Biotechnology has played an essential role in the development of the healthcare chemical industries. The range of products includes diagnostic prophylactic and therapeutic agents. The pharmaceutical industries are the largest manufacturers of these agents with chemicals in the latter two categories accounting for major markets. The search for on discovery of new drugs almost always involves a biological model of action related to disease. The discovery of a potentially active compound starts a sequence of exhaustive chemical and biological testing that may culminate in manufacture of the agent or an improved analog. The role of biotechnology in this complex path to regulatory approval and marketing is diverse often starting with preparation of the agents to be screened or the biological targets for action. Scale up to commercial manufacture of the compound or its procurers often involve biological processes as the route of choice to complex chemistries.
The Pharmaceutical Industries
The world market for healthcare drugs in 1981 was nearly $25 billion and rose to approximately $35 billion by 1983. Over 110000 tonnes of bulk medicinals were produced in the United States in 1980 relative to research and development expenditures of nearly $1.9 billion. Research and development expenditures for ethical products produced by the major US pharmaceutical manufacturers could exceed $2.7 billion in 1985.
Products in the pharmaceutical industry are typically high potency low volume and high value. The amount of product in an effective dose may range from less than a microgram per dose for live virus vaccines to tens of grams per patient day for antibiotic therapy. Annual manufacturing level for signification market penetration ranges from a few grams of live virus vaccine to about 100 000 kg of an antibiotic such as tetracycline. The value of a drug an article intended for use in the diagnosis prevention treatment mitigation or cure of disease in humans or animals may range to over $1 million per gram for a new biologic (e.g. a vaccine).
The path to product approval and licensure by the regulatory authority is arduous and long typically requiring seven to ten years to reach licensure of human healthcare products. This process is research intensive with major efforts spent on extensive clinical testing and development of commercial process technology. Establishment of proprietary rights protected by patent or license (usually with an exclusivity clause) is often a prerequisite for corporate commitment to develop a new drug. A primary goal of drug discovery programs is to identify new unique drugs that are safe effective and broadly patent able.
The general strategy is marketing human and animal healthcare drugs is to achieve market entry first with a new drug of high quality priced relative to the cost/benefit value of the treated disease. Market share in the human drug category can usually be taken from an established product by one with qualities that are clearly recognized as improved. Product differentiation and the manufacturer s reputation are major factors in establishing market share between similar competing there poetics. While the manufacturer s reputation may be the largest factor in establishing the market share for similar competing biologics first entry in the market place usually dominates the impact of early product differentiation. Although product characteristics (absence of side effects supplemental label claim color and form purity etc) seem to control the human healthcare drug market the animal healthcare market for food production seems to be clearly more sensitive to the price for benefit received.
Common features in the evolution of products and processes
Table 1 lists 11 classes of drugs of major commercial importance in human healthcare and five categories of importance in the animal healthcare market. Biological synthesis is the preferred route to most products of complex chemistry because of its efficiency selectivity and remarkable propensity for continued yield improvement. While biology and biotechnology have played important roles in discovering and developing drugs in virtually all of these classes biotechnology for commercial production is currently limited to relatively few of the marketed compounds (roughly 23% of the total sales for the human healthcare markets shown in Table 1). The largest single class of drugs made entirely or in part through biological processes is the antibiotics comprising nearly 90% of the system is anti-infective sales. Antibiotics are the largest selling group of drugs in the human healthcare market with cardiovascular agent and antiinflammatories running a distant second and third.
Most of the biological routes to pharmaceuticals involve aerobic submerged cultivation of microorganisms (fermentation). (The term fermentation refers in this context not to anaerobic cultivation as in the alcoholic beverage industry but to the general cultivation of microorganisms in liquid media.)
Fermenters in the pharmaceutical industry are low pressure vessels designed for high horse power agitation to facilitate dissolution of oxygen in the growth medium. Although many of the production fermenters in the industry are well over 20 years old the continual refinement of these vessels has kept them quite serviceable. The relatively short commercial period of patent protection for healthcare products combined with the high capital cost of building new fermenters has led the industry toward multiple use facilities rather than construction of new dedicated factories. The continual drive toward higher volumetric productivity has led to high oxygen demand. While most of the larger fermenters were designed on the basis of oxygen transfer modification of existing fermenters maybe facing new constraints. Higher volumetric productivity has often meant increased broth viscosity (with increased cell mass) and increased that load (increased power transfer from higher horsepower agitators and increased metabolic rates). Fluid builk mixing and heat transfer are becoming increasingly more important parameters for achieving high fermenter productivity particularly in older units. Wang provide a detailed analysis of fermenter design.
Product characteristics such a purity and form are controlled by purification and final isolation procedures. Human healthcare products are generally of high purity (usually greater than 95% and often over 98% purity). Where possible crystalline products are sought as a means of achieving high purity and desirable form (color stability dissolution rate). The ultimate use of these products contraindicates use of toxic separation agents in the final steps of product isolation. Fermentation products often have limited chemical and thermal stability. Generally the larger the molecular weight of the product the more gentle must be the recovery.
In 1928 Alexander Fleming s curiosity was around by zones of inhibition surrounding mold colonies contaminating aroused Petri dish. On further investigation he concluded that the mold had secreted a compound which further investigation showed to have activity against several pathogenic bacteria. The lytic filtrate of the mold Penicillin notatum he called infection and has led to the discovery and served as a model for the development of numerous pharmacologically active compounds produced by fermentation when penicillin manufacture began in the United States the process was to grow the P. notatum on the surface of a simple medium for 5 10 days and use the liquid underlying the culture which contained the penicillin in concentrations of 10 20 Oxford units ml-1 (an activity equivalent to 0.006 0.012 mg pure benzyl penicillin Na salt). A solvent extraction procedure was used to isolate material for clinical pharmacological and chemical characterization.
Experiments at the USDA Northern Regional Research Laboratory in Peoria; IL led to the development of the submerged culture method using a medium containing starch lactose and corn steep liquor and also discovered new strain P. chrysogenum NRRL 1951 (from a moldy cantaloupe) which led to all modern production strains. Moyer discovered that the addition of phenlacetic acid to penicillin producing cultures increased the antibiotic yield and changed the side chain from a mixture to predominantly penicillin G. The basic understanding of process kintetics and essential nutritional requirements was established in defined medium studies of Johnson.
Many different penicillins are produced by P. chrysogenum provided the appropriate carboxylic side chain is added to the medium in sufficient quantity. The original stimulatory effect of corn steep liquor was in part due to its providing additional quantities of a mixture of carboxylic acids which has the free methylene to the carboxyl which is necessary for incorporation by the organism. Many biosynthesized penicillins all having the structure shown in (I) have been identified but only penicillin G (R = Ph) and penicillin V(R = PhO) are important the therapeutically.
Both have a similar spectrum (Gram positive bacteria) and activity but whereas penicillin G is degraded by stomach acid and must be administered parenterally penicillin V is more acid stable and is orally administered. Penicillin V is relatively inactive against gonococci and penicillinase producing bacteria. See Table 1 for molecular weights and relative activities.
The technology of the manufacturing process is still regarded as highly proprietary by the major commercial firms even though excellent fermentation technology and organisms have been publicity avaliable for purchase for many years from Panalab Genetics and other. Similarly purification of these compounds is throughly described in the literature. The distinguishing aspects of commercial processes are of an operational character and cost advantages result from which of the well understood alternatives are implemented and with what degree of optimization and attention to detail.
The process involving culture maintenance fermentation and isolation is presented in Figures 1 and 2. Nutrient utilization and accumulation of microbial mass and penicillin are presented in Figure 3. In analyzing data of the type presented in Figure 3a quantitive approach which clearly presents rates of utilization and production is essential to understanding process behavior. Penicillin fermentation is such that substantial apparent improvements may result in little or no decrease in unit manufacturing cost and it is not always obvious where process improvement dollers should be expended. In this author s experience it is difficult to maintain a competitive position without some effort in each area of the process strain fermentation and separation.
Figure 1. Penicillin production fermentation
Basically the process involves cultivation of P. chrysogenum in vessels of increasing size up to 200 000 liters beginning with a slant or a vial of frozen vegetative mycelium. At harvest the batch is filtered to remove the mycelium and the filtrate is extracted and crystallized or acid precipitated.
The Culture Strain Development
Strain maintenance and initial seed
As shown in Figure 1 the process begins with lyophilized spores of a production strain. Alternatives are to store the master or secondary cell bank in the form of spore suspensions in liquid nitrogen or as frozen vegetative material with glycerol and/or lactose as suspending agents at either 70°C or under liquid nitrogen. Additional details regarding storage and maintenance of a master cell bank are given. Frozen spores (a standard number) are inoculated on slants and allowed to sporulate then are transferred to vegetative cultures in Erlemeyer flasks containing a vegetative medium generally similar to the production medium. Cultures stored as frozen mycelium avoid the introduction of variability which may result from sporulation.
Figure 2. Penicillin purification process of Gist Brocades
Figure 3. The time course of carbohydrate nitrogen penicillin and biomass concentrations during a penicillin fermentation.
Regardless of the culture maintenance details. Eventually the master lot will be exhausted and so before this occurs a secondary lot is prepared. It is common for a sporulation cloning and natural selection procedure to be carried out in which colonies are evaluated for their production capability and similarity to the parent in other criteria often including the degree of sporulation and some morphological traits. These evaluations include shake flask and tank fermentations and new lots are often run side by side against the parent in production trials before full approval is given by those responsible for production results.
It is not uncommon for some natural selections to exhibit slightly greater product formation than the parent but such increases are generally small. Because of the many factors which may affect the process and because small changes often seem to be ameliorated by a selection procedure a minimal strain maintenance/improvement effect is deemed essential to the long term success of a production effort.
Lein presents various culture presentation reisolation assay procedures and both seed and fermentation media used in a program in the early 1970s.
Lein diagramatically represented the pathway to secondary metabolites as DNA RNA enzyme primary metabolities + enzymes secondary metabolites calling attention to the complex multigene pathway which is strongly influenced by genetic and physiological factors. In practice antibiotic synthesis has been correlated with various complex physiological factors such as sporulation and growth rate and levels of various intermediates with in the empirical and various semi empirical approaches to mutation and screening must result in part from these characteristics and from the fact that even as we work out the details of the metabolic pathways isolate the enzymes involved and clone most of them (a task that will likely be complete with in one or two years) we remain ignorant of which are rate limiting in a particular industrial strain under the current physiological conditions enforced in the production fermenters. Until the middle 1960s UV irradiation and nitrogen mustard were the preffered mutagens in penicillin strain improvement studies as seen in Figures 4 and 5. Table 2 lists various mutagens which have been used. In recent years NTG a mutagen providing extremely high mutation rate relative to its killing effect has become the preffered compound for this reason and simply because its mechanism differs from the techniques previously emphasized NTG has the potential thereby of inducing different groups of mutational events.
Screening (e.g. examining all members of a population which has been mutagenized has been the most significant technique in raising penicillin yields to date. Table 3 lists media used for screening in the Panlabs program. One approach to improving the eficiency of this procedure is the potency index agar plate technique used in the improvement of high yielding cultures In this case the zone size was reduced by incoporating penicillinase in the agar to reduce the sensitivity to a useful range. Additional details on this and other approaches to strain improvement are presented by Ball.
