advantages of the use of microbial biotransformation in the field of natural products. The microbial models for mammalian drug metabolism and applications in drug studies

1. By : Radwa Ahmed Taha

2.  Definition of microbial biotransformation
 Examples of industrially relevant
biotransformation and
biocatalytic processes
 Advantages of microbial biotransformation of
bioactive compounds

3.  It is the specific modification of a definite
compound to a distinct product with
structural similarity, by the use of
biological catalysts including
microorganisms like fungi or bacteria.
 The biological catalyst can be described
as an enzyme, or a whole, dead
microorganism that contains an enzyme
or several enzymes produced in it.

4.  Biotransformation is also known to comply
with the green chemistry strategy today.
 Green chemistry is a term used for
sustainable chemical industrial
manufacturing processes towards
achieving minimal waste production and
energy consumption

5. year process
5000 BC Vinegar production
800 BC Casein hydrolysis with chymosin for cheese production
1670 Orlean process for the industrial bio-oxidation of ethanol to acetic
acid
1934 Regioselective biooxidation of sorbite to sorbose for Reichstein
Vitamin C
synthesis
1950 Bioconversion of steroids
1970 Hydrolysis of penicillin to 6-aminopenicillanic acid
1974 Glucose to fructose isomerisation with immobilized glucose
isomerase
1990 Hydrolysis by protease (trypsin) of porcine insuline to human
insulin
1995 biotransformation of nicotinonitrile to nicotinamide

6. 1. The process of microbial transformation can operate at
near neutral pH, ambient temperatures and
atmospheric pressures, while chemistry often requires
extremes of these conditions which are not exactly
environmentally friendly and industrially undesired
2. extreme pH, temperature and pressure may provide
harmful effects towards personnel operating the harsh
procedures and may also affect community surrounding
the areas.
3. Regiospecificity and stereospecificity of the microbial
biotransformation process which allows the production
of chiral products of racemic mixtures.

7. 4. the ability of microorganisms like fungi and
bacteria produce large amounts of biomass
and a great variety of different enzymes in a
short time. Their small size has by far the
largest surface-to-volume ratio ever living on
the earth
5. Concerns regarding transmission of animal
based-diseases such as bovine spongiform
encephalopathy (BSE), scrapie , Kuru and
Creutzfeld-Jacob syndrome
ex : growth hormone purified from cadaver
pituitaries for dwarfism is now produced by a
recombinant Escherichia coli

8. 6. Several individual reactions can be
combined by single microbial reaction
7. Sometimes it is cheaper to use a
microorganism to prepare an organic
compound than to synthesize it chemically
8. Production of novel metabolites

10.  Importance of mammalian models
 General strategy for use of microbial models
 Biochemical basics for microorganisms as models for
drug metabolism studies
 Modes of bioconversion
 Advantages of microbial systems as models for drug
metabolism
 Microbial model of steroid metabolism
 Microbial oxidative metabolism of diclofenac
 Biotransformation of celecoxib using microbial
cultures

11.  Microbial biotransformation systems can be used
to complement mammalian drug metabolism
studies
 There is now increased availability of genetically
engineered microorganisms expressing human
drug-metabolising enzymes
 The use of microorganisms as metabolite
factories is a useful approach for biosynthesis of
regioselective and stereospecific products instead
of difficult chemical reactions

12. 1. There are difficulties in carrying out
metabolic studies in mammals because of
small amount of metabolites obtained by
these systems.
2. Such small amounts of mammalian
metabolites do not allow full structural
elucidation or biological evaluation.
3. it is not possible detect highly nascent
intermediate

13. General strategy for use of microbial models
-Suitable microorganism and culture conditions are
determined.
-A two-stage fermentation protocol used for study

15. 1. Cytochrome P-450 monooxygenases in
mammals:
Oxidative biotransformation in mammals
are mostly achieved by Cyt-P 450 linked
monooxygenases.
It mediates aromatic and aliphatic
hydroxylations, N,O,S, dealkylations, S, N,
oxidations ….. etc.
 Monoxygenase enzymes of fungi are similar
to those of mammals.

16. 2. Several fungi displayed NIH shift during
aromatic hydroxylation similar to hepatic
microsomes with the following differences:
a-Ortho in fungi ------- Vs para in hepatic
microsomes
b-Polar substrates are better utilized by fungi
while non polar substrates are better utilized by
hepatic microsomes.
3. These similarities are considered as support
for microbial models of mammalian
metabolism which can be defined as use of
microorganisms to facilitate study of drug
metabolism by mammals.

