2 ANTIBIOTIC DEVELOPMENT
The
latest progressive trend in the logistic features of antibiotic development may
be expatiated by the following sequence of objectives, namely:
(a)
To screen and evaluate different types of sources of microorganisms for
detection of purposeful antagonism.
(b)
To identify and select modified versions of microbial mutants, establish
optimal environmenta and nutritional conditions, and to develop appropriate
methods for recovering antibiotics from cultures.
(c)
To induce the production of particular desired metabolites.
(d)
To improve upon and modify the fermenatative metabolites either by the help of
chemical or biological manipulations to accomplish more useful antibiotic
products (compounds).
(e)
To develop detailed methods for ‘total synthesis’ of antibiotics from ab
initio for a feasible economic advantage, and
(f )
To make use of an adjunct agent to distinctly enhance the impact or
availability of an ‘antibiotic’.
2.1 Quest for New Antibiotics
In the
quest for new antibiotics, rather simpler, standardized and quicker
procedures have been developed and established for screening viable
microorganisms having antibiotic-yielding capability.
In
actual practice, however, the soil samples are the choicest candidates towards
an endeavour to identify the microbes for the simplest logical reason that they
are considered as the richest source of antibiotic-producing organisms.
Interestingly, majority of these organisms happen to be the bonafide members of
a specific class of branching, procaryotic microorganisms which essentially
retain a coveted status in their morphologic characteristic features between
bacteria and fungi. A survey of literature reveals that between early fifties
to late seventies the microbial sources of antibiotics discovered in Japan and
USA mainly comprise of actinomycetes (85%), fungi (11%), and bacteria (4%).
The
following are the summary of the most prominent genera and their taxonomic
relations.
In
general, nowadays a great deal of emphasis is being focused upon the pathogens
responsible for causing mostly incurable fungal and viral infections, besides
the bacterial infections, such as: methicillin-resistant Staphylococcus
and Pseudomonas species.
Following
are the various steps involved in the so called ‘general method’ for the
methodical screening of newer antibiotics, namely:
Step
I: Treatment
of the soil sample (or sample from other sources) by an antifungal chemical antibiotic,
cycloheximide which specifically checks the growth of interfering
bacteria and fungi but nevertheless affects the actinomycetes. Besides,
a diluted solution of phenol (1 : 140) may also be used as an antibacterial
agent.
Step
II: The treated sample, in their varying known dilutions are
subsequently streaked on agar plates containing medium (nutrients) which
augments and accelerates the growth of actinomycetes.
Step
III: The streaked agar plates are incubated for 3 to 7 days
between 25-30°C; and examined carefully for their characteristic colonies of
actinomycetes. After due physical identification these colonies are selectively
transferred onto fresh medium aseptically.
Step
IV: Well grown big cluster of colonies of the above selected
organisms are cut in such a manner that the ‘plugs’ comprise of both the
organisms and the underlying agar.
Note:
In case, the isolated organisms produces an antibiotic, it must normally
diffuse into the agar medium.
Step
V: The
‘plugs’ are meticulously removed and placed on an agar plate which has
already been seeded with a specific ‘test organism’ that clearly shows a
positive indication of the potential effectiveness and usefulness of the
antibiotic in question.
Step
VI: All the ‘test plates’ are duly incubated for a
stipulated temperature and duration required for the maximum (optimum) growth
of the ‘test organisms’. In case, there exists a clear zone of inhibition
around the ‘plug’ of the actinomycete, it may be inferred that an ‘antibioticcomponent’ is present in the ‘plug’ which obviously inhibited the
growth of the ‘test organisms’.
2.2 Large-Scale Production
Always,
the ultimate decision to carry out the large-scale production of a ‘new
antibiotic’ is based on several cardinal qualifying factors, such as: (a)
its chemical properties, (b) its physical characteristics, and (c)
its detailed biological activities.
However,
there are two extremely vital requirements for production, namely:
(i)
The organism should produce the ‘new antibiotic’ most preferably, in a
submerged culture as opposed to a surface culture, and
(ii)
The organism should liberate and excrete the ‘new antibiotic’ right into
the prevailing culture medium.
There
are, of course, some other important considerations also for the large-scale
production of a ‘new antibiotic’ that are of rather minor nature, such
as:
(i)
A few ‘antibiotics’ are produced in the cells of the organisms and
therefore, requires altogether special cost-involving extraction procedures for
their final recovery.