The proven success of the empirical approach (mutation and screening for increased potency) notwithstanding this is never the less a tedious procedure. A specific selection procedure designed to identify those organisms possessing a desired phenotype (generally by allowing only that group to survive) can greatly increase the effectiveness of the program provided the trait selected for is related as expected to improved yield. One company Panlab Genetics Inc. established a multiclient penicillin strain development program in 1973. Participants paid a fee for access to strains developed by Panalab. Tables 4 and 5 present the result of the Panlab strain improvement program to 1980 in Oxford units (1667 Ou pen G Na mg 1 or 1595 Ou pen V Na ml 1). The strains were provided with protocols for their use in flaks and in pilot fermenters of up to 3000 liters. Lein has presented the program in considerable detail. Several different and clearly quite successful selective techniques are described. The improved yields presented in the tables were achieved largely through the sequential application of selective procedures involving high concentrations of amino acids biosynthetic intermediates and amino acid analogs. These selective techniques presumably lead to organisms with higher levels of amino acids and intermediates. Specific selections leading to the improvements in Table 4 and 5 are presented by Lein. Changes in colony morphology during the program are presented in Figure 6. Note the more clumpy character of later strains presumably due to rapid hyphal branching.
An important contminant of the high potency broth turned out to be the oxidized parahydroxy form of phenylacetic acid which when incorporated interferes with the semisynthetic chemistries. A clever selection technique used by Panlab involved choosing for further evaluation small colonies from plates with phenylacetic acid as sole carbon source except for just enough glucose to produce a small colony. Thus the small colonies selected were those imparied in the ability to oxidize the side chain precursor. The technique was successful and several commercial users of P14B 4 and its progeny no longer are concerned about interference by p hydroxy penicillin G. Also this loss of precursor is eliminated.
Novel b Lactam Antibiotics
From 1952 to 1975 novel b lactam antibiotics came almost exclusively from modifications to either the penicillin molecule or the cephalosporin molecule and more recently the cephamycin C molecule. Since that time the situation has changed. Several important new classes of naturally occuring b lactam antibiotics have recently been discovered which include thienamycin clavulanic acid norcardicin olivanic acid and the monobactams.
A combinations of reasons lies behind this sudden change in events. The nocardicins were discovered only after a test strain was developed which was hypersensitive to b lactam antibiotics. Both clavuanic acid and some of the olivanic acids were discovered using an enzymatic screen to detect inhibitors of b lactamase Thienamycin was discovered using a screen for detecting inhibitors of peptidoglycan synthesis. Only two cultures are known to produce thienamycin and these occur very infrequently in the soil. Coupling its rarity with the chemical instability of thienamycin served to prolong the purification and recognition of this important new antibiotic. Moder analytical techniques for determining structures from small amounts of materials have also played a crucial role in the discovery of some of these compounds because broth titers have often been very low in the original culture.
These discoveries stand as a testimony to the remarkable versatility of microorganisms to elaborate novel types of compound. The naturally occurring bicyclic b lactam antibiotics have only the b lactam ring in common. To this is fused a thiazolidine ring in the penicillins a dihydrothiazine ring in the cephalsoporins and cephamycins an oxazohdine ring in clavulanic acid and a pyrroline ring in the carbapenems. The carbapenenems have neither a sulfur nor an oxygen atom in their ring structure.
Fig.1. Structure of thienamycin molecule
Theinamycin was discovered by Kahan as a result of a screening procedure in which soil microorganisms were tested for their production of inhibitors of peptidoglycan synthsis. It is an antibiotic with an unusual and highly desirable antibiotic spectrum against both Gram positive and Gram negative bacteria. Its activity is undiminshed when tested against organisms which produce b lactamases and are consequently resistant to many penicillins and cephalosporins.
It is produced in the culture filtrate of Strpetomyces cattleya as a component of a complex of b lactam antibiotics including penicillin N cephamycin C and the N acetyl derivative of thienamycin. This species is rare in Nature and has been found only once in more than 10 000 isolates from soil. Until recently S cattleya was the only organism known to produce thienamycin. A recent patent claimed that S. perenifacins also produces this antibiotic.
Thienamycin is a zwitterionic compound with an acidic dissociation constant of ca. 3.1. The first derivatives were prepared when only small amounts of partially purified antibiotic were available. Using field desorption mass spectra of the antibiotic (MH+ 273) and high resolution mass spectra of certain derivatives the elemental composition was determined to be with a molecular weight of 272.
In contrast to well known penam and cephem antibiotics it contains no sulfur atom in the ring system and the two b lactam ring protons are trans to one another. The carbapenem nucleus was unknown prior to the discovery of thienamycin.
Thienamycin decomposes in dilute aqueous solution by apparent first order reaction and has greatest stability at neutral pH. However the decomposition accelerates as the antibiotic concentration is increased. This behavior is believed to be due to aminolysis of the lactam by the primary amine of a second theinamycin molecule. The semisynthetic derivative of N formimidoylthienamycin (Imipemide) overcomes this stability problems whicle retaining the full biological activity of thienamycin.
Imipemide appears to be the most potent and broad spectrum b lactam antibiotic reported. Minimum inhibitory concentration (MIC s) of 10 mh ml 1 and less were reported for both Gram positive and Gram negative bacteria including Psedomonas. It is fully effective against b lactamase producing strains at the same levels. Imipemide is not absorbed orally but is effective subcutaneously at levels of 0.005 0.2mg kg 1 for Gram positive and 2 10 mg kg 1 for Gram negative bacteria in mice.
There are a number of potential chemical synthesis routes for this product. Although the molecule is small it has not been easy to make chemically becuase of two main problems the construction of the bicyclic nucleus and the introduction of systituents at the 2 and 6 positions. Despite these difficulties remarkable progress has been made on this front and chemical synthesis may become the preferrd route for production.
Biosynthesis and Regulation of Thienamycin
The thienamycin molecule is illustrated in Figure1. The pyrrolinecarboxylic acid portion was found to be derived from glutamate.
In a typical fermentation after the rate of accumulation of thiemnamycin has fallen to zero resuspension of washed cells in buffer alone again permits antibiotic synthesis to resume. When antibiotic synthesis ceases in 36 48 h the cells can again be made productive by resuspension in fresh buffer. This phenomenon indicates that thienamycin may regulate its own synthesis by feedback inhibition. Experiments to prove this beyond doubt have not been clear cut. The natural tendency of the molecule to degrade creates a practical difficulty and contradictory evidence has been found in a resting cell system and in a thienamycin synthesizing protoplast system.
Lilley in chemostat studies found that thienamycin production by S. cattleya was highly growth dissociated and was only produced in detectable amounts during phosphate limited growth. Evidence of the growth dissociated synthesis can be seen in Figure 2 in which no thienamycin was produced during the growth phase (as measured by carbon dioxide evolution). Thienamycin synthesis only starts when rapid growth ceases and when the readily assimilable carbon source (in this case glycerol) is completely utilized. There appears to be both growth regulated synthesis and carbon catabolite repression of synthesis. The cessation of thienamycin synthesis and carbon catabolite repression of synthesis. The cessation of thienamycin synthesis coincides with a large increase in ammonium ion. Therefore there may be a correlation between ammonium concentration and thienamycin regulation in the range of ammonium concentration observed here (> 500 mg1 1 as ammonium). Alternatively this could be a result of chemical degraduation or an indication of cell lysis.
Thienamycin and cephamycin C are b lactam antibiotics produced by S. cattleya. CO2+ was found to have no effect on the growth of the culture but was found to be essential for thienamycin synthesis and had little effect on cephamycin synthesis. Foor et al. also found that FeCl3 and Na2SO4 were essential for thienamycin synthesis.
Foor demonstrated using studies on agar that cellular differentiation and antibiotic synthesis were not associated with one another. Aerial mycelial formation pigment formation and sporulation occurred at pH < 6.2 whereas antibiotic synthesis occurred in the pH range 6.5 7.5. In liquid fermentation studies if the pH is allowed to rise above 7.0 then very little antibiotic is accumulated. This is probably due to very high rates of theinamycin chemical degradation which occur in an alkaline environment.
Lilley have reported that synthesis of the antibiotic is controlled by inorganic phospate. Other reported results are less definitive. Perhaps becuase the high rate mutants were used for the studies. Foor et al. found that using phosphate at the concentration which just satisfied growth requirements (about 5 10 mM) resulted in maximum production of thienamycin. Further increases in phosphate concentration resulted in a decrease in production. The decrease however correlated quite well with a decrease in half life of the antibiotic. More recent data have provided a different interpretation of the role of phosphate in this fermentation and will be described in a future publication.
Foor determined that glucose supported growth equal to that seen with glycerol while ribose galactose D xylose mannitol gluconate and arabinase were increasingly less effective as carbon sources. No growth occured with glucose 6 phosphate fructose sorbitol or the disaccharides lactose maltose sucrose cellobiose or melibiose. Glutamate was fair as a carbon citrate succinate fumarate and malate failed to support growth.
Classical Fermentation Process
Imipemide is not yet commercially available and so is not being manufactured on a production scale. However information can be given for those procedures which were used to make thienamycin producer for use in clinical trials and this involved running fermentations at the 50 000 liter scale.
These are shown in Figure 3.
The thienamycin fermentation has proved to be unusually difficult to optimize for a variety of reasons. Under most conditions of growth absolutely no product is formed presumably as a result of carbon catabolite repression. Thienamycin synthesis also appears to be inhibited toward the latter part of the fermentation cycle by a yet to be determined mechanism. This limited the amount of carbon which could be added to the fermentaion and while feeding of glycerol had a stimulatory effect in the synthetic medium this was never obtained in the complex medium fermentation.
The media developed to date fall into two extreme categories. The complex medium of choice is particularly variable and difficult to work with whereas the synthetic medium while more elegant and invaluable for biochemical regulation studies is vastly more expensive becuase of a requirement for isoleucine. The complex medium was used for making product and pimarily consisted of solulac proflo corn steep liquor and glycerol. A comparison is made between the two media in Table1.
A detailed analysis of a typical fermentation batch at the 50 000 liter scale is provided in Figure 2. The kinetics of the fermentation are very unusual and under most conditions product formation is completely non growth associated. The batch cycle can be divided almost exactly into two parts. During the first half of the cycle rapid cell growth occurs and the viscostiy of the broth increases in parallel with increase in cell mass of S. cattleya. The phosphate concentration decreased to 80 Mgl 1 and the ammonium concentration decreases to zero. As the viscosity increases the KLa decreases to a minimum by the time of maximum oxygen transfer requirements. At this point the KLa is 60% of that available in the beginning of the cycle . At the beginning of the cycle cell growth presumably occurs using proteinaceous carbon and the culture switches to glycerol. Growth continuous until the glycerol is depleted. The whole fermentation radically changes at this point. The carbon dioxide evolution rate immediately drops by 16 relatively short time and then abruptly stops. This coincides with a rapid increases in ammonium concentration. The phosphate level is above 100 Mgl 1 during the period of rapid synthesis.
Streptomycin was the first member of an entirely new group of antibiotics called the aminoglycoside (aminocyclitol) antibiotics. The antibiotic was isolated from a species of Streptomyces by Waksman s group in 1944. Since that time many other aminoglycoside antibiotics have been isolated as fermentation products of soil microorganisms; neomycin kanamycin paromomycin gentamicin ribostamycin tobramycin spectionomycin micronomicin (formerly sagamicin) astromicin (formerly fortimicin A) validamycin etc. The number of antibiotics belonging to this class has been increased further by addition of semisynthetic derivatives which have biological activity. Many aminoglycoside antibiotics have been used successfully as therapeutic agents. Table 1 shows 13 aminoglycoside antibiotics available for therapeutic use at present. Among others astromicin is now in clinical trials. In 1981 the estimated world wise sales were approximately 500 million dollars. Gentamicin sales comprise about one half of this total. Figs 1 6 show the chemical structures of clinically important aminoglycoside antibiotics.
Aminoglycosides antibiotics are among the most potoent antibiotics known. They are broad spectrum active against both Gram positive and Gram negative bacteria as well as mycobacteria although they have no useful activity against anaerobic bacteria or fungi. The aminoglycosides are limited to parenteral routes of administration for the treatment of systemic infection. They are not absorbed when given by mouth but are excreted unchanged in the faeces. To varying degrees all of the aminoglycosides have some potential for toxicity to the kidney (nephrotoxicity) and the inner ear (eight nerve or otoxicity).
