Enzyme Inhibition Studies of Antipyrine & Aminopyrine

Abstruct
This report investigated the ability of Antipyrine & Aminopyrine to inhibit CA
& ChE enzymes. And the purpose and objectives of choosing CA and ChE
enzymes for this investigation. An introduction for pyrazolone was presented in
addition to antipyrine and aminopyrine drug characteristics. The results showed
that Aminopyrine was a better inhibitor of these enzymes compared with
antipyrine. Molecular docking results obtained from online docking server
swissdock supported the inhibition activity results.

Introduction
The Antipyrine and Aminopyrine drugs was used to inhibit the CA and ChE
enzymes. The figure 1 showed the structure for Antipyrine and Aminopyrine.

Figure 1. Structures of Antipyrine and Aminopyrine

The CA enzyme, Carbonic anhydrases (CAs) are enzymes responsible for life.
The sixteen (CA) isoforms found in humans are involved in numerous
physiological and pathological process. This make them important target for the
design of inhibitors and activators to treat wide range of disorders. The
treatment achieved by inhibition of (CA) was, edema, attitude sickness, obesity,
cancer, glaucoma, neuropathic pain, epileptic seizures.
The ChEs enzymes, Cholinesterases (ChEs) are enzymes which hydrolyze the
neurotransmitter acetylcholine (Ach). Two main types of (ChEs) are found in
vertebrates namely, acetylcholinesterase (AChE) & butyrlcholinesterase
(BuChE), which differ in their tissue distribution, substrate specify, kinetic
properties. In neurological disorders such as Alzheimer’s Desease (AD), the
levels of (ACh) in the brain is substantially minimized, which leads to impaired
communication between nerve cells and thus significant loss cognitive and
behavioural function. Cholinesterase inhibitors (ChEIs) prevent the rapid
breakdown of (ACh), allowing more of the neurotransmitter to be available for
neurotransmission which improve symptoms of (AD).
The objective is to quantificationally evaluate the inhibitory activity of
Antipyrine & Aminopyrine against the:

Human (CA)
isoform ChEs

hCA I AChE

hCA II BuChE
1.1 Pyrazolone
Is the name given to the Heterocyclic five membered ring which consist of two
Nitrogen atoms and Ketonic group. Since the end of the 19 th century, there has a
continuous interst in Pyrazolone derivatives. Compounds possessing the
Pyrazolone skeleton have more attention due to their wide rang biological
activities. The Pyrazolone derivatives include Antipyrine & Aminopyrine which
are well known as anti-inflammatory, analgesic and antipyretic drugs. These
drugs have been meticulously studied, their (CA), (AChE) and (BuChE)
inhibition activities have not been reported.

1.2 Antipyrine Drug

The drug can be identify according to properties listed in the Table 1.
Properties Description
Chemical Formula C11H12N2O
Pharmacodynamics Antipyrine is an analgesic and antipyretic that has
been given by mouth and as ear drops. Antipyrine is
often used in testing the effects of other drugs or
diseases on drug-metabolizing enzymes in the liver.
(From Martindale, The Extra Pharmacopoeia, 30th ed,
p29).
Mechanism of Action Antipyrine is thought to act primarily in the CNS,
increasing the pain threshold by inhibiting both
isoforms of cyclooxygenase, COX-1, COX-2, and
COX-3 enzymes involved in prostaglandin (PG)
synthesis.
Toxicity Not reported.
Side effect May occur redness, cough but no acute symptoms
expected.
Molecular weight 188.23 g/mol.

1.2.1 Pharmacokinetic of Antipyrine

 The Metabolism:

Antipyrine > Edaravone

Antipyrine Cytochrome P450 2C9 Endaravone Reaction Type
Cytochrome P450 2C8
Demethylation
Cytochrome P450 2C19
Cytochrome 450 1A2
 Antipyrine > 3 – Hydroxymethylantipyrine
Antipyrine Cytochrome P450 1A2 3 – Hydroxymethylantipyrine Reaction Typ
Cytochrome P450 2C9 3-Hydroxylation

Antipyrine > 4 - Hydroxyantipyrine

Antipyrine Cytochrome P450 3A4 4 – Hydroxyantipyrine Reaction
Type
Cytochrome P450 1A2 4-
Hydroxylation

