PERMEABILITY

In-vitro, in-situ and In-vivo method

Presented by : Samir S. Chavhan

[Link] II Sem
Industrial Pharmacy
Sudhakarrao Naik Institute of Pharmacy
 PERMEABILITY
Permeability is the ability of a drug (or any molecule) to pass through a biological
barrier, like cell membranes, intestinal wall, skin, etc.

High Permeability
If the drug is fat-loving (lipophilic) → it passes through the membrane easily.

Low Permeability
If the drug is water-loving (hydrophilic) → it struggles to pass.
 TYPES OF PERMEABILITY
(Based on crossing mechanism)

A) Passive Diffusion
• No energy needed.
• Drug moves from high concentration to low concentration naturally.

B) Facilitated Diffusion
• Uses special carrier proteins, but still no energy needed.

C) Active Transport
• Needs energy (ATP) to push drug across against the concentration gradient.
 ABSORPTION
Absorption is the process by which a drug moves from the site of administration
(like mouth, skin, or muscle) into the bloodstream.

STEPS INVOLVED IN ABSORPTION

1. Disintegration of dosage form (e.g., tablet breaks into smaller pieces)
2. Dissolution of the drug (drug particles dissolve in fluids)
3. Permeation through biological membranes
4. Entry into Bloodstream (systemic circulation)
PERMEATION IS A PART OF ABSORPTION.

ABSORPTION = PERMEATION + MORE STEPS.

(Transport, Metabolism At The Membrane, etc.)
Different Method Of Determining Drug Permeation
1. In vitro method
2. In vivo method
3. In situ method
 In-Vitro Method
In vitro" means experiments done outside a living body, usually in test tubes, petri
dishes, or laboratory instruments — not inside humans or animals.

In vitro methods help predict how a drug will behave before testing in living
organisms.

Purpose of In Vitro Methods:

To study solubility, permeability, and absorption properties.
To save time, cost, and reduce animal testing.
To optimize formulations before in vivo studies (animal/human studies).
 1. Shake Flask Method
Purpose:
To measure the partition coefficient (P or log P), which tells us how much a drug prefers
oil (lipid) vs. water (aqueous) environments.

Method:
a) A known amount of drug is added to a mixture of two immiscible solvents (usually n-octanol
and water).
b) The mixture is shaken vigorously and then allowed to settle.
c) Drug concentration is measured in both layers.
Partition coefficient (P) = Concentration in octanol / Concentration in water.

Importance:
Higher partition coefficient = Higher lipophilicity = Better membrane permeation.
 2. Equilibrium Solubility Method
Purpose:
To determine the maximum amount of drug that can dissolve in a solvent under equilibrium
conditions.

Method:
a) Excess drug is added to a solvent (water, buffer, etc.).
b) The mixture is stirred for several hours at a constant temperature.
c) After reaching equilibrium, the solution is filtered and the concentration of drug in the filtrate
is measured.

Importance:
Helps to understand if a drug will dissolve properly in biological fluids for absorption.
 3. Franz Diffusion Cell Method
Purpose:
To measure drug permeation across a biological or synthetic membrane.

Method:
The diffusion cell has two chambers:
1. Donor chamber (where the drug is placed)
2. Receptor chamber (where the drug that permeates is collected)
A membrane separates the two chambers.
Samples are collected from the receptor side at intervals to measure drug permeation.

Importance:
Commonly used for studying transdermal (through skin) drug delivery systems.
 4. PAMPA
(Parallel Artificial Membrane Permeability Assay)

Purpose:
To evaluate passive permeability of drugs through an artificial membrane.

Method:
A lipid-like artificial membrane is placed between two compartments (donor and acceptor).
The drug diffuses passively through the membrane.
Concentration in the acceptor side is measured over time.

Importance:
Predicts intestinal absorption and blood-brain barrier permeability without using real cells.
 5. Caco-2 Cell Model

Purpose:
To study intestinal permeability and predict oral drug absorption.

Method:
Caco-2 cells (derived from human colon carcinoma) are cultured to form a tight, monolayer sheet
similar to the human intestine.
Drug is added to the apical (top) side.
Its transport across the cell layer to the basolateral (bottom) side is measured.

Importance:
Gold standard for studying active and passive transport across the intestinal wall.
 6. Everted Gut Sac Method
Purpose:
To measure drug absorption across the intestinal wall using animal intestine.

Method:
A section of small intestine (usually from rats) is taken and turned inside out (everted).
It is filled with a buffer and placed in a drug-containing solution.
The drug’s transport into the sac is measured over time.

Importance:
Mimics in vivo intestinal conditions and allows study of absorption and metabolism.
 7. Artificial Membrane Permeation Assays
Purpose:
To study drug permeability using synthetic membranes instead of biological tissues.

Method:
Synthetic membranes (like cellulose acetate or PVDF) are used to mimic a biological barrier.
Drug diffusion across these membranes is monitored.

Importance:
Provides a simple, reproducible method for early-stage permeability screening.
 AT A GLANCE
1. Shake Flask = Partition Coefficient

2. Equilibrium Solubility = Solubility

3. Franz Cell = Skin/Barrier Permeation

4. PAMPA = Passive Membrane Permeability

5. Caco-2 Cells = Intestinal Absorption

6. Everted Gut Sac = Intestinal Absorption (animal tissue)

7. Artificial Membrane Assays = Synthetic Permeability Tests

 In Vivo
"In vivo" means "within the living.

"It refers to experiments conducted inside a living organism (animals, humans, plants).Purpose:To
study the biological effect of a drug, formulation, or treatment under natural physiological
conditions.

Importance
Provides realistic biological data.
Helps predict safety and efficacy before human use.
Essential for pharmacokinetics, toxicity testing, and drug development.
Types of In Vivo Studies
Pharmacokinetic Studies:(ADME - Absorption, Distribution, Metabolism, Excretion)
Pharmacodynamic Studies:(Effect of drug on the body)
Toxicity Studies:(Single-dose, repeated-dose, carcinogenicity)
Efficacy Studies:(Therapeutic action evaluation)

Common Animal Models

Rodents: Rats, mice
Non-rodents: Rabbits, dogs, monkeys
Special models: Zebrafish, fruit flies (for genetics)
 In Situ Method

In situ" means "at the original place.

"In situ methods involve conducting experiments directly at the site where the phenomenon
occurs, without removing the tissue or organ from the organism.

Purpose:
To study biological processes under near-physiological conditions but with better control than in
vivo.
Importance
Bridge between in vivo and in vitro studies.
Allows better observation and manipulation of specific organs/tissues.
Reduces variability compared to whole-body (in vivo) studies.

Common In Situ Techniques

1) In Situ Perfusion:
(e.g., Perfusing isolated organs like liver, intestine, kidney)
2) In Situ Hybridization:
(Detecting specific DNA or RNA sequences inside tissues)
3) In Situ Gel Formation:
(Drug delivery systems that form gels at the application site)
THANK YOU