Table 1. Aminoglycoside Antibiotics used in Therapy
Figure 1. Neomycin and promomycin
Figure 2. Ribostamycin xylostasin and butirosin
All aminoglycoside antibiotics experience bacterial resistance usually following several years of extensive use. In many species of bacteria this resistance is determined by the presence of R factors which direct the synthesis of antibiotic inactivating enzymes. These enzymes inactivate aminoglycosides by three separate mechanisms; acetylation adenylation and phosphorylation. An understanding of these resistant mechanisms at a molecular level has led to the preparationof some semisynthetic derivatives resistant to enzyme inactivation. This approach has yielded two semisynthetic aminoglycosides amikacin and dibekacin already in clinical use and netilmicin in clinical trials.
Figure 3. Kanamycin and tobramycin
Figure 4. Gentamicin micronomicin and sisomicin
Figure 5. Steptomycin and dihydrostreptomycin
Figure 6. Astromycin
Streptidine and Deoxystreptamine
Glucose provides the carbon atoms of streptidine and deoxystreptamine the aminocyclitol units of streptomycin and many deoxystreptamine antibiotics respectively. However there are marked differences between the biosynthesis of streptidine and that of 2 deoxystreptamine. Streptidine was shown to be derived from myo inositol. The biosynthetic sequence from myo inositol to streptidine has been established by cell free enzymic studies as well as by istopic competition studies. Streptidine appears to be produced by two consecutive series of five similar enzymic steps from myo inositol oxidation amination phosphorylation carbamidinylation dephosphorylation. The mechanism of myoinositol formation has recently been elucidated in the biosoynthetic studies of actinamine which is also derived from myo inositol. Glucose 6 phosphate is first oxidized to a 5 ulose intermediate and the following aldol type cyclization starts with a stero specific abstraction of the prop R proton of the C 6 methylene group and leads to myo inosose phosphate. Subsequent education and hydrolysis gives myo inositol.
Exogenous myo inositol cannot be utilized to form the deoxystreptamine moiety of various antibiotics. Carbons 1 and 6 of D glucose label carbons 1 and 2 respectively of deoxystrepta Deoxy scyllo inosose (compound 1 in fig 7) is utilized by deoxystreptamine requiring idiotrophs (D mutants) to make gentamicin micronomican and butirosins and is probably the first cycle intermediate. Deoxy scally inosamine (compound 2 in fig 7) was shown to be the second cycle intermediate both by feeding experiments and by isolation of the intermediate using blocked mutants of the butirosin producer Bacillus circulans and a micronomicin producer Micromonospora sagamiensis. Thus it seems likely that the route involves amination of deoxy scyllo inosose to form deoxy scyllo inosamine followed by similar oxidation and amination at the C 1 position of deoxystreptamine. The formationof deoxy scyllo inosose has been shown to be completely different from that of myo inositol. Kakinuma proposed that the overall reaction from D glucose to deoxy scyllo inosose is a dehydration condensation sequence involving a hypothetical enrol (or enroll phosphate) intermediate. Another possible pathway including vibo quericitol is also proposed.
Figure 7. Proposed biosynthetic pathway for 2 deoxystreptamine and blocked steps of the D mutants of M. sagamiensis M.purpurea and M. inyoensis
The enzymic sequences involved in streptomycin biosynthesis have been extensively studied by cell free systems. And now the biosynthetic sequence of streptidine and streptose has been established in details. Though enzymes involved in the synthesis of N methylglucosamine have not yet been identified it can be estimated that about 28 enzymes take part in the conversion of glucose into streptomycin.
Figure 8. General scheme of streptomycin biosynthesis showing branch between idiabolic and trophabolic pathway and sites of experimental interventions. DSM dihydrostreptomycin; SD streptidine; Eth ethionine; A 5 Demain s mutant; NDP nucleoside diphosphate
Neomycin Paromomycin Ribostamycin and Butirosin
The extensive work of Rinehart and his group has shown that the individual subunits of neomycin and paromomycin are first synthesized and then subsequently joined together to form the complete molecule. The mode of assembly of the subunits is still a matter for conjecture. Pearce have demonstrated that neamine (which consists of neosamine coupled to a deoxystreptamine unit) can be incorporated into the neomycin molecule in S. rimous forma paromomycinus. From the postulated precursor neamine two possible routes to neto neomycin are available one by addition of aribose unit thus forming ribostamycin prior to conversion to neomycin the other by addition of a molecule of one obiosamine to produce neomycin directly. Baud have provided evidence in favour of the route via ribostamycin although the 13C studies by Stroshane have demonstrated the concurrent synthesis of neamine and neobiosamine during fermentation thus indicating the viability of the alternative route. In the biosynthesis of ribostamycin and butirosin in S. ribosidificus and B. cirulans ribose seems to be added last. More recently Autisser proposed a biosynthetic pathway of neomycin and paromomycin including new intermediate 6 deamino 6 hydroxy derivatives of neomycin and paromomycin which were accumulated in the culture broths of S.fradiae and S.rimosus forma paromomycinus. Putting together both the neomycin biosynthetic pathway postulated by Autisser et al. And the butiorosin pathway proposed by Takeda a biosynthetic pathoway for ribostamycin neomycin paromomycin and butirosin may be postulated as shown in figure 9.
Gentamicin Micronomicin and Sisomicin
Biosynthetic pathways of gentamicin and sisomicin have been proposed form the biotransformation experiments using idiotrophic mutants of M. purpurea suggest that gentamicin biosynthesis involved a branched point at gentamicin X2 with one branch leading to gentamicin (micronomicin) and the other leading to gentamicin C1 and C2. The identify of the biotransformation products was indicated by chromatographic evidence alone and should be confirmed by other techniques. The authors investigated the biosynthesis of micronomycin and gentamicin by using mutants blocked in various steps in the biosynthetic pathway. A variety of compounds was isolated form the culture broths or biotransformation mixtures with the mutants and identified from the spectral data. Based on the analyses of biotransformation and biochemical blocks in the mutants a biosynthetic pathway of micronomicin and gentamicin was proposed. In this scheme sisomicin group antibiotic (sisomicin verdamicin and antibiotic G 52) which are usually not produced by gentamicin and micronomicin producing microorganisms are involved in the biosynthesis of micronomicin and gentamicin. Actually a mutant which appeared tobe blocked at step L in fig.10 (4 5 dehydrogenation) was isolated from M. sagamiensis and found to produce antibiotic G 52 and sisomicin. Additionally a novel aminoglycoside antibiotic 6 N methylverdamicin was isolated form the transformation products of JI 20B or verdamicin with D mutants of M. sagamiensis.
Fortamine and Fortimicins
The biosynthetic pathway of fortimicins and fortamine containing the 1 4 diaminocyclitol unit of the fortimicin class of compounds has recently been elucidated by use of techniques similar to those used in the experiments on micronomicin biosynthesis. Odakura isolated a number of mutants which were blocked in fortimicin biosynthesis from astromicin (formerly fortimicin A) producing M. olivasterospora. Biosynthetic intermediates produced by the mutants were isolated and identified KY11554 KY11581 KY11583 Ky11556 KY11560 KY11555 KY11557 and KY11582 produced fortimician FU 10 AO KL1 KK1AP KH KR and B as a main product respectively. Based on cosynthesis and biotransformation analyses by use of the mutants and the silated compounds the biosoynthetic sequence was proposed as follows; FU 10AO KL1KK1 APKH KR B astromicin.
Tylosin is a 16 membered antibiotic produced commercially by strains of Streptomyces fradiae. Production of tyrosine by S.rimosus and by S. hygroscopius has also been reported. The initial isolation of two tylosin producing strains of S. fradiae from soil samples obtained from Nongkhai in the north eastern part of Thailand has been described by Hamill. Tylosin is composed of a branched lactone (tylonolide) and three sugars; mycarose mycaminose and mycinose.
Tylosin is used exclusively for animal nutrition and veterinary medicine. Its use is permitted as an improver of feed efficiency as a growth promoter for medicinal use in chickens and swine and for medicinal use in cattle. There have not been any reports detailing volume of sales of tyrosine but Perlman lists it amongst products with sales volumes greater than $50 million per annum. Perlman also lists Eli Lilly and Co. Indianapolis USA and Dista Products Ltd. Liverpool UK as two producers.
Structure of Tylosin and Related Compounds
The structure of tylosin is shown in Figures 1 and 2. Morin was the first to describe the structure of the antibiotic as consisting of a central 16 membered lactone ring with the three sugars mycarose mycaminose and mycinose attached. Mycinose is projected from C 14 of the lactone ring while mycarose is attached to the basic sugar mycaminose the resulting disaccharide being attached to the oxygen at the C 5 position of the lactone ring. Tyosin has been classified in the tylosin chalocomycin group) other members of the group including ciramycin A rosamycin angolamycin and V 58941.
Figure 1. Structure of tylosin
Figure 2. Structure of tylactone and related compounds (top) and of tylosin and related compounds (bottom)
During tyrosine production by Streptomyces fradiae other structurally related compounds were found to accumulate in particular macrocin relomycin and desmycosin. The structure of these compounds and other related compounds found on the biosynthetic pathway are shown in figure 2. Proof of the absolute configuration of tylosin has recently been provided by Jones.
The tylosin biosynthetic pathway can be divided into three main sections viz synthesis of tylactone synthesis of the sugar mycarose mycaminose and mycinose and the terminal stages in the pathway involving the conversion of tylactone to tylosin.
Synthesis of the tylactone is the initial stage in the pathway followed by addition of the sugars and modification of the lactone ring. Studies on the incorporation of BC precursors into tylosin indicated that the carbon skeleton of tylactone is derived form five propionates two acetates and one butyrate. Corcoran studied the biosynthesis of the lactone ring of the 14 membered macrolide eryhromycin and found that it was synthesized by a multienzyme synthase complex. Based on the evidence for the erytthromycin lactone ring. Masamune proposed that tylactone is formed by a mechanism similar to the synthesis of saturated long chain fatty acids. The work of Omura on the inhibition of lactone formulation by the antibiotic cerulenin adds further weight to this hypothesis.
The formation of mycarose is best documented of the three sugars occurring in tylosin. Pape showed that TDP mycarose is synthesized from TDP D glucose and S ade nosyl l methionine in Streptomyces rimosus fermentation s producing tylosin. The reaction required NADPH and has TDP 4 keto 6 deoxy D glucose as an intermediate together with a second methylated TDP sugar the structure of which is unknown . Formatioin of TDP 4 keto 6 deoxy D glucose is catalyzed by the enzyme. TDP D glucose oxidoreductase. Pape showed that the methyl group at C 3 or mycarose is transferred from methionine. The other two sugars mycaminose and mycinose are also derived form glucose and the methyl groups are transferred from methionine has suggested that the synthesis of mycaminose is similar to mycarose and has proposed a pathway.
Work in recent years has elucidated the terminal stages in the tylosin biosynthetic pathway. These workers made extensive use of mutants blocked in various steps in the pathway cofermentations using the blocked mutants bioconversion efficiencies for tylosin intermediates using mutants blocked in tylactone formation but with the remainder of the pathway either partially or wholly intact and analysis of the O methylation reactions in wild type and mutant extracts. The preferred biosynthetic pathway from tylactone to tylosin is shown in Figure 3. Following the fomation of the tylactone the first step is the addition of mycinose to C 5 followed by oxidation reactions of C 20 and C 23. Mycinose is then added to produce demethyllactenocin which is then glycosylated with mycarose to form demethylmacrocin. Demethylmacrocin is then methylated to macrocin whichis subsequently methylated to tylosin. Omura have propsoed a pathway where the route was from tylactone to O mycaminosyl tylactone to either 23 deoxy O mycaminosyl tylonolide or 20 deoxy 2 dehydrodemycinosyl tylosin both of which can be converted to O mycaminosyl tylonolide which is converted to desmycosin and then to tylosin. In a subseqent paper it was concluded that O mycaminosyl tylactone is first hydroxylated at C 20 which is oxidized to formyl followed by the hydroxylation of C 23.