Antipyrine > 3 – OH – Methylantipyrine

Antipyrine Cytochrome P450 1A2 3 – OH - methylantipyrine Reaction Type
Cytochrome P450 2C9 Aliphatic
hydroxylation

 Absorption, Distribution and Excretion:

Antipyrine is used in the test to provide an index of hepatic drug metabolizing

capacity. Properties of Antipyrine that make it suitable are as follows: rapid and
complete absorption from the gastrointestinal tract after oral administration.
(Federal register/ vil.49 no.168/ notices).
At clinical doses Antipyrine concentrations in plasma and saliva are identical
(first order kinetics) so that the Antipyrine test can be used noninvasively by
simple measuring salivary concentrations. (Federal register/ vil.49 no.168/
notices).
The apparent volume of distribution was calculated by given dose by the
extrapolated value of Antipyrine concentration at time zero. The metabolic
clearance calculated according to below equation:

Where :
T1/2: Antipyrine half-life
Vd : volume of distribution

(M. DANHOF & D.D. BREIMER, STUDIES ON THE DIFFERENT METABOLIC

PATHWAYS OF ANTIPYRINE IN MAN I. ORAL ADMINISTRATION OF 250,
500 AND 1000 MG TO HEALTHY VOLUNTEERS, Department of Pharmacology,
Subfaculty of Pharmacy, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The
Netherlands).
Following i.v and oral administration of Antipyrine 500mg to 6 healthy volunteers.
results from plasma and saliva showed that the oral bioavailability of Antipyrine given
as an aqueous solution was completed. the saliva/ plasma concentration ratio was
constant with time from about 3 h onwards. it is concluded the pharmacokinetic
parameters of Antipyrine can be satisfactory established on the basis of saliva results,
although the volume of distribution and clearance values are then slightly too high.
( M. Danhof, A. van zuilen, J. K. Boeijinga, and D. D. Breimer, Studies of different
metabolic pathways of antipyrine in man, europen journal of clinical pharmacology).
Four metabolites of Antipyrine were detected in the urinary excretion, 4-Hydroxy-
antipyrine, Norantipyrine, 3-Hydroxymethyl-antipyrine, 3-carboxy-antipyrine. (M.
DANHOF & D.D. BREIMER, STUDIES ON THE DIFFERENT METABOLIC
PATHWAYS OF ANTIPYRINE IN MAN I. ORAL ADMINISTRATION OF 250,
500 AND 1000 MG TO HEALTHY VOLUNTEERS, Department of Pharmacology,
Subfaculty of Pharmacy, Sylvius Laboratories, P.O. Box 9503, 2300 RA Leiden, The
Netherlands).
1.3 Aminopyrine
The drug can be identify according to properties listed in the Table 1.
Properties Description
Chemical Formula C13H17N3O
Mechanism Action Aminophenazone is pyrazolone metabolized very slowly
by normal new born babies. In older infants, a higher
amount of exhaled 13-CO2 is observed.
Pharmacodynamic Aminophenazone exhibits analgestic, anti-inflammatory,
and antipyretic properties.
Molecular weight 231.29 g/mol
Drug indication Formerly widely used as an antipyretic and analgesic in
rheumatism, neuritis, and common colds. Currently used
to measure total body water.
Pharmacology Aminophenazone exhibits analgesic, anti-inflammatory,
and antipyretic properties.
Toxicity Not reported
Side effect AMIDOPYRINE is known to cause
AGRANULOCYTOSIS in man, but it has been found
impossible to reproduce this effect in the dog & other
laboratory animals.