(ii)
Some other minor but equally important related considerations are, namely: minimum
inhibitory concentration (MIC) against the strains of pathogenic organisms,
chemical stability, activity in vivo, and lastly the toxic
manifestations in mammals.
The
most intricate, diligent and marvellous exploitation of the wisdom of the man
in the application of the in-depth knowledge of microbiology, biotechnology,
pharmaceutical chemistry, and engineering has ultimately opened the
flood gate towards the development of ‘newer antibiotics’ and their
commercial production to curtail the existing human sufferings.
The
various important sequential procedural steps that are essentially required for
the largescale production of antibiotics are stated as under:
(i)
Invariably requires growth of the producing organisms in aerated stainless
steel tanks with a capacity to hold thousands of gallons of the respective
nutrient medium.
(ii)
The fermentation process is duly initiated with the help of spores or
occasionally, vegetative growth from a pure stock culture** of the
organism.
(iii)
The inoculation of the huge fermentation tanks are normally accomplished by
carrying out successively the transfer of the organism to increasingly greater
volumes of nutrient. The major advantages of making use of a large standard
inoculum are as stated below:
(a)
Considerable reduction in the total incubation time required for the normal
production of
the
antibiotic,
(b)
Reduces importantly the slightest possible chance for undesired costly
contamination by foreign microorganisms, and
(c)
Caters for the best ever possible scope and opportunity for the entire control
and management of subtle nutritional and environmental factors that vitally
influence the ultimate yield of the antibiotic.
------------------------------------------------------------------
*
Based on the chromatographic, physico-chemical properties, antibiotic spectrum
and comparing the same to a database
of
previously identified compounds.
**
Stock Cultures: These are maintained very carefully (e.g., by
lypholization) that essentially require transfer as
infrequently
as possible, as repeated transfers may ultimately select only those cells of
the organism which are rather
poor
generators of antibiotic.
2.2.1 Phases in Fermentative Process
In
fact, there are two important and distinct phases normally encountered
in the fermentative process, namely:
(a)
Growth Phase of the Organism: It is also sometimes referred to as the ‘trophophase’;
wherein the number of organisms per unit time increases progressively, and
(b)
Idiophase of the Organism: In the idiophase there is a substantial
antibiotic production; and hence, invariably termed as the ‘antibiotic
production phase’.
The
above mentioned two phases in the fermentative process may be further
explained with the help of the following diagram:
In
this particular instance, both the growth phase and the idiophase in
the course of a typical ‘penicillin fermentation’ performed in a
culture-medium consisting of:
(i)
Source of carbon nutrition: – e.g., lactose and glucose;
(ii)
Nitrogen sources: – e.g., corn steep liquor; and
(iii)
Phosphate buffer: – to provide P in the medium and also to
maintain the pH of the medium.
The
observations from the above diagram are as follows:
(a)
The growth of microorganisms is shown in the above diagram by the curve
indicating an enhancement of mycelial nitrogen (Mycelial N). This particular
phenomenon continues right from the beginning (0 hours) of the culture period
to nearly one day (24 hours).
Note:
In the ‘growth phase’, the culture becomes thick by virtue of the formation of ‘aggregates
of fungal cells’ usually known as mycelium.
(b)
Glucose is preferentially consumed as compared to lactose specifically in the ‘growth
phase’, as it may be employed as a prime source of C directly.
(c)
Ammonia (NH3) gets liberated also in the ‘growth phase’ which
is caused due to the deamination of various amino acids present in the corn-steep
liquor (medium).
(d)
Release of NH3 evidently increases the pH of the medium from acidic
to almost 7 (neutral). Thus, the ideal and optimum pH necessarily required for
the stability of ‘penicillin’ is 7, which is maintained by adding
adequate ‘phosphate buffers’ into the medium.
(e)
The ‘penicillin production’ happens to rise very progressively and
rapidly between 24-48 hours.
Note:
Just in the initial stage of ‘penicillin production’, glucose gets fully
utilized, and
subsequently
the fungus makes use of ‘lactose’ as a source of C.
(f )
Interestingly, no additional growth takes place as the lactose cannot be used as
such unless and until it gets converted to glucose and galactose via hydrolysis.