Growth of Producer Microorganisms
A scheme along the lines of the following is usually adopted for the production of tylosin by Streptomyces fradiae
Several different formulations for agar slant media have been described. The slant medium described had the following composition (gl 1) glucose (10.0) phytone (10.0) agar (25.0) biotin (0.001) sodium this sulfte (1.0). Slants of this medium were incubated for 10 days at 28°C and then stored at 4°C until used. It has been reported that slant cultures held under refrigeration for longer than six weeks before use showed decreases in antibiotic yields. Spore suspensions obtained from agar slants are used to inoculate liquid vegetative medium which is formulated to provide consistent amounts of mycelial growth a terminal PH near neutrality and good yields of the antibiotic in the resulting tylosin production medium. Of the various vegetative media which have been described the most commonly described consists of(gl 1) glucose (15.0)cornsteep liquor (10.0) yeast extract (5.0 6.25) calcium carbonate (3.0 3.8). Aerobic growth on the vegetative medium was carried out for 48 hours and the resulting suspension of vegetative mycelia was used to inoculate the tylosin production medium.
Both complex and defined media have been described for the production media. Initial reports on tylosin production described a complex medium consisting of molasses nutrisoy flour distillers soluble and calcium carbonate. A more recent publication described a complex medium consisting of (gl 1) beet molasses (20.0) corn meal (15.0) fish meal (9/0) corn gluten (9.0) NACl (1.0) (NH4)2HPO4(0.4) calcium carbonate (2.0) crude soybean oil (30.0) (Baltx and seno 1981). Despite the lack of any detailed reports on the optimization of complex media it would seem from the limited information available that the following ingredients need to be present in a complex medium; a source of readily assimiable carbohydrate and a source of carbohydrate in the form of starch; an insoluble protein source; a source of mineral salts; and a source of lipid to supply energy and precursors during tylosin synthesis.
Stark published a detailed study on the effects of carbon source amino acids methylated fatty acids and inorganic components on mycelial growth and tylosin biosynthesis. The optimum medium developed by these workers contained the following (gl 1 except where stated) NaCl (2.0) MgSO4 (5.0) CoCl2.6H2O (0.001) iron (III) ammonium citrate (1.0) ZnSO4.7H2O (0.01) CaCO3 (3.0) Glycine (7.0) L alanine (2.0) L valine (1.0) betaine (5.0) glucose (35 0) (0 01) methyl oleate (25.0 ml l 1) K2HPO4 (2.3). Gray and Bhuwapathanapun (1980) modified the above medium by the substitution of calcium chloride for the calcium carbonate and sodium glutamate instead of glycine valine and alanine resulting in a soluble medium suitable for use in continuous culture if the methyl oleate is fed separately. Madroy have described a synthetic medium for tyrosine production consisting of glucose betaine glycine potassium nitrate magnesium sulfate calcium carbonate methyl oleate potassium phosphate buffer and trace elements.
There have been no reports on the kinetics of growth and tylosin production in high yielding fermentations on optimized complex media. Most reports on tylosin producing fermentations incubate the cultures at 28 30°C under aerobic conditions for 7 10 days in batch culture. Initial PH is usually in the range 7.0 7.8 with final pH values when quoted in a similar range.
Product Recovery and Purification
Tylosin can be recovered from fermentation broth by applying either adsorption or extraction techniques. Extractants which can be used include water immiscible polar organic solvents such as ethyl acetate and amyl acetate chlorinated hydrocarbons such as chloroform and water immiscible alcohol s ketones and ethers. For recovery of tylosin by adsorption a range of adsorbents and ion exchange resins can be used and then the tylosin can be eluted with an organic solvent. The organic solvlent extract can then be either evaporated to dryness to provide a crdud tylosin or concentrated in vacuo and a precipitant added. The formation of various salts of tylosin has also been described. The isolation of compounds after 23 deoxy 2 dehydro O mycaminosoyl tylonolide on the tylosin synthetic pathway (Figure 3) by methods suitable for organic solvent extractable basic compounds has been descirbed by Kirst. The fermentation broths were filtered extracted using amyl acetate or ethyl acetate at pH 9.0 9.5 and then back extreacted into water at about pH 4.0 to separate the products from organic solvent soluble neutral compounds.The pH of the aqueous phase was carefully controlled so as to prevent the hydrolysis of mycarose which is cleaved under acidic conditions from the products. The products were then either crystallized directly from the aqueous solution or extracted into a volatile solvent such as methylene chloride or ethyl acetate at pH 9.0 9.5 Evaporation of the solvent yielded a dry product which could be used direclty or further purified by crystallization. Further details on the separation by multistage countercurrent extraction of closley related compounds proudced by mutant strains of S. fradiae are described by Kirst. Vasileva described the recovery of tylosin from filtered S. fradiae fermentionatn liguor byextraction into either chloroform or dischloromethane at pH 9.5 and 2 5°C with at 95% efficiency. The same workers studied the effect of different extracting solvents and temperature on the efficiency of extraction of tyrosine and examined the reextraction from butyl acetate back into water at pH 4.0.
The isolation of tylactone and 5 0 mycarosyltylactone has been described by Jones. A mutant strain of Streptomycin fradide which accumulated the two compounds was grown on a complex medium. The broth was extracted with petroleum ether and the extract concentrated to an oil. The oil was dissolved in ethyl acetate heptane was added and the ethyl acetate allowed to evaporate slowly to permit crystallization. The crystals consist of a mixture of the two compounds which were separated by silica gel chromatography.
Figure 3. The preferred biosynthetic from tylactone to tylosin. The compounds listed in sequence are tylactone O mycaminosyl tylactone 23 deoxy 20 dehydro O mycaminosyl 23 deoxy O mycaminosyl tylonide O mycaminosyl tylonolide demethyllactenocin demethylmacrocin macrocin tylosin
The present modern era of antibiotics research is characterized by advanced screening procedure aiming specifically not only at antibacterial or antifugals but at compound like enzyme inhibitors immunomodulators and antitumor drugs. New sources like rare microorganisms (actinomycetes pseudomonales) or various marine organisms are currently at the beginning of exploitation. Significant improvements have been obtained by semisynthetic derivatives of natural compounds. Concerning peptide antibiotics b lactams still have the major part of the market. Since these have been treated earlier in this volume we focus on recent trends of the extended concept of antibiotics and biosynthesis implication for the production of modified compounds. Less attention will be given to chemical aspects of drug improvement.
CURRENT APPLICATIONS OF PEPTIDES
Peptides that are currently produced on an industrial scale are listed with their applications in Table 1. To illustrate a few general approaches in product evaluation some notes on blasticidin S (agriculture antibiotic improvement by antagonists) bleomycin (antitumor agent improvement by semisynthesis) bestatin (immunoenhancer used in combination with bleomycin) and cyclosporin (isolation from rate microorganisms) have been included.
Blasticidin S an agricultural antibiotic
Blasticidin S the first successful Japanese agricultural antibiotic is effective against the rice blast pathogen Pyricularia oryzae. Its properties have been summarized in a review on agricultural antibiotics by Misato. The benzylaminobenzenesulfonate being less phytoxtoxic to the host plant has been produced industrially.
Blasticidin S is produced by several strains of Streptomyces first isolated from griseochromogenes in 1958. It is certainly not a peptide but a related product leucylblasticidin S can be considered a modified dipeptide. Another biogenetically related compound cytomycin also exhibits antitumor properties like blasticidin S. The obvious precursors are cytosine glucose and arginine as has been shown by in vivo incorporation studies. Biochemical studies in cell free translation systems indicate an interaction with the peptidyl transferase center. Resistance to blasticidin S in P. oryzae may arise from reduced mycelial permeability.
Figure 1. Structures of the agricultural antibiotic blasticidin S (1;R = H) leucylblastidn S (2;R = Leu) and cytomycin (3) a related antitumor compound also produced by Streptomyces griesochromogenes. Detoxin (4) from Streptomyces caespitosus has been used as an antogonist to eliminate side effects of blasticidin S
Research for agricultural antibiotics had been started to replace organic mercurials as rice blast controls. With blasticidin S fields are sprayed at 10 20 p.p.m. From studies of environmental metabolism rapid breakdown has been observed. Toxicity to fish is rather low so it can be used in the paddy field. Elimination of toxic effects on mammals like conjunctivitis upon accidental eye contact or severe inflammation of mucous membrane or injured skin has been attempted many times by chemical or enzymatic modifications. The discovery of selective antagonists the detoxins from Streptomyces caespitosus led to combinations with reduced phytotoxicity and eye irritation properties. At the same time it was found that simple addition of calcium acetate selectivey reduced the eye effect so that this combination is now in use.
Bleomycin and bestatin peptides used in anticancer therapy
Since many antimicrobials able to suppress or retard the growth of tumors screening for cytostatics had already started in the 1950s and has led in the meantime to more than 1000 compounds. As has been pointed out by Oki establishment of chemotherapy of cancer and more recently applications in combination with surgery radiotherapy of cancer and more recently applications in combination with surgery radiotherapy and immunotherapy have led to a remarkable expanding market. Currently the most promising directions are modification of established compounds as well as the application of various biochemical screens. Such screens as has recently been discussed by Aoyagi for enzyme inhibitors acting on proteinases esterases or phosphatases have led to compounds with immunomodulating properties that may also find application in cancer treatment. To illustrate these concepts the examples of bleomycin and bestain have been selected.
The structures of several blemomycin tape glycopeptides are summarized in Figure 2. Phleomycins and already been discovered as Cu2+ containing antibiotics by Umezawa and inhibition of Ehrlich carcinoma with a high therapeuticindex was found soon afterwards. However it was not clinically tested since it showed irreverisible nephrotoxicity in the dog. The search for phleomycin analogs led to the isolation of bleomycin (BLM) in 1966. It was also a blue Cu2+ complex and Cu2+ essential for its fermentative production from Streptomyces verticillus. BLM caused reversible hepatotoxicity but was not nephrotoxic. Its current main clinical use is in the palliative treatment of squamous cell carcinoma of the head and neck as a mixture of natural BLMs a first generation agent . In the 1970s hundreds of semisynthetic analogs were prepared by directed biosynthesis and chemical transformation. In order to modify the terminal amine moiety the preparation of bleomycinic acid has been accomplished first enzymatically and then chemically.
Figure 2. (a) Structures of some bleomycin tape compounds. The structure shown with R representing various amine compounds like NH (CH2) SOCH3 NH(CH2) 4NH2 etc. is bleomycin. In phleomycins the C (31) C (32) doubled bond is missing. Zorbamycins (YA 56x) contain an extra CH2OH at methyl group 20 an extra methyl group at 25 no double bond 31 32 an no hydroxy group at 42. IN tallysomycin an extra amino sugar is fond at 28 a hydroxy group at 29 and the methyl group at 22 is missing. Obviously variations are restricted to certain regions of the compound. (b) Structure of the active Fe2+/O2 complex involved in DNA double strand scission. The indicated amino group is essential for this reaction and its removal by BLM hydrolase leads to inactivation. The hydrolase concentration is low in tumor cells.
