PHARMACEUTICAL BIOTECHNOLOGY

Name: Saumya S
Reg. no.: 15BBT0054
Date: 18/9/17
Assignment 1
INDIAN PHARMACOPEIA

BACLOFEN:

Molecular Formula: C10H12ClNO2

Mol. Wt.: 213.7
IUPAC: DL-4-Amino-3-P-chlorophenylbutanoic acid

INTRODUCTION:
Baclofen is a gamma-amino-butyric acid (GABA) derivative used as a skeletal muscle
relaxant. Baclofen stimulates GABA-B receptors leading to decreased frequency and amplitude
of muscle spasms. It is especially useful in treating muscle spasticity associated with spinal cord
injury. It appears to act primarily at the spinal cord level by inhibiting spinal polysynaptic
afferent pathways and, to a lesser extent, monosynaptic afferent pathways.

Pharmacodynamics:
Baclofen is a muscle relaxant and antispastic. Baclofen is useful for the alleviation of signs
and symptoms of spasticity resulting from multiple sclerosis, particularly for the relief of flexor
spasms and concomitant pain, clonus, and muscular rigidity.
Targets: Gamma-aminobutyric acid type B receptor subunit 1 and subunit 2 -
Component of a heterodimeric G-protein coupled receptor for GABA, formed by
GABBR1 and GABBR2. Within the heterodimeric GABA receptor, only GABBR1 seems to
bind agonists, while GABBR2 mediates coupling to G proteins. Ligand binding causes a
conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins)
and modulates the activity of down-stream effectors, such as adenylate cyclase.

Fig 1: Spinal cord and muscle showing the action of muscle relaxants. Black lines ending in
arrow heads represent chemicals or actions that enhance the target of the lines. Blue lines ending
in squares represent chemicals or actions that inhibit the target of the line.

Although Baclofen is an analog of the putative inhibitory neurotransmitter gamma-

aminobutyric acid (GABA), there is no conclusive evidence that actions on GABA systems are
involved in the production of its clinical effects. In studies with animals, Baclofen has been
shown to have general CNS depressant properties as indicated by the production of sedation with
tolerance, somnolence, ataxia, and respiratory and cardiovascular depression. Baclofen is rapidly
and extensively absorbed and eliminated. Absorption may be dose-dependent, being reduced
with increasing doses. Baclofen is excreted primarily by the kidney in unchanged form and there
is relatively large intersubject variation in absorption and/or elimination.

Mechanism of Action:
Baclofen is a direct agonist at GABAB receptors. The precise mechanism of action of
Baclofen is not fully known. It is capable of inhibiting both monosynaptic and polysynaptic
reflexes at the spinal level, possibly by hyperpolarization of afferent terminals, although actions
at supraspinal sites may also occur and contribute to its clinical effect.
Fig 2: Mechanism of Action of Baclofen.

Absorption:
Rapidly and almost completely absorbed from the GI tract.
Metabolism:
~ 15% of the dose is metabolized in the liver, primarily by deamination. 70-80% of the
dose is excreted unchanged or as metabolites in urine and the remainder is excreted in feces.
Half-life:
2.5-4 hours
Route of elimination:
In a study using radiolabeled baclofen, approximately 85% of the dose was excreted
unchanged in the urine and feces. Baclofen is excreted primarily by the kidney as unchanged
drug; 70 - 80% of a dose appears in the urine as unchanged drug. The remainder is excreted as
unchanged drug in the feces or as metabolites in the urine and feces.

IDENTIFICATION:
A. Determine by infrared absorption spectrophotometry: Compare the spectrum with
that obtained with baclofen RS. When examined in the range 220 nm to 320 nm, a 0.07
per cent w/v solution shows three absorption maxima, at 259 nm, 266 nm and 275 nm.
The specific absorbance at these maxima are 9.8 to 10.8, 11.5 to 12.7 and 8.4 to 9.3,
respectively.