Hence the prevailing decreased availability of C in the medium obviously offers
a ‘triggering mechanism’ in the production of penicillin.
2.2.2 Enhancing Yield in Large-Scale Production
During
the past half-a-century an enormous volume of intensive and extensive research
has been duly carried out by different groups/individuals across the world to
determine and establish the optimal nutritional and environmental parameters
required necessarily for antibiotic production. In reality, these conditions
are certainly not quite similar to those required for maximum vegetative growth.
The various factors that exert vital impact upon the qualitative and
quantitative antibiotic production are enumerated below:
* Sources
of nutritional C and N
* Ratio
of C/N in nutrients
* Mineral
composition of medium
* Temperature
of incubation
* Initial
pH, control and management of pH during the entire course of fermentation.
* Aeration
mode and rate
* Time-phase
for addition of special growth and antibiotic enhancing materials.
Empiric
Observations The selection of optimal fermentation parameters is not only
based on certain empiric observations but also serve as critical factors.
Examples
1. A
few strains of microorganism Bacillus subtilis give rise to the maximum
yields of bacitracin* at a C & N ratio of 1 : 15; but at a lower
ratio 1 : 6 it forms licheniformin** which happens to be a structurally
related but an undesired commercial antibiotic.
-----------------------------------------------------------
*
Bacitracin: Its antibacterial actions are similar to those of penicillin,
including Gram + ve cocci and bacilli and some
Gram
–ve organisms. Because of its toxicity when used parenterally, it is normally
used topically in ointment form.
**
Licheniformins: These are antibiotic substances usually produced by Bacillus licheniformis.
2. Phenylacetamide
or related substances when added to the culture medium of penicillin
production though exhibits a very negligible effect on the yield of penicillin
compounds, yet shows a very significant improvement upon the ultimate
composition of the penicillin mixture.
3. Phenylacetic
Acid Derivative’s inclusion as a part and parcel in the nutrient mixture
composition is observed to influence favourably the production of Penicillin
G; and this particular vital step has considerably minimised the tedious
problems with regard to the use of either unknown or variable composition of
mixtures; besides, the significant cost, time and energy involved unnecessarily
in separating the individual antibiotic substances.
4. Acyl
Moieties: The application of different acyl groups so as to achieve the
fermentative production of certain other penicillins, for instance:
phenoxy-methylpenicillin (or Penicillin V) could not achieve appreciable
feasible success in large-scale production; but surprisingly, the various
semisynthetic techniques evolved not only superseded this specific line of
action but also greatly enhanced the production of specialized penicillins.
5. Mercaptothiazole:
The incorporation of mercaptothiazole in cultures of Streptomyces aureofaciens
certainly approves the doctrine that certain ‘chemical additives’ might be
useful without necessarily being introduced into the antibiotic molecule
partially or fully.
6. Effect
of Enzyme Induction: It has been proved beyond any reasonable doubt there
are certain ‘chemical additives’ that may enhance the antibiotic
production by means of an enzyme induction effect.
Example:
Methionine when added to a cephalosporin C fermentation process,
during the growth phase of the organism (i.e., ‘trophophase’)
there is an apparent stimulation observed in the actual production of the
antibiotic. As methionine does not behave as a precursor to the antibiotic in
its biosynthetic process, in comparison to the performance of phenylacetic acid
in the biosynthesis of Penicillin G, one may conclude and infer with rather
stress and emphasis that methionine stimulates the ultimate production of
cephalosporin C biosynthetic enzymes.
7. Inhibition
of Antibiotic Production: Lysine exhibits an inhibition of penicillin
fermentation by its presence in the culture medium which ultimately retards the
antibiotic production. This particular phenomenon may, however, be explained by
the fact that both lysine and penicillin are the end products of a branched
biosynthetic pathway wherein the alpha-amino adipic acid serves as a ‘commonprecursor’. The production of ‘lysine’ is regulated and monitored by
two processes, viz., repression or inhibition of the requisite enzymes
needed for the production of alpha-aminoadipic acid. Hence, lysine puts a hault
of alpha-aminoadipic acid formation which finally causes a decrease in the
production of penicillin.