Since all available proteases did not remove agmatine from BLM B2 a screening was initiated based on the low antimycobacterials activity of bleomycinic acid (5% of BLM B2). An acylagamtine amidohydrolase was then detected in a strain of Fusarium anguidoides and has been used in the preparation of semisythetic BLMs. The first second generation BLM peplomycin (with ethyl 3 diaminopropane as terminal amine) introduced clinically in 1981 has several advantages such as high distribution in the stomach inhibition of gastric carcinoma and lower lung toxicity it is now used in the treatment of prostatic cancer.
During their study of mammalian cell membrane enzymes. Umezawa investigated the mechanism of influenza virus. They found activites of aminopeptidase alkaline phosphatase and esterase located on the host cell membrane and transferred of the virus envelope. In a screening for aminopeptidase B bestatin was obtained from the broth of Streptomyces olvoretculi a competitive inhibitor also of leucine aminopeptidase but not of aminopeptidase A or endopeptiases. Labelled bestatin has been used to detect aminopeptidases on the surface of various mammalian cells including lymphocytes and tumor cells. It has been found to enhance the immune response in mice and to exhibit a suppressive effect on some slowly growing tumors in mice. First clinical studies were reported at the Bestatin Conference in Tokyo in 1979. A metabolite of orally given bestatin p hydroxybestatin has been found to be a more effective immunostimulator. These compounds should be useful in cancer treatment in the annihilation of minimal residual tomors and in the treatment of infections of immunodeficient patients.
Figure 3. Bestatin an aminopeptidase inhibitor produced by Streptomyces olivoreticuli with immunoenhancing properties
Cyclosporin an immunosuppressor
Another immunomodulator not with enhancing but with promising suppressive properties is cyclosporin. The cyclic 11 peptide was isolated in 1970 from two new strains of Fungi Imperfecti Cyclindrocarpon lucidum and Tolypocladium inflatum. Since then the unusual structures of nine analogs have been established. These and other analogs have been prepared by directed biosynthesis transformation and chemical synthesis. Since screens for cytostatic and immunosuppressive drugs were developed in the 1970s immunosuppressive effects of cyclosporin were recognized together with its antifungal properties (narrow spectrum e.g. Aspergillus and Neurospora). Extensive clinical studies have been carried out and conferences on cyclosporin were held in 1981 and 1982. The peptide has a remarkable affinity for lymphocytes. An interference with an early event during lymphocyte transformation has been suggestd. There have been numerous encouraging results in clinical transporations. However long term effects and reversible nephrotoxicity have not been studied in sufficient detail to permit general applications.
Figuer 4. Structure of cyclosporins. So far nine cyclosporins have been identified the differences being located inthe unusual unsaturated C 9 amino acid (C9A) and the amino acids 2 and 11. A unique DL replacement has been found in 11
Structural Types of Peptides
Peptides and compounds containing peptide structures have been classified by Perlman into linear and cyclic types the latter being subdivided according to the mode of ring closure with either peptide or ester bonds. In a systematic approach Berdy lists 77 groups of amino acid derivatives peptides and peptolides together with protein and proteide types of antibiotic. This classification is based on structural pecularities of well known compounds and contains 780 entries analogs and double citations included. It has the advantage of being a quite comprehensive compilation the data being available in computerized form. The user has to be familiar with the structure of individual groups but does not have the possibility of searching for similar constituents ring sizes or modifications. A more general approach has been suggested by us from the biogenetic viewpoint. A scheme based on the terms linear cycle or lactone appears to be of limited use since many compounds carry two or all of these characteristics. Any cyclic structure however can be linearized to a precursor structure which is then modified by anzymatic catalysis. Thus sequence data and constituents could be compared and localized within families or groups.
Figure 5. The major structural types of cyclic peptides 1. tentoxin cyl 2; 2 cyclochlorotine; 3. capreomycine tuberactinomycin 4. destruxin. echinocandin ferrichrome; 5 ilamycin ulcylamid; 6 bactracin; 7 octapeptins; 8 polymyxins; 9 ulithiacylamide; 10 cycosubtilin; 11 tyrocidin gramicidin S; 12 cyclosporin; 13 gratisin; 14 mycobactillin; 15 enduracin; 16 AM toxin; 17 ostreogrycin A; 18 brevigellin; 19 actinomycin; 20 isarin destruxin globomycin; 21 ostreogrycin B; 22 esperin; 23 A 43 F; 24 etamycin; 25 viscosin; 26 stendomycin; 27 surfactin; 28 lipopeptins; 29 griselimycin mycoplanecin; 30 LL AO 341; 31 telomycin; 32 polypeptins; 33 A 21978. 34 brevistin; 35 pyridomycin; 36 enterobactin; 37 angold erratamolide; 38 sporidesmolides; 39 ennatins beauvercin; 40 triostin echinomycin; 41 bassianolide; 42 valinomycin.
This approach has not been realized and many types of modification reaction have not yet been characterized. The limited number of approximately 300 peptide structures evaluated so far still shows surprisingly little structural variance compared to the synthetic organic chemist s possibilities but also surprisingly few structural homologies regarding sequence data. To illustrate the limited structural variance the principal types of structure are shown in Figure 5. As far as sequence data are concerned convenient short notations are not available for all nonprotein amino acids identified so far. For example more than 10 differently stereochemical arrangement and a possible D configuration it quite difficult to design say a three letter notation compared to the one letter characterization of protein amino acids. Considering the biochemistry of enzymes involved many more homologies seem to evolve than would be expected from the peptide structure.
BIOSYNTHESIS OF PEPTIDE ANTIBIOTICS
Ribosomal and Nonribosomal Mechanisms
From structural considerations the nonribosomal origin of the majority of known peptide antibiotics is obvious. Nonprotein components like D amino acids and fatty acids cyclic or modified structures are linked to antibiotic properties. It should be realized however that conventional screening producers using compounds isolated from culture broth and detection of growth inhibitory properties might well miss large peptides not being transported through the membrane in either way. A good reason to select low molecular weight compounds is their possible lack of antigenicity.
Knowledge of the biogenetic origin of a compound is essential to direct efforst towards cultural improvement and possible directed biosynthesis. Structural genetic manipulations should rely on a detailed knowledge of biosynthetic events. Proposals for manipulations presented so far consider multistep enzymic procedures involving a large number of discrete functions. A single function changed or knocked out could then lead to a modified product. Biosynthetic studies of several peptide antibiotics with in the past decade have led to new types of enzymic organization the multifunction enzyme type. Although not yet definitely proved a single gene cluster may provide the massage for a single polypeptide catalyzing a multistep sequence of reactions. Changes with in a single function could then deteriorate the functioning of the multi enzyme.
Figure 6. Structures of the food preservatives subtilin (bottom) (B. subtilis) and nisin (Streptococcus lactis) which are presumably of ribosomal origin
The famous examples of presumably ribosomal origin are nisin and subtilin two modified peptides of 34 and 32 amino acid residues produced by streptococcus lactis and Bacillus subtilis ATCC 6633. The structures have been postulated to originate from an all L precursor of protein amino acids. Biosynthesis in vivo of both nisin and subtilin has been found to be sensitive to chlorampenicol. Ring closure reactions leading to a compact peptide structure may proceed by inversion with a final D configuration in the peptide. Both antibiotics have antibacterial (also Streptococcus) antifungal antiprotozoal and antimalarial activity and are used a preservatives in the milk and canned food industries.
Reactions Involved in Enzymatic Peptide Formation
The topic of enzymatic formation has been reviewed quite extensively. So here after a brief discussion of functional organization of enzyme systems we just focus on the types of reaction involved from the point of view of possible applications in manipulated biosynthesis.
Streptomycin and Commercially Important Aminoglycoside Antibiotics
GENERALITIES ON AMINOGLYCOSIDE ANTIBIOTICS
The antibiotics of clinical importance which are commonly designated as aminoglycosides from an extremely large group. As their name indicates all these products possess one or more aminosugars. However this characteristics alone is not sufficient to define them accurately as a considerable number of antibiotics would also be include resulting in a group whose structural and therapeutic features are very different from one another.
The story of aminoglycosides started two important discoveries by Waksman and coworkers in the USA the discovery of streptomycin isolated from the fermentation broths of and that of neomycin produced by Streptomyces fradiae. The outstanding properties of these two products prompted manufactures to undertake research in the same field in order to isolate more active aminoglycsides with broader spectrum and less noticeable secondry effects. The last 30 years have been marked by regular announcements of discoveries of new aminosides of natural origin endowed with great therapeutic value kanamycin (1957) paromomycin (1958) spectinomycin (1961) gentamicin (1963) tobramycin (1967) lividomycin (1968) ribostamycin (1969) sisomicin ( 1969) and sagamicin (1973) to mention only the main ones which have been commercialized. The aminosides which have been isolated in recent years differ little from the point of view of their biological properties from the best of the above listed products although some of them (fortimicins istamycins etc.) possess original structures.
Although far from exhaustive a fuller list of natural glycosidic aminocyclitols is show in Table 1. There are in total over a hundred of them mostly obtained from Streptomyces cultures but also from other Actinomycetales (Micromonospora Dactylosporangium Sacharo polyspora) and even from Bacillus. This family of antibiotics has also been gradually enriched with semisynthetic and synthetic products whose remarkable biological activity has enabled them to supplant in certain cases in clinical use the original natural products; for instance amikacin and dibekacin which derive from kanamycins or netilmicin derived from sisomicin.
Table 1 Principal Aminoglycosides from Natural Origins
Structure of Different Classes of Aminoglycoside Antibiotics
The aminosides are usually classified according to the structure of their aminocyclitol unit. They fall into three main groups. (1) The aminosides whose aminocyclitol is a streptamine derivative; the only clinically important representatives are streptomycin and dihydrostreptomycin which possess the streptidine ring. Bluensomycin also belongs to this category. Its aminocyclitol bluensidine differs from the streptidine in possessing a carbamoyl group instead of a guanidino group. (These are the most numerous aminosides possessing a 2 deoxystreptamine ring which is sometimes N substituted. With most of them two of the hydroxy groups of the ring are linked to amino sugar moieties either in positions 4 and 5 (neomycins paromomycins lividomycins ribostamycin butirosins) in positions 4 and 6; the latter include the clinically most important aminoglycoside antibiotics (kanamycins tobramycin gentamicins and sisomicin and their semisynthetic derivatives amikacin dibekacin and netilmjcin). With others the 2 deoxystreptamine ring is only linked to one amino sugar residue either in position 4 (apramycin) or in position 5 (destomycins hygromycin B). (3) The other aminosides (fortimicins etc.) and the antibiotics which strictly speaking are not aminosides but which are related by similarities of structures or very close biological properties (spectinomycin kasugamycin hygromycin A validamycins). Among those products only spectinomycin has had a commercial outlet owing to its being particularly active against gonorrhoea.
Figure 1 Structure of streptamine streptidine and 2 deoxystreptamine
Microbiological Activity and Clinical Use
Aminoglycosides are among the most potent antibiotics known so far. Having a broad spectrum they are active both against Gram positive and Gram negative bacteria and against mycobacteria. They are therefore clinically widely used despite their tendency to provoke serious secondry effects. Their use finds its justification in that they inhibit a great number of pathogenic microorganisms which cannot be effectively treated with other antibacterial agents despite the recent discoveries in the b lactam field.
The antibacterial action of aminoglycosides is mostly of a bacterial type. Their activity is particularly noticeable against aerobic Gram negative bacteria such as Escherichia coli Klebsiella Proteus and Enterobacter. Some of them can inhibit Pseduomonas aeruginosa. Although a lot of them are highly active against Staphylococcus aureus in vitro their clinical efficacy when dealing with severe Staphylococcus infections is not guaranteed. They are ineffective against streptococci. Finally their activities against Mycobacterium tuberculosis are variable streptomycin being still the product most widely used against that germ.
No aminoside is active against anaerobes or against aerobic bacteria growing in anaerobiosis. Their activity is null against anaerobes or against fungi viruses and protozoa.