1.3.1 Pharmacokinetic of Aminopyrine

 Metabolism
Aminopyrine > [4 – (Dimethylamino) – 5 – methyl – 2 – phenyl – 1, 2 – dihydro –
3H – pyrazol – 3 – one]*
Aminopyrine Cytochrome P450 2C19 [ ]* Reaction Type
Cytochrome P450 2C8 N-dealkylation
Cytochrome P450 2D6
Cytochrome P450 2C18
Cytochrome P450 1A2
 Aminopyrine > N – Desmethylaminopyrine
Aminopyrine Cytochrome P450 1A2 N – Desmethylaminopyrine Reaction Type
Cytochrome P45 3A4 N-dealkylation
Cytochrome P450 2C18
Cytochrome P450 2C19
Cytochrome P450 2C8
Cytochrome P450 2C9
Cytochrome P450 2D6

 Absorption, Distribution and Excretion:

It is absorbed rapidly following oral admin..Excreted in urine uncharged or

conjugated with glucuronic & sulphuric acids. (Jones,L.M., et al. veterinary
pharmacology & therapeutics. 4th ed. Ames: lowa state university press, p.363).
Slightly bound to plasma protein. (goodman, L.S., and A. Gilman.(eds) The
pharmacological basis of therapeutics. 5th ed. New York: Macmillan publishing
Co., Inc., p347).
Three metabolites of Amiopyrine were detected in the urine, serum, and saliva
of health subjects after ingestion of Aminopyrine. (werner M, Werner D;
Agents Actions supplaas 10( Trends inflammation Res 2): 99.
2. Martials used
 Distilled water used for all experiments
 AChE lyophilisate from the electric eel.
 BuChE lyophilisate from equine serum.
 Antipyrine, Aminopyrine, solvents and all the compounds needed for
inhibition assays were purchased from Sigma Aldrich corporation and
used without purification.

2.2 Factors depended in Experiment and Methods used

 IC50: Concentration required to produce 50% inhibition. It is the
functional strength of the inhibitor and is not an indicator of its affinity.
 Ki : Reflects the binding affinity of the inhibitor.
  (IC50) was calculated from activity (%) vs [inhibitor] graphs. The
inhibition constants (Ki) were calculated from the Cheng Prussoff
equation (CHENG et al. 1973).
 The esterase activity for CA I & CA II was determined
spectrophotometrically (Thermo Scientific Evolution 200 Series UV-VIS
Spectrophotometer) according to method reported by Verpoorte and co-
workers (VERPOORTE et al. 1976).
 The inhibiting abilities of compounds 1 and 2 against AChE and BuChE
were evaluated by a slightly modified version of the method reported by
Ellman and co-workers (ELLMAN et al. 1961).

3. Molecular Docking
 The online docking server SwissDock (EADock DSS, Swiss
Institution of Bioinformatics, Switzerland, www.swissdock.ch).
 SwissDock is a protein ligand docking web service which effectively
predicts the molecular interactions that may arise between a protein
and ligand (small molecule).

4. Results and discussion

 hCA I and hCA II inhibitory activity

- The results indicated that 2 was approximately 1.3-fold stronger at

inhibiting hCA I and hCA II compared with 1.
- On the other hand, compounds 1 (IC50 = 42.20 nm) and 2 (IC50 = 29.90
nm) were 1.21-fold and 1.71-fold more effective at inhibiting hCA II.

 AChE and BuChE inhibitory activity

 - Compound 2 was a better AChE and BuChE inhibitor compared with 1.
- Compound 2 was 2.7-fold more potent than compound 1 against AChE
(IC50 = 96.10 nm for 1 and IC50 = 36.20 nm for 2).
- Compound 1 was a less active inhibitor of both ChEs.
- Similarly, compound 2 was less active at inhibiting BuChE compared
with the control drug.
- Compound 2, however, was approximately 1.7-fold more active
compared with rivastigmine (IC50 = 60.21 nm) against AChE.

 Molecular docking studies

- SwissDock identify the potential interactions of compounds 1 and 2 with

the amino acid residues within the CA I, CA II, AChE and BuChE
active sites.
- The estimated binding energy values (ΔG) for these interactions are
summarized in Table 3.The lowest free energy of binding was observed
for compound 2 with hCA II and BuChE

5. Results Summary
- Antipyrine & Aminopyrine were evaluated for their ability to inhibit hCA
I, hCA II, AChE and BuChE by depending on IC50 and Ki.
- Aminopyrine was a better inhibitor of these enzymes compared with
antipyrine.
- Molecular docking results obtained from online docking server swissdock
supported the inhibition activity results.
- The finding of this study is encouraging and these results may be taken
into account for the development of improved CA and ChE inhibitors.
- The study shed light on pyrazolone compounds as potential inhibitors of
CA or ChEs.