8. Mutation
and Strain Selection: Mutation* influenced and persuaded by virtue of
exposure of the parent-strain to uv-light, X-rays, or a host of mutagenic
chemical substances e.g., analogues of purines and pyrimidines, nitrogen
mustards (viz., mechlorethamine hydrochloride, mephalan,
cyclophosphamide, chlorambucil)** is widely recognized as the most virile
and versatile means for the selection of improved strains.
-------------------------------------------------------
*
Mutation: A change in a gene potentially capable of being transmilted to
offspring.
**
Kar, A., Medicinal Chemistry, New Age Internatural (Pvt) Ltd, New Delhi, 4th edn, 2006.
It is,
however, pertinent to mention here that a constant search across the globe of
natural sources for either newer wild-type(s) or various diversified species of
organisms that afford to yield the ‘antibiotic’ in much higher
percentage than the original one. In the particular instance of induced mutations,
lethal levels of the mutagen are adjusted in such a manner so that nearly
90-99% of the cells of the organism are destroyed (killed). Thus, the
high-antibiotic-yielding mutants are selected meticulously from the remaining
surviving cells.
Example:
Production of Penicillin: Initially, a penicillin antagonism was
noticed from a culture of Penicillium notatum Westling, that yielded a
meagre 4 mg L–1 of penicillin from its culture medium.
In
other words, no mutation of Penicillium notatum were ever observed in
the early selection process which could have given a significant yield of
penicillin in the submerged fermentation technique. In 1944, there was an
unique breakthrough in research whereby through the natural selection, a strain
of Penicillium chrysogenum Thom was invented that raised the yield of
penicillin almost by 10 times i.e., 40 mg L–1. Later on, with the help
of vigorous modification of mutation techniques amalgamated with
strain-selection, the ultimate yield of penicillin has been successfully
enhanced to 21,000 mg L–1.
The
recent quantum advancement in the field of molecular biology there has
been a tremendous expansion with specific reference to the knowledge of
molecular regulation related to antibiotic biosynthesis. In this manner perhaps
one may accomplish greater heights in the antibiotic production through such
measures as:
* Rational
manipulation(s) of the antibiotic-producing organisms to enhance its yield
significantly.
* Deregulating
the particular rate-limiting biosynthetic enzymes.
* Introduction
of additional ‘copies of genes’ matching the rate-limiting steps.
* Rational
implementation of specific genes for parallel/alternate biosynthetic routes.
* Production
of ‘hybrid-antibiotics’ through the fermentatively-generated structural
analogues of the natural antibiotic molecules.
2.2.3 Separation and Isolation of Antibiotics
Generally,
the large-scale-produced antibiotics are released rapidly right into their
nearest environment i.e., the nutrient medium, where they get
accumulated. However, there are some other instances e.g., the peptide
antibiotics, wherein the specific antibiotic is stored endocellularly (within
the cells); the fermentation is maintained unless and until the cells
accomplish an advanced matured physiologic age, the process of fermentation is
arrested (ceased) whereby majority of the cell membranes have either lost their
selective retention characteristic property or have undergone lysis—thereby
releasing the antibiotic into the surrounding medium. In other words,
therefore, the isolation process of various antibiotic substances is nothing
but purely a recovery from the culture broth. The various standard operating
procedures (SOPs) essentially comprise of: selective precipitation,
specific adsorption, or finally the chosen extraction with an immiscible solvent.
In
fact, in an ideal situation the very first isolation process must be as crisp,
selective and efficient as possible so as to achieve the maximum yield, besides
to help in subsequent purification without any cumbersome method. However, the
particular chemical characteristic feature of an antibiotic shall be the
ultimate determining factor, and also their accompanying metabolites to guide
and dictate the manipulative procedures which may be adopted effectively in any
particular instance.
Obviously,
a balanced compromise procedure that is economically viable and feasible shall
be the ‘ideal procedure’ for all practical purposes.
The
various means of extraction and purification of ‘antibiotic substances’ may
be accomplished through a number laid-down, tested and tried techniques that
shall now be discussed briefly as under:
(a)
Liquid-Liquid Extraction: Invariably the application of certain
water-immiscible organic solvents e.g., chloroform, solvent ether,
carbon tetrachloride etc., are exercised for the extraction of most
antibiotics. This particular process has evidently two major
disadvantages, namely:
(i)
Lacks high-degree of selectivity because majority of solvents, which are fairly
cheap and hence economical, tend to be employed on a large-scale production,
and
(ii)
Comparatively inefficient as most of the known ‘antibiotic substances’
are generally highly polar molecules.