Aminoglycoside activity in vitro can be only roughly estimated since the answers vary considerably according to whether the strains examined have already been in previous contact with the antibiotic that is to say according to whether they contain one or several plasmids carrying resistance factors. However the gradual improvement in potentialities of six major aminosides discovered from 1944 to 1970 clearly appears from the results in Table 2.
Sensitivity of clinical Gram negative isolates to the same antibiotics varies in the same positive way. Moellering reports the particularly telling results of a survey of several thousands of strains in that example the respective sensitivities to streptomycin kanamycin gentamicin and tobramycin are 64 85 99 and 98% respectively in the case of E. coli 52 91 100 and 100% in the case of Samlmoella and 5 4 78 and 94% with Pseudomonas aeruginosa.
Thirty years have passed since the discovery at Oxford England of the first cephalosporin B lactam cephalosporin C. From this compound present in such trace amounts in the original cultures as to be below the limits of detection then available has developed a multi million dollar industry. Scientists from many different disciplines ranging from geneticists to chemical engineers and working in Britain USA Europe and Japan have by their combined efforts produced an impressive array of drugs to combat infectious disease and unravelled many of the mysteries surrounding the biosynthesis of the cephalosporin antibiotics.
The cephalosporions are just part of a large group of lactams produced by microorganisms. The story of their discovery and development would be incomplete without reference to the earlier discovery of penicillin. Alexzender Fleming made his rather fortuitous discovery in 1929 with the observation that growth of bacteria on an agar plate was inhibited by a mould contaminant which turned out to be Penicillium notatum. Years elapsed before this discovery was finally exploited by Florey and Chain who isolated the active compound and initiated a new era in the treatment of infectious disease. Penicillin G however appeared to be limited to use against Grampositive bacteria; its relatively low acid stability precluded oral administration; a small percentage of patients developed allergic reactions and perhaps more important there was an increasing number of penicillin resistant isolates appearing.
Like penicillin before it the cephalosporin C story had a rather curious beginning. Giuseppe Brotzu working at an institute at Cagliari on the island of Sardinia isolated from seawater near a sewage outlet an organism which had antibiotic activity against both Gram positive and Gram negative bacteria. The organism was identified as a Cephalosporium species and was sub sequently sent to Oxford in 1948. The discovery of cephalosporin C was still five years into the future. The early work with the Brotzu isolate revealed two compounds which probably account for the observations made in Sardinia. Cephalosporin P had activity against Gram positive bacteria and subsequently proved to be less interesting than cephalosporin N which had Gram negative activity. Cephalosporin N was found to be a b lactam with the fused b lactam and thiazolidine rings of penicillin and was therefore renamed penicillin N. The compound was first purified by Abraham and its structure elucidated by Newton. During the purification of crude preparations of penicillin N a similar type of compound was detected which had strong UV absorption was relatively acid stable and was resistant to the enzyme penicillin s which destroys penicillin N. Thus in the autumn of 1953 a new chapter in the blactam story began. The cephalosporins industry has been founded largely on derivatives of the cephalosporin C produced by Cephalosporium acremonium. A mutant strain M8650 isolated at Clevedon Antibiotics Research Station England permitted work on the isolation of the new antibiotic to proceed and led to the publication of its structure by Abraham.
Figure 1. Structural formulae of penicillin N and cephaslosporin C
For many years it appeared that the blactam antibiotics were confined to just a few species of organisms all of them fungi. However a new class of cephalosporins the cephamycins were isolated from streptomycetes by two groups working independently. The cephamycins are characterized by the presence of a 7 a methooxy grouped attached to the B laetam ring. More recently novel blactam structures have been isolated from a range of microorganism including bacteria. Streptomycin clavuligerus produces a bb lactam which is a potent b lactamse inhibitor clavulanic acid. Other streptomycetes produce novel carbapenems such as the olivanic acids and thinamycin. A Nocardia species produces a monocyclic B lactam nocardicin and certain bacteria produce monocyclicb lactates now known as the monobactams. Many of these compounds are not cephalosporions and belong to the broader category of b lactams. However their structures are of interest in particular with respect to the mode of action and structure activity relationships of the cephalosporin antibiotics. The relationship between the cephalosporins and other important class of b lactams is shown in Table 1.
Table 1 Structural formlae and producers of inportant b lactams
MODE OF ACTION OF CEPHALOSPORINS
Structure and Biosynthesis of Bacterial Cell Wall
The successful application of the cephalosporins and the b lactam antibiotics in general for the treatment of infectious disease is due in no small way to their unique mode of action. It was recognized at an early stage that these antibiotics had as their target the biosynthesis of the bacterial cell wall. As inhibitors of a part of metabolism unique to bacteria they are consequently relatively non toxic tot he animal host.
The Gram positive bacterial cell wall comprises layers of peptidoglycan and teichoic acid polymer outside the cell membrane. The peptidogly can accounts for about 50% of the wall dry weight. The outer envelope of the Gram negative bacteria is more complex. Again peptidoglycan is the important structural polymer in the wall but there is an additional outer membrane composed of proteins lipoproteins lipopolysaccharices and phospholipids. This layer presents a barrier to the uptake of small molecules including the antibiotics. The common feature in all bacterial cell walls is the peptidoglycan polymer. The glycan polymer consists of a repeating disaccharide unit comprising N acetyleglucosamine and the related sugar N acetylmuramic acid; Spot peptides can be linked to the muramic acid residues. These oligopeptide chains usually contain one diamino amino acid (e.g. lysine or diaminopimelic acid) which is the basic for cross linked peptidoglycan is the structural polymer which protects the smotically fragile bacterial memebrane. In Staphylococcus aureus the cross linked peptide has five glycine residues whilst in Escherichia coli the terminal amino acid of one peptide chain is directly cross linked to the free amino group of diaminopimelic acid in the adjacent peptide chain. The presence of D amino acids especially D alanine is characteristics of the bacterial cell wall peptidoglycan.
Cell wall biosynthesis occurs by a series of enzyme catalyzed reactions some occurring inside and some on the surface of the cell. The first building blocks is a UDP N acetylglucosamine sugar nucleotide which is also the precursor of the second component of the disaccharide N acetyllmuramic acid. In E. coli attachment of a pentapeptide unit (L ala D glu DAP D ala D ala) through the amino group of L alanine tot he muramic acid derivative occurs within the cell. A transglycosylase combines the two sugar derivative to yield a disaccharide with its pentapeptide substituent which becomes the repeating sequence in the peptidoglycan polymer. These peptidoglycan precursors are transported across the lipophilic memebrane by combination with a lipid phosphate carrier. The incorporation of newly synthesized units into the pre existing or nascent polymer occurs at the outer surface of the cell membrane. The lipid phosphate carrier is released to capture and transport a new submit.
The final step in wall biosynthesis and the step implicated in b lactam inhibition is the cross linking of the oligopeptide residues to yield the final polymer matrix. This occurs by the reaction between the penultimate D alanine residue of one peptide chain and the free amino group of a glycine (S. aureus) or DAP residue (E. coil) in an adjacent peptide chain with concomitant release of the terminal D alanine. The reaction is driven by the energy in the D ala D ala peptide linkage and overcomes the lack of an energy source as ATP outside the cell. It is the inhibition of this final step catalyzed by transpeptidfase enzymes which forms the basic of the lethal action of b lactam antibiotics. Other enzymes such as carboxypeptidase which removes terminal D alanine residues endopeptidase which cleaves the cross linked peptides and muramidase which splits the disaccharide bond are also present in the bacterial memebrane. Their detailed function is not yet completely understood but they certainly play an important role in the expansion of the cell wall surface as the bacterium increases in size allowing insertion of new wall material elongation and septum formation. Cell wall biosynthesis is illustrated in Figure 2.
Figure 2. Biosynthesis of E. coli peptidoglycan
Sensitivity and Resistance
The action of cephalosporins and other B lactam antibiotics on bacteria such as E. coli results in various morphological responses including inhibition of cell division formation of filaments and spherial cells. By using radioactivity labelled benzylpenicillin it was shown that a series of proteins within the cell memebrance reacts covalently with the B lactam antibiotics. In some cases these so called penicillin binding proteins (PBPs) have been allocated specific enzyme activities in cell wall biosynthesis such as transpeptidase and carboxypeptidase activity. Inhibition of these enzymes by the B lactam involves the formation of a stable covalent enzyme inhibitor complex which can subsequently break down to regenerate the active enzyme with concomitant release of inactive B lactam breakdown products. The efficacy of particular antibiotic often depends upon the speed of formation of the complex and its stability. Changes in these reaction rates can result in resistance to the antibiotic.
Tipper proposed that penicillin inhibited the transpeptidase reaction by virtue of its structural analogy with the terminal D ala D ala in the peptidoglycan peptide chain. Modification of the side chain substituent appears to change the reactivity of the antibiotic. Inhibition of the transpeptidase or carboxypeptidase alone does not account for the bacteriocidal effect of the b lactams and resulting cell lysis. It appears that other autolytic reactions are triggered which bring about the final demise of the cell.
Bacteria vary in their sensitivty to b lactam antibiotics in general and in their response to individual penicillin or cephalosporin derivatives. Three main factors have been recognized to account for this variable response. First the antibiotic must penetrate the cell wall in order to reach the target in the cytoplasmic membrane. In Gram positive bacteria the cell wall seems not to be a very effective barrier to the penetration of the low molecular weight pencillins and cephalosporins. However in Gram negative bacteria the outer membrane is sometimes responsible for exclusion of the antibiotic and therefore resistance to it. This seems to be the prime cause of resistance to cephalosporins in the pseudomonads where most derivatives fail to penetrate easily via the points channels for diffusion of these antibiotics through the outer memebrance. The second important barrier to effective action of the cephalosporins is the presence of B lactamase enzymes. These enzymes are secreted into the medium by Gram positive bacteria and are found in the periplasmic space of Gram negative bacteria. The b lactamases are widely distributed and were observed when resistant organisms were first encountered. The evolutionary origin of these enzymes is not yet clear although there is often some structural homology with PBPs. Much of the effort in synthesizing the bewildering range of cephalosprin derivatives has been directed at finding compounds resistant to the B lactamases. The occurrence of new resistant strains especially in the hospital environment and the interspecific transfer of resistance factors on plasmid vectors have presented a never ending challenge to the organic chemist to synthesize new potent derivatives. The third factor determining the efficancy of the cephalosporin is the sensitivity of the target itself that is the transpetidase enzyme or other PBP in the cell membrane. Modification of the relative rates of formation and breakdown of the enzyme inhibitor complex ocean lead to changes in susceptibility of the bacterium.
Although penetration the presence of B lactamases and target specificity are the important factors determining in vitro activity of the cephalosporin other factors are important when the antibiotic is used for the clinical treatment of infectious disease. These come under the heading of pharmacokinetic propitious. The ideal antibiotic has a very high antibacterial activity a long half life in the blood stream and is subsequently recovered unaltered in the urine. These properties can be influenced by the various substitutents of the cephem nucleus. Cephalosporins retaining the 3 acetoxymethyl substituent of the parent compound cephalosporin C e.g. cephalothin are attacked by acetyl esterases to yield the less active desacetyl derivative. In the first generation of cephalosporins the replacement of the 3 acetoxy group by pyridine to give cephaloridine overcame this problem but led to another its nephrotoxicity.
Structure/ Activity Relationships
The classical cephalosporins can be modified at three positions on the cephem nucleus 7b 7ba and 3.
It was assumed correctly that replacement of the a aminoadipyl substituent of cephalosporin C by a hydrophobic group would increase the antibacterial activity. This came by straight analogy with the penicillins benzylpenicillin being much more active than penicillin N. A thienylacetyl group proved to be more active than the phenylacetyl and led to the firsty generation compounds cephalothin and cephaloridine which had the advantages over penicillins of Gram negative activity and resistance to b lactamases from Gram positive organisms. As mentioned earlier the replacement of the 3 acetoxy by pyridine prevented deactivation by mammalian esterases.
