume of the product is not more than 10% of the volume of the medium  suitable method for aqueous solutions, oily liquids, ointments an creams

Scheme for sterility test by membrane filtration Scheme for sterility test by direct inoculation

 wide applications  a large volume can be tested with one filter  smaller volume of culture media is required  applicable to substances for which no satisfactory inactivators are known  neutralization is possible on the filter  subculturing is often eliminated  shorter time of incubation compared with direct inoculation SSelection of the samples SSample size

I Love The Rabbit!

Pyrogenic - means producing fever Pyrogens - fever inducing substances – Having nature Endogenous (inside body) Exogenous (outside body) – Exogenous pyrogens – mainly lipopolysaccharides bacterial origin, but not necessary Pyrogens PProduced mostly by gram-negative bacteria EEndotoxin - complex of pyrogenic lipopolysaccharide, a protein and inert lipid; llipid part of the lipopolysaccharide is the main pyrogenic agent; polysaccharide part increases solubility

Generalized structure of Endotoxins

 solvent - possibly the most important source  the medicament  the apparatus  the method of storage between preparation and sterilization  thermostable  water-soluble  unaffected by the common bactericides  non-volatile  These are the reasons why pyrogens are difficult to destroy once produced in a product  Test for pyrogens = Rabbit test  Bacterial endotoxins

 Reproducible pyrogenic response  Other species not predictable  Rabbit vs. dog as model?  Rabbits: false positives  Dogs: false negatives  Similar threshold pyrogenic response to humans  Rabbits must be healthy and mature  New Zealand or Belgian Whites used  Either sex may be used  Length of use  >48 hours within negative result  >2 weeks within a positive result  Must be individually housed between 20 and 23°C

 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 (glassware, syringes, needles)  retaining boxes (comfortable for rabbits as possible)  thermometers (standardized position in rectum, precision of 0.1°C)

 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 0.6  C is not used in the main test  main test:  group of 3 rabbits  preparation and injection of the product:  warming the product  dissolving or dilution  duration of injection: not more than 4 min  the injected volume: not less than 0.5 ml per 1 kg and not more than 10 ml per kg of body mass  determination of the initial and maximum temperature  all rabbits should have initial T: from 38.0 to 39.8  C  the differences in initial T should not differ from one another by more than 1  C

No.of RabbitsIndividual Tempt. rise (°c) Tempt. Rise in group (°c) Test 3 rabbits0.61.4Passes If above not passes 3+5 = 8 rabbits Passes If above test not passes perform the test again If above test not passes, the sample is said to be pyrogenic

 to detect or quantify endotoxins of gram-negative bacterial origin  reagent: amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).  The name of the test is also Limulus amebocyte lysate (LAL) test  the test is based on the primitive blood-clotting mechanism of the horseshoe crab enzymes located with the crab's amebocyte blood cells endotoxins initiation of an enzymatic coagulation cascade proteinaceous gel

 avoid endotoxin contamination  Before the test:  interfering factors should not be present  equipment should be depyrogenated  the sensitivity of the lysate should be known  Test:  equal V of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube  incubation at 37°C, 1 hour  remove the tube - invert in one smooth motion (180°) - read (observe) the result  pass-fail test  Three different techniques:  the gel-clot technique - gel formation  the turbidimetric technique - the development of turbidity after cleavage of an endogenous substrate  the chromogenic technique - the development of color after cleavage of a synthetic peptide-chromogen complex

 6 methods with different steps of accuracy of LAL test results:  Method A: gel-clot method: limit test  Method B: gel-clot method: semi-quantitative test  Method C: turbidimetric kinetic method  Method D: chromogenic kinetic method  Method E: chromogenic end-point method  Method F: turbidimetric end-point method  In the event of doubt or dispute, the final decision is made upon Method A unless otherwise indicated in the monograph.  allows detection or quantification of endotoxins  clotting of the lysate in the presence of endotoxins.  1.Preparatory testing  Confirmation of the labeled lysate sensitivity  Tests for interfering factors

 2. Limit test (method A)  procedure described on page. 24  a firm gel - positive result.  an intact gel is not formed - negative result.  the interpretation of the results  3. Semi-quantitative test (method B)  quantification of bacterial endotoxins in the test solution by titration to an end- point.  procedure is similar as in the limit test  The results are expressed as concentration of endotoxin as less, equal or greater than (labeled lysate sensitivity).  photometric test to measure the increase in turbidity  end-point test (Method F): quantitative relationship between the endotoxin concentration and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period.  kinetic test (Method C): a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of turbidity development.

 measuring the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the lysate  end-point test (Method E): is based on the quantitative relationship between the endotoxin concentration and the quantity of chromophore released at the end of an incubation period  kinetic test (Method D): a method to measure either the time (onset time) needed for the reaction mixture to reach a predetermined absorbance, or the rate of color development  Fast - 60 minutes vs. 180 minutes  Greater Sensitivity  Less Variability  Much Less False Positives  Much Less Expensive  Alternative to Animal Model  cheaper,  more accurate than other  is performed in the pharmaceutical laboratory  specific for endotoxins of gram-negative origin  particularly useful for:  Radiopharmaceuticals and cytotoxic agents  Products with marked pharmacological or toxicological activity in the rabbit (e.g. insulin)  Blood products which sometime give misleading results in the rabbit  Water for injection where LAL test is potentially more stringent and readily applied

Particulate Matter Monitoring  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 (A- 125)  Applicable for:  100 ml or more volume containers of single dose LV given by IV infusion  Not applicable for:  Multidose injections  Single dose SVP  Injectable solutions constituted from sterile solids Particle size in micrometer Max.No.of particles (equal to or larger than) per ml Nil

 Contamination  Contaminant  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…  Visual method  Coulter counter method  Filtration method  Light blockage method

 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.  Filtration method:  It is used for counting the particles in hydraulic fluids.  Sample passed thr’ filter  Material is collected on filter  Evaluated under microscope.  Disadvantage:  Skilled & trained person is required  Light blockage method:  Used for hydraulic oils  Allows stream of fluid under test to pass between a bright white light source & photoiodide sensor.

 Microscopy  X- ray powder diffraction  Mass microscopy  Microchemical tests  Electron microscopy etc…  Its presence may causes:  Septicemia  Fever & blockage of blood vessels  Quality of product may affect  As per USP  LVP : NMT 50 particles/ ml (size 10 or more than 10 micrometer) & 5 particles/ ml (size more than 25 micrometer)  SVP: 10,000 particles/ container of size 10 micrometer or greater & NMT 1000 particles/ container greater than 25 micrometer.

Refrences: Indian Phamacopoeia. Pharmaceutical Product Development by NK.Jain.

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