Interestingly,
in most instances the above two serious drawbacks are easily
circumvented by adopting a chemical-engineered-flow process; but even
then the highly polar ‘antibiotics’ fail to separate in which the
partition-coefficient obviously favours the aqueous phase.
(b)
Recovery through Adsorption: Extremely polar antibiotics i.e., the
aminoglycoside antibiotics, such as: neomycin, streptomycin,
paromomycin, kanamycin, amikacin, gentamycin, tobramycin, netilmicin, are
normally recovered from the culture medium through adsorption on certain
appropriate adsorbent. It has been observed that—
* Most
adsorbents remove highly polar antibiotics from culture media with varying
degree of selectivity.
* Selecting
a suitable adsorbent offers major limitations by virtue of the fact that while
applying reversal of the adsorption process for recovering the antibiotic(s)
very careful and moderate conditions be applied so as to avoid its possible
denaturation/destruction.
* Ideally,
the application of controlled-activity grade charcoal as an adsorbent, and
subsequent elution with a dilute mineral acid (H2SO4) is
normally employed as an universal method of choice.
(c)
Chromatography-Recrystallization-Standard Manipulations: It is, however,
pertinent to state here that as soon as one is able to lay hands onto the ‘crude
antibiotic’ recovered from the culture medium (or nutrient broth), it
becomes absolutely necessary to accomplish the said product in its purest form
within the permissible attainable limits of purity. In order to achieve this
the ‘crude product’ is subjected to various advanced techniques of chromatography,
followed by meticulous recrystallization procedure, and ultimately subjected to
the standard manipulative operations using specific skill and wisdom.
Salient
Features Some of the salient features required to cause a suitable
extent of purification are:
1. The
attempt to achieve a very high degree of ‘chemical purity’ is neither
practicable nor necessary for therapeutic purposes.
2.
Foreign proteins i.e., extraneous metabolites, responsible for
undesirable side-effects are excluded automatically through the process of
purification.
3.
Complete separation/elimination of closely structurally related antibiotic
substances is invariably unfeasible.
4.
Antibiotics derived from various fermentative procedures most frequently
employed in therapy are, in true sense, admixtures of very intimately related
chemical entities having one of the metabolites predominantly present in the
mixture.
5.
Reproducible therapeutic response is of prime importance, which must be
attained through permissible practical limits due to the fact that a given
antibiotic compound always constitute a major component of the mixture.
6. It
also furnishes the economic viability of antibiotic substances in various drug
formulative operations by virtue of the fact that the inefficiency and total
expenses involved for complete separation of closely related chemical
substances having unequal relative concentrations, may be avoided to a great
extent.
Example:
Chlortetracycline present upto 6% in the commercial tetracycline fairly
represents an actual realistic and practical approach of such purification
considerations.
Note:
The overall accepted standards of purity for antibiotics and other antibiotic
formulations (i.e., dosage forms) are strictly controlled and monitored by the
pharmacopocia of various countries, such as: USP; B.P.; Eur. P.; Int. P.; Ind.
P.; Japanese P., etc.
(d)
Purity of Antibiotic: The highest attainable purity of an antibiotic is
an absolute necessity so as to minimise its undesirable side effects.
Example:
Vancomycin is a glycopeptide antibiotic particularly effective for the
treatment of endocarditis* caused by Gram +ve bacteria. However, its
wide application and usefulness was grossly restricted due to its nephrotoxicity.**
Interestingly, upon much improved purity status of vancomycin not only reduced
nephrotoxicity significantly but also raised its position in the therapeutic armamentarium.
(e)
Antibiotic Masking of Microbial Contaminants: The parenteral
preparations need to be guaranteed for their stringent sterility test(s) in
the presence of an antibiotic. Therefore, it has become almost necessary to
assess the masking of the very presence of the microbial contaminants by means
of the bacteriostatic action exerted by the prevailing antibiotic.
There
are, in fact, three basic approaches, which are not only vital but also
fundamental in nature, that may be employed so as to eliminate as far as
possible the ‘antibiotic masking’ of microbial contaminants, such as:
1. All
antibiotic formulations (dosage forms) which are essentially inactivated
promptly either by chemical or biological methods must be suitably treated
before carrying out the test for sterility.