The spread of b lacxtamase producers and the regular occurrence of resistant pathogenic strains was the impetus behind the search for new cephalosporin derivatives with b lactamase resitance. The penalty for having increased resistance to b lactamase is often that the antibacterial activity is the penalty for having increased resistance to b lactamase is often that the antibacterial activity is reduced. Of the three possible sites for modification of the classical b lactams those at positions and 7 b and 7a have the main impact on b lactamase resistance. Modification at position 3 can affect antibacterial activity and pharm acokinetic properties of the cephalosporin. A major breakthrough occurred with the discovery in the streptomycetes of the cephamycins cephalosporins having a 7a methoxy substituent. The methoxy substituent confers nearly complete resistance to b lactamases. Other bulkier 7a substituents e.g. 7a ethoxy also confer resistance to b lactamases but at the expense of drastically reduced antibacterial activity. The first cephalosporin derivative incorporating the 7a methoxy group was cefoxitin which combines resistance to b lactamase and broad spectrum activity.
Changes in the 7b group also affect the antibacterial activity and b lactamase sensitivity. Steric effects are important here in regulating the reactivity of the b lactam ring. By substitution in the a carbon of the side chain significant improvements were obtained over the straight substituted acetic acid derivaties such as cephalothin. Compounds of this second generation of cephalosporin derivatives include cephamandole and cefuroxime. These compounds although having a broad spectrum and improved b lactamase stability are still relatively inactive against pathogenic pseudomonads and must be administered parenterally. Despite their acid stability in comparison with the penicillin s the cephalosporins in general are not readily absorbed from the gut. The exception is cephalexin. Here the 7b substituent is the D phenyglycine residue. This or a slightly modified analogue is common to all orally absorbed cephalosporins. Cephalexin has a methyl substituent at position 3 on the cephem nucleus. Although this group occurs naturally in desacetoxycephalosporin C an intermediate in the biosynthesis it is produced in relatively low yields even in blocked mutants.
Cephalexin is therefore normally produced by chemical ring expansion from pencillin V followed by deacylation to 7 aminodesace toxycephalosporanic acid (7 ADCA) and re acylation with the phenyglycine substituent. Cefaclor with a 3 chloro substituent and cefoxadine having a 3 methoxy group are new orally active derivatives which have improved antibacterial activity compared with cephalexin.
A third generation of cephalosporin derivatives is already working its way through the clinics and challenging the existing market leaders. Worthy of mention are cefotaxime ceftizoxime and ceftazidime the latter having activity against the pseudomonads. One of the most interesting derivatives at present in the clinic is moxalactam. This cephalosporin has an oxygen atom to replace the sulfur atom present in the cepheming of all the other derivatives. This confers increased antibacterial activity. The 7b methoxy substituent and the substituted malonic acid side chain result in resistance to many b lactamases.
Table 2 shows the structures of some of the important cephalosporin derivatives in current therapeutic use and some new compounds undergoing clinical trials.
An estimate of the worldwide systemic antibiotic market for 1980 showed that the cephalosporins occupied first place with a market value of approximately US $1750 million (equivalent to 29%). The ratio of oral to parenteral forms was 44 56.
Amongst the oral products cephalexin had an 83% market share and was the biggest single best seller of the cephalosporins. Amongst the parenteral products well established derivatives such as cephalothin and cephazolin had a large share of the market (over 20% each). However the trend for these products is downwards as the second and third generation products take a larger bite of the cake. In the immediate future it is probable that products such as cephamandole cefoxitin and cefaclor will take an increasing share of the market.
Commercial production of CEPHAMYCIN ANTIBIOTICS
Penicillin and cephalosporin antibiotics are clinical products which were first marketed in recent decades for the treatment of bacterial infections and animals. These products have generally been made via a fermentation process which various fungal species are grown in large agitated vessels and used to synthesis the desired product. The Penicillin or cephalosporin produced by these organisms has been isolated from the fermenter broth and then purified and modified to produce a clinical product.
Since the original introduction of cephalosporin derivatives and penicillin a large number of new b lactam products with surperior antibacterial activity have been marketed. The great majority of new products have been chemical derivatives of the original penicillin and cephalosporin compounds. These new products are manufactured by first preparing penicillin or cephalosporin via an existing fermentation process. In this way new antibiotics have been introduced without the need to develop wholly new fermentation processes. This chemical modification approach has resulted in the clinical availability of several generations of antibiotic products exhibiting antibacterial spectra which far exceed those of microably synthesized penicillin or cephalosporin compounds. More recently however new classes of broadly active b lactam antibiotics have been discovered which are not chemical derivatives of penicillin or cephalosoporin. The cephamycins for example represent such a class of microbially synthesized b lactam antibiotics. The molecular structure of these compounds resembles that of the cephalosporins. However two important characteristics. b lactamase resistance and the nature of the producing species distinguish the cephamycins from the cephalosporins. The cephamycins are resistant to the action of b lactamase enzymes whereas cephalosporins are inactivated by lactamases. Since many clinical pathogens synthesize lactamase they are resistant to both penicillin and cephalosporin. Frequently such bacteria are susceptible to cephamycins. As a result cephamycins can be used to treat a more broad spectrum of human infections. The taxonomic classification of synthesizing microorgansims also distinguishes cephamycins from cephalosporins. Cephamycins are synthesizing microorgansims also distinguishes cephamycins from cephalosporins. Cephamycins are synthesized only by the filamentous bacteria actinomycetes. In contrast the vast majority of observed penicillin and cephalosporin producing organisms are eukaryotes principally fungi.
The differences in the metabolic requirements of fungi and actinomycetes required development of new fermentation processes for the cephamycins. Biochemical similarities do exist between the b lactam biosynthetic pathways in fungi and actinomycetes and these similarities were used as the basis for development of the initial cephamycin fermentation processes. The intent of this chapter is to review the metabolic pathways in penicillin cephalosporin and cephamyci producing organisms which are relevant to cephamycin production and to discuss means by which this information was used to formulate the current cephamycin production processes.
CEPHAMYCIN PRODUCT DESCRIPTION
The penicillins were the b lactam antibiotics to become commercially available in the United States. Following their introduction into clinical use this class of compounds was found to be capable of controlling many human infections for which there had previously been no effective treatment. Upon further investigation it was observed that there were several naturally occurring penicillin derivatives which were lethal to a wide variety of Gram positive bacteria. However there were certain classes of Gram positive bacteria which were resistant to the effect of penicillin. In addition the spectrum of penicillin activity against Gram negative organisms was not as broad.
Figure 1. Naturally occuring cephamycin antibiotics
A continuing effort was then made to find naturally occuring compounds whose bactericidal spectra complemented or surpassed those exhibited by the existing penicillins. This was accomplished by randomly screening marine and soil samples containing a wide variety of bacterial and fungal species. New compounds were identified by testing the antibacterial activity of these samples toward a group of indicator organisms. Among many other discoveries Brotzu isolated a novel activity produced by a previously unknown fungal species. The producing culture was originally classified as Cephalosporium acremonium. This organism attracted great attention because it produced penicillin N a compound which is more active against certain Gram negative bacteria than were existing penicillins. Of even greater importance however was the finding that this organism produced another b lactam product which was highly active against Gram negative bacteria. This novel product was termed cephalosporin C. The chemical structure of cephalsoporin was determined by Abraham and Hodgkin. Like penicillin the basic cephalosporin C nucleus was found to be bicyclic but differed from the penicillin nucleus by the presence of a six membered ring in place of the five membered thiazolidine ring.
Since the discovery of cephalosprorin C a number of species have been isolated which produce a variety of related antibiotics products. In all over 1000 naturally occuring or chemically modified antibiotics products have been reported of which over 100 are commercially produced. One important class of microbially produced compounds the cephamycins was separately isolated in the early 1970s by Merck and co. Inc and by Eli Lilly and Co. The first member of this family to be isolated was cephamycin C. It is produced by both Nocardia Lactamdurans and Streptomyces clavuligerus as well as several other actinomyces. The basic structure for this compound is illustrated in Figure 1 as are the structure for other naturally occuring cephamycins. The cephamycin family of compounds is produced only by the filamentous bacteria actinomycetes. A characteristic methoxy substitution at the 7a position of the bicyclic ring structure distinguishes between cephamycins and cephalosporins.
Mode of Action
As with all b lactam compounds the antibacterial activity of the cephamycins derives from their ability to bind to a receptor protein in the susceptible organism and then to inhibit further cell wall synthesis. This inhibition leads to a loss of viability in the affected cell. The b lactam structure specifically inhibits activity of the transpeptidase enzymes involved in peptidoglycan biosynthesis. As a result assembly of the cell envelope in both Gram positive and Gram negative organisms is prevented.
The primary means by which microorganisms express resistance to b lactam antibiotics is by synthesizing enzymes which have the ability to cleave the b lactam ring. Occurrence of such lactamase activity is widespread. Therefore in order to control proliferation of organisms which synthesize lactamase it was necessary to identify structure which would resist lactamase mediated breakdown. Two approaches were taken. In one chemical modifications of existing antibiotics were made with the intent of conferring resistant to lactamase. A second approach was to identify microbially synthesized product which are resistant to lactamase. Cephamycin C was first identified as an inhibitor of cell wall synthesis. Subsequently it was found that this compound is particularly resistant to lactamase activity. It was determined that the characteristic 7 a methoxy group of the cephamycins was the substituent which confers lactamase resistance. This group acted to stabilize the b lactam ring structure reducing lactamase sensitivity.
While cephamycin C is particularly active against Gram negative organisms it is not as active against Gram positive organisms. As a result this molecule was chemically modified to enhance its antibacterial spectrum. The intent was to alter the existing side chains of the cephamycin C molecule such that a wide spectrum of Gram positive antibacterial activity would be added without sacrificing the Gram negative activity or in vivo stability of the parent molecule.
Figure 2. Structure function relationships of cefoxitin
Replacement of the aminodipoyl side chain at the 7b position of cephamycin with a thienylacetyl substitution similar to the that in cephalothin had the desired effect. The resulting semisynthetic b lactam compound cefoxitin has a particularly wide antibacterial spectrum. The substituents of the cefoxitin molecule which contribute various specific activities toward the overall spectrum are also indicated in Figure 2. Cefoxitin is currently manufactured and marketed by Mark and Co. Inc.It has found a number of uses in preventation and treatment of human infection. This product is widely used in preventing postoperative infection by penicillin resistant bacteria. An additional attribute of the cefoxitin spectrum is its unusual activity against a variety of anerobic bacteria.
In purified form this compound is a white powder which is quite soluble in water; it is slightly soluble in ethanol and dimethyl sulfoxide. Ultraviolet (UV) proton nuclear magnetic resonance (NMR) and infrared (IR) spectra of this material have been constructed but are not reproduced here.
Cephamycin C Assay Techniques
As in the case with most antibiotics quantitative analysis of microbially produced cephamycin C is normally accomplished by either high performance liquid chromatographic (HPLC) or biological assay techniques. HPLC analysis of filtered aqueous samples of cephamycin C has been demonstrated by Kamogashira. Concentration of product are measured relative to those of freshly prepared standard solutions of cephamycin dissolved in distilled water or an appropriate buffer.
Bioassays of cephamycin concentration in aqueous solution can also be made utilizing disc plate techniques. Such assays involve use of agar Petri plates containing an appropriate medium as well as a suitably grown indicator organism (Vibrio percolans) as the basis for assay. Filter paper discs saturated with cephamycin solutions are set on the assay plates and incubated. Resulting zones of inhibition of Vibrio growth are then measured. Inhibition zones can be compared with analogues zones produced by standard solutions of cephamycin and can be used for quantitative assessment of antibiotic concentration.