B. Determine by thin-layer chromatography, coating the plate with silica gel G.

Mobile phase: A mixture of 5 volumes of anhydrous formic acid, 5 volumes of water, 20
volumes of methanol, 30 volumes of chloroform and 40 volumes of ethyl acetate.
Test solution: Dissolve 10 mg of the substance under examination in the mobile phase
and dilute to 10 ml with the mobile phase.
Reference solution: A 0.1 per cent w/v solution of baclofen RS in the mobile phase.
Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the
 plate and spray with ninhydrin solution until the plate is slightly wet. Place the plate in an
oven maintained at 100 for 10 minutes. Examine in daylight. The principal spot in the
chromatogram obtained with the test solution corresponds to that in the chromatogram
obtained with the reference solution.

TESTS:

1) Appearance of solution: Dissolve 0.5 g in 1 M sodium hydroxide and dilute to 25 ml

with the same solvent. The freshly prepared solution is not more intensely coloured than
reference solution BY5.
2) Chromatography technique
3) Assay: Weigh accurately about 0.15 g and dissolve in 50 ml of anhydrous acetic acid.
Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically. Carry
out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02137 g of
C10H12ClNO2.

TYPES:

I. Baclofen Oral Solution:

Baclofen Oral Solution is a solution of Baclofen in a suitable aqueous vehicle. Baclofen
Oral Solution contains not less than 95.0 per cent and not more than 105.0 per cent of the
stated amount of baclofen, C10H12ClNO2.

Identification:-

Determine by thin-layer chromatography, coating the plate with silica gel G.

Solvent mixture: 35 volumes of acetonitrile and 65 volumes of water.
Mobile phase: A mixture of 20 volumes of glacial acetic acid, 20 volumes of water and
80 volumes of butan-1-ol.
Test solution: Dilute a volume of the oral solution containing 5 mg of Baclofen to 100
ml with the solvent mixture.
Reference solution: A 0.005 per cent w/v solution of baclofen RS in the solvent mixture.
Apply to the plate 5 l of each solution. Allow the mobile phase to rise 10 cm. Dry the
plate in air. Place an evaporating dish containing a mixture of 4 ml of water, 1 ml of 7 M
hydrochloric acid and 0.5 g of potassium permanganate in a chromatography tank, close
the tank and allow to stand for 2 minutes. Place the plate in the tank, close the tank and
leave the plate in contact with the vapour for 1 minute. After removal of the plate, place it
in a current of cold air until an area of coating below the line of application shows only a
faint blue colour on the addition of 0.05 ml of potassium iodide and starch solution.
Spray the plate with potassium iodide and starch solution and examine in daylight. The
chromatogram obtained with the test solution exhibits a spot that corresponds to the spot
in the chromatogram obtained with the reference solution. In the Assay, the principal
peak in the chromatogram obtained with the test solution corresponds to the peak in the
chromatogram obtained with the reference solution.
II. Baclofen Tablets:
Baclofen tablets contain not less than 90.0 per cent and not more than 110.0 per cent of
the stated amount of baclofen, C10H12ClNO2.

Identification:-

Determine by thin-layer chromatography, coating the plate with silica gel G.

Solvent mixture: 4 volumes of absolute ethanol and 1 volume of glacial acetic acid.
Mobile phase: A mixture of 80 volumes of butan-1-ol, 20 volumes of glacial acetic acid
and 20 volumes of water.
Test solution: Shake a quantity of the powdered tablets containing 20 mg of Baclofen
with 20 ml of the solvent mixture for 30 minutes and filter.
Reference solution: A 0.1 per cent w/v solution of baclofen RS in the solvent mixture.
Apply to the plate 5 l of each solution. After development, dry the plate in air, spray
with ninhydrin solution and heat at 100 for 10 minutes. The principal spot in the
chromatogram obtained with the test solution corresponds to that in the chromatogram
obtained with the reference solution.

REFERENCES:

1. Indian Pharmacopeia
2. https://www.drugbank.ca/drugs/DB00181
3. http://www.uniprot.org/uniprot/Q9UBS5
4. https://www.ncbi.nlm.nih.gov/pubmed/1621492