Examples:
(a)
Inactivation of the enzyme penicillinase by Penicillin G, and
(b)
Inactivation of hydroxylamine hydrochloride by Streptomycin.
-------------------------------------------------------------
*
Endocarditis: Inflammation of the lining membrane of the heart. It may be due
to invasion of microorganisms or an
abnormal
immunological reaction.
**
Nephrotoxicity: A toxic substance that damages specifically the kidney tissues.
2.
Most parenteral antibiotic preparations, particularly those having the
relatively more stable ones, may be evaluated conveniently by subjecting the
preparations to such a level of dilution so that the ‘antibiotic level’ is
definitely below the minimum threshold concentration for its activity, and
3.
Physically removing, at the very first instance, any possible microorganisms by
the help of a sterile Millipore filter in such a manipulative manner such that
the organisms (undesired) are evidently separated from the antibiotic.
2.2.4 Sophisticated Skillful Antibiotic Preparations
A lot
of wisdom, skill and knowledge has been rightly incorporated in accomplishing
fairly stable sophisticated antibiotic preparations. There are various ways and
means that have been explored meticulously in order to achieve these
objectives, namely:
(a)
Shielding of relatively less stable antibiotics in gastric juice (acidic)
through various chemical and physical approaches,
(b)
‘Prodrug Approach’: Usage of rather insoluble corresponding antibiotic
analogues so as to get rid of objectionable taste, and thus make it more
patient-friendly especially in certain vital oral formulations.
Example:
Chloramphenicol Succinate/Palmitate—The bitter taste of
chloramphenicol is completely masked by preparing its corresponding esters for
use in suitable pareutral preparations.
(c)
Soluble/Insoluble Derivatives: The preparation of various soluble or
insoluble derivative of antibiotics are afforded so as to make it convenient
for its desired delivery at a particular site in vivo.
Example:
Gentamycin
sulphate, Neomycin sulphate, Tetracycline Hydrochloride, Penicillin G sodium
etc. These salts are more readily absorbed in vivo and hence enhance
their therapeutic efficacy.
It is
pertinent to cite here certain classical examples highlighting the
sophisticated skillful antibiotic preparations, namely:
(i)
Use of ‘buffers’ in oral penicillin G formulations significantly
minimise its loss of potency due to gastric juice,
(ii)
Enteric coating of erythromycin tablets with synthetic polymers, definitely
protect the macrolactone ring present in it, till it sails through the
entire distinctly acidic environment of the stomach (i.e., gastric
juice) and subsequently makes it pass into the long small intestinal canal
where it eventually gets absorbed.
Example:
The
two commonly used modified versions of erythromycin are, namely:
(a)
Erythromycin ethylsuccinate, and
(b)
Erythromycin estolate (i.e. the lauryl sulphate salt of the propionyl
ester).
These
two salts are very much insoluble than the parent macrolide antibiotic; and
provide dual usefulness in oral pareteral suspensions viz., first,
to refrain of their very bitter taste due to poor solubility; and secondly,
to protect their safe journey till the lower end of the intestine.
(iii)
Enhancing the solubility characteristics of erythromycin for allowing it to be
given intravenously could be accomplished by making its glucoheptonate and
lactobionate salts.
(iv)
Benzathine penicillin G possesses insoluble property, and this contributes
heavily as a stability factor for its usage in oral suspensions.
(v)
Penicillins give rise to insoluble procaine and benzathine salts that are used
extensively through IM route for prolonged and sustained effects.
(vi)
Probenecid is invariably employed as an adjunct substance to the penicillins;
and this affords two vital classical plus points: first, it
checks the tubular excretion of penicillins; and secondly, to accomplish
significant sustained blood levels of these antibiotics.
(vii)
Amoxicillin and Ticarcillin supplemented with a β-lactamase
inhibitor, clavulanic acid, in various preparations usually offers an
expanded therapeutic spectrum.
In
short, the classical examples enumerated above from (i) through (vii) paints a
beautiful rosy picture which further testifies the reality that a constant
research in the applications of different aspects of pharmaceutical technology
with a very strong bearing on the basic fundamental knowledge of medicinal
chemistry shall ever open the limitless boundaries of ‘wonderful drug
formulations’ to save the mankind of its sufferings.
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