17. 1. Prospective: carried out in
microorganisms first and then
extrapolated to mammals.
2. Retrospective: carried out in
animals first and then in
microorganisms.

20. 1. Maintenance of stock cultures is simple and
cheap
2. Screening requires large number of strains to
metabolize the drug and is a simple
repetitive process, requiring only a periodical
sampling of incubation media.
3. metabolic capabilities of microorganisms
can be high, requiring the use of higher
concentrations of the drug. This allows easier
detection, isolation and structural
identification

21. 4. Novel metabolites can be isolated with
new or different activities.
5. There is a possibility of predicting the
most favored metabolic reactions.
6. The models can be scaled up easily for
the preparation of metabolites for
pharmacological and toxicological
evaluation.

22. 7. These models can be utilized in the
synthetic reactions where tedious steps
are involved.
8. The models can be useful in cases
where regio- and stereo-specificity is
involved.
9. In most cases relatively mild
incubation conditions are used.

25. production
of 4’-hydroxydiclofenac using
Epiccocum nigrum IMI354292

26.  The study aimed to use microbial
fermentatios to generate the 4’-hydroxylated
metabolite of diclofenac, a major metabolite
of this drug in man
 An initial screen of 11 microorganisms was
carried out (50 ml scale) to identify the
organism best suited to the regioselective
conversion of diclofenac to its 4’-
hydroxylated metabolite.
 the fungus Epicoccum nigrum IMI354292 was
selected as the most suitable microorganism.
2 g diclofenac was added to 30 L fermenter

27. Mammalian phase I metabolism of diclofenac

28.  4’- hydroxydiclofenac was found to be
the predominant metabolite produced
by the organism investigated as well as
in man according to in vivo studies.

30.  Celecoxib , a non-steroidal anti-
inflammatory drug (NSAID), is the first
specific cyclooxygenase-2 (COX-2)
inhibitor approved by FDA for the
treatment of osteoarthritis, rheumatoid
arthritis, and familial adenomatous
polyposis.
 Celecoxib is extensively metabolized by
the CYP2C9 isozyme in humans and rats to
produce the major metabolites by methyl
hydroxylation and its further conversion to
carboxylic acid.

32.  The metabolic pathway involves
oxidation of methyl group to produce
hydroxymethyl metabolite, which is
further converted to carboxylic acid.
 Since the drug celecoxib is lipophilic in
nature, it should be eliminated
predominantly by metabolism, and
hence, the study of metabolic pathways
is important.

33.  Bacterial, fungal, and yeast cultures were
employed in the present study to elucidate
the metabolism of celecoxib
 HPLC analysis of biotransformed products
indicated that majority of the metabolites are
more polar than the substrate celecoxib.
 The major metabolite was found to be
hydroxymethyl metabolite of celecoxib, while
the remaining metabolites were produced by
carboxylation, methylation, acetylation, or
combination of these reactions.

34.  The methyl hydroxylation and further
conversion to carboxylic acid was known to
occur in metabolism by mammals.
 The results further support the use of
microorganisms for simulating mammalian
metabolism of drugs.

35.  The media used for biotransformation studies were:
1. dextrose broth for fungi
2. nutrient broth for bacteria
3. MGYP (Malt Glucose Yeast extract broth) for
yeast
 The first stage culture was initiated in 50-ml culture
flasks containing 10 ml of sterile liquid medium and
inoculated with a loop of culture scratched from
freshly grown agar slant. The culture flasks were
orbitally shaken then placed at 30°C in refrigerated
shaker incubator
 The second stage cultures were added with 2 mg
each of celecoxib (in 100 μl methanol) to obtain a
final drug concentration of 0.2 g/l.

36.  after 10 days of incubation with celecoxib,
were extracted with ethyl acetate. The
combined ethyl acetate layers were
evaporated, and the dried samples were
reconstituted with HPLC grade methanol.
 The samples were centrifuged , the
supernatants were used for HPLC diode
array detection (HPLC-DAD) and liquid
chromatography tandem mass spectrometry
(LC–MS/MS) analyses.

37.  HPLC analysis of the extracts of the
cultures showed that 23 out of 39
cultures were able to metabolize
celecoxib to produce one or more
metabolites