A third assay technique for quantitative measurement of cephamycin has been reported. This method utilize one dimensional thin layer chromatography (TLC) applied to precoated silica get plates. Spotted plates are developed in a solvent and scanned in a high speed TLC scanner at 273 nm. This technique permitted quantitative analysis of samples containing 1 g of antibiotic.
Most patented processes for the biological production of antibiotics and other secondary metabolic products consists of a batch biological reaction in which a producing organism is inoculated into a stirred aerated fermenter vessel containing an aqueous mixture of organic raw materials. These medium ingredients are often quite complex and sometimes consists of nonhomogeneous by products of other manufacturing processes. On first inspection complex mixtures of which most classical fermentation media are composed might seem ill begotten. However most frequently medium constituents result from a rational from a rational attempt to satisfy the complicated metabolic requirements of the producing organism.
Any given microorganism has specific requirements for nutrient supplies of carbon hydrogen nitrogen phosphorus and sulfur as well as trace requirements for a host of other elements. Traditionally complex materials such as corn steep liquor and distillers solubles which are rich in a variety of nutrients have been used to satisfy these requirements. Use of such complex raw materials has been considered desirable for several reasons. Since the cost of these materials is usually quite low they provide an inexpensive supply of nutrients. In addition when precise minimum nutrient requirements are not known levels in excess of predicted minima can be added to assure optimal product synthesis without a major cost effect on the final product. Further corn steep liquor or yeast extract often contain trace levels of components which stimulate product synthesis. The stimulatory component is often an amino acid or trace metal which constitutes only a small fraction of the raw material. Corn steep liquor may also be a source of phosphate. However until the active ingredients is identified the entire complex product is added to the medium to insure inclusion of the stimulant.
A certain amount of background information is important for an understanding of how and why lincomycin is produced. Therefore this section providing supplementary information is included.
Lincomycin an antibiotic with mainly Gram positive antibacterial activity was first reported in 1962. The original patent was granted in 1963 to The Upjohn Company. The antibiotic is produced by a soil streptomycete that was designed as Streptomyces lincolnensis. However it has also been reported that the antibiotic is produced by a number of other organisms.
The chemical structure of lincomycin as determined by Hoeksema is shown in Figure 1. Cleavage of lincomycin at the amide bond was found to give a methythio amino sugar and an amino acid. The stereochemistry of the carbohydrate moiety was studied; and assigned as methyl 6 amino 6 8 dideoxy 1 thio D erythro a D galactoacto pyranoside which was given the trivial name methylthiolinocsaminide (MTL).
The amino acid portion of lincomycin was shown to be trans N methyl 4 n propyl L proline the trival name for this compound is propylhygric acid (PHA). It was found to be of key importance to have the natural trans isomer of PHA since the cis isomer of lincomycin was only about one half as active as lincomycin.
Figure 1. Lincomycin atructure and biosynthetic origin
The antimicrobial activities of lincomycin and several of its analogs have been described. Lincomycin was found to possess excellent activity against clinical strains of staphylococci streptocicci and diplococci both in vitro and in vivo. The antibiotic is effectic is effective when administered either subcutaneously or orally and has a low order of acute toxicity. In mice CD50 value of 8.0 mg kg-1 and 3.8 mg kg-1were obtained with Streptococcus hemolyticus when the antibiotic was administered subcutaneously and orally respectively. The antimicrobial spectrum of lincomycin is generally similar to that of erythromycin but linomycin is not cross resistant with any other major antibiotic. However a phenomenon designated as the macrolide effect or dissociated cross resistance has been reported to occur between lincomycin and erythromycin; this effect will be discussed in more detail later in this report. Analogs of lincomycin have been described that prossess altered and extended activities.
In one study the incubation of Streptomyces rochei in the presence of lincomycin resulted in the formation of lincomycin 3 phosphate. This derivative was inactive in vitro but was apparently hydrolyzed in vivo giving lincomycin. Other important derivatives will be considered in more detail below.
Mode of Action
It was first reported by Josten that lincomycin inhibits the incorporation of 14C lysine into log phase cells of Staphylococcus aureus by 87%. Further analysis indicated that incorporation of labeled lysine into protein was inhibited by 92% while incorporation into cell walls did not appear to be inhibited. Under the test conditions protein synthesis was shown to cease immediately after lincomycin addition to the growing culture while RNA synthesis stopped 15 minutes later and DNA synthesis was unaffected for 60 minutes. These suggested that lincomycin acts by inhibiting protein synthesis.
Since lincomycin was known to inhibit Gram positive but not Gram negative organisms it was of interest to determine whether this difference is because of differences in protein synthesizing mechanisms or in cell permeabilities. For these studies the effect of lincomycin on the incorporation of 14C phenylalanine into protein by cell free systems of Escherichia coli and Bacillus stearothermophilus was examined. It was noted that lincomycin had little effect on the incorporation of 14C phenylalanine into protein by the Gram negative organism E. coli. On the other hand incorporation into protein by the B. stearothermophilus was strongly inhibited by lincomycin. These data suggest that the protein synthesizing system of E. coli is insensitive to lincomycin. By mixing ribosomal and supernatant fractions of E. coli and B. strearothermophilus in various combinations it was shown that the inhibition of protein synthesis by lincomycin (10-4M) occurred only when ribosomes of B. stearothermophilus were used. Studies were then conducted with 30S and 50S ribosomal subunits from E. coli and B stearothermophilus in an effort to determine which subunit was sensitive to lincomycin. The mixing of the ribosomal subunits of each organism in various combinations showed that the incorporation of 14C phenylalanine into protein was inhibited by lincomycin only in systems containing the B. stearothermophilus 50S subunit. Thus inhibition of protein synthesis by lincomycin appeared to occur at the 50S subunit. Further studies on the effect on the effect of lincomycin on binding of 14C phenylalanine 1RNA to the ribosomal poly U complex were conducted. The results were consistent with the lincomycin mode of action being inhibition of binding of aminoacyl tRNA to the mRNA ribosome complex at the 50S subunit.
Dilution experiments that the lincomycin ribosome complex can dissociate at 25°C suggesting that the binding is reversible. Erythromycin antagonism of lincomycin was reported by Griffith. It was found that erythromycin resistant and lincomycin sensitive strains of S. aureus have greatly reduced sensitively to lincomycin in the presence of erythromycin. This phenomenon was referred to earlier in this report as dissociated cross resistance.
The work on lincomycin mode of action has been reviewed by Chang and Weisblum . Most of the data suggest that the mode of action of lincomycin is inhibition of aminoacyl tRNA binding to ribosomes. More specially lincomycin stimulated the hydrolysis of formylmethionyl tRNA while inhibiting esterification of formylmethionine and peptide bond formation.
Lincomycin Assays for Fermentation Development and Production
Both biological and chemical assays have been developed for lincomycin. The biological assay is accomplished by using the diffusion disc plate assay procedure. Penassay seed agar is inoculated with Sarcina lued and added to Petri plates. Samples and standards are then spotted onto12.7mm assay discs placed on the agar. Incubation is at 30°C for 16 18 h. This assay has a standard deviation of about 8%. The biological assay has been modified in order to determine the lincomycin content of animal feeds. In addition the disc plate assay provides the basis for an automated turbidometric bioassay.
The chemical assay is based on the liberation of methanethiol by the acid hydrolysis of linocmycin. The thiol is then distilled from the sample solution and reacted with color reagent a 0.01% solution of 5 5 dithiobis (2 nitrobenzoic acid) in water. The color develops almost instantaneously at room temperature and is then read spectrophotometrically at 420 nm. This procedure has been adapted for use with an automatic analyzer. The standard deviation of the assay is about 5%.
An assay for lincomycin based on gas liquid chromatography (GLC) has also been described. By the use of the GLC procedure the ratio of lincomycin to lincomycin B may be determied since the two components are easily resolved. Quantitation was achieved by the use of an internal standard. More recently high performance liquid chromatography assays for lincomycin have been developed that are quite accurate and also resolve the two components.
The structure of lincomycin is presented in Figure 1.The molecule is composed of two moieties an amino acid (PHA) and a sugar (MTL) joined by a peptide bond. Seven of the nine carbons and the nitrogen in the amino acid moiety are derived from L tyrosine. The N methyl carbon and the third carbon in the side chain have L 3 Hydroxytrosine or L 3 4 dihydrox phenlalanine (L DOPA) is a more immediate Precursor to lincomycin than L tyrosine. The carbons derived from (L DOPA) is a more immediate precursor to lincomycin than L tyrosine. The carbons derived from L DOPA are numbered in Figure 1 using the order in which they are found in the parent compound.
Most of the biosynthetic work has been based on fermentation using chemically defined medium (CDM). Media composition and incubation conditions are summarized in Table 1.
Seed cultures were incubated at 28 °C for 48 hours on a rotary shaker. Generally the seed culture was centrifuged and the mycelium was washed three times with sterlic deionized water prior to use as inoculum. The synthesis medium was inoculated at a rate of 2% (V/V) with washed mycelium and was incubated at 28 °C on a rotary shaker for periods up to 6 8 days.
The more successful approach to the eludation of the lincomycin biosynthetic pathway to date is the use of either radioactively labled or stable isotopes of precursor candidates followed colnesis resulting in a 14.1% incorporation in to lincomyin A. Lincomycin A. Lincomycin A. Lincomycin B lacking the methionine derived carbon in the side chain of the amino acid moiety also incoporated radioactivity. After degradation 96% of the radioactivity of the amino acid moiety was assigned to the carboxyl carbon. Thus this carbon is derived from C 1 of L tyrosine. Feeding experiments using radioactively labeled L DOPA also showed that this amino acid is a precursor to lincomycin.
A similar experiment using L[U 14C] tyrosine as a precursor showed a seven fold increase in incorporation of radioactivity when compared to the use of L[1 14C] tyrosine. Essential all the radioactivity was found in the amino acid moiety. These two experiments suggest that seven of the nine carbons in the amino acid moiety were derived from L tyrosine.
Experiments using 15N labeled tyrosine demonstrated a higher level of incorporation into the nitrogen of the amino acid moiety than into the one found in the peptide bond when analyzed by mass spectrometry.
Another experimental avenue that confirms and extends these results involves the use of 13C NMR studies using suitably precursors of lincomycin. Deuterated carbons reduce the 13C NMR signal and produce a multiplet corresponding to the resonance of the assigned carbon. In this work (3 5 2H) L tyrosine was fed to S. lincolnensis resulting in the formation of a monodeuterated lincomycin. A suggesting that one of the carbon deuterium bonds is lost in the course of biosynthesis. The remaining carbon deuterium bond appeared in the b position of the side chain of the amino acid moiety of lincomycin (designated 5 in Figure 1.)
Feeding experiments using (2 5 6 2H) L DOPA further clarified the events in the biosynthetic pathway. A trideuterated lincomycin resulted from these experiments. The 2 5 and 6 positions of L DOPA corresponded identically to the identified position in Figure 1. These findings suggest that carbons 3 and 4 of L DOPA are lost during biosynthesis. These data are also consistent with the result of the feeding experiments with educated and L[1 14C] labeled tyrosine. The original location of the L DOPA derived carbons retained in the antibiotic can be inferred from this group of experiments. These results also suggest that L tyrosine is converted to L DOPA in an early step of biosynthesis. Hurley have found evidence for similar pathways in the biosynthesis of anthramycin tomaymycin and sibibromycin that share the same precursors (L tyrosine and L DOPA) and obtain acid moieties related to PHA in the antibiotics.
Pharmacologically Active and Related Marine Microbial Products
Marine microorganisms have been little studied for their application to human needs. Early research into marine microorganisms was concerