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.
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* 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.
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* 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.
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* 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.
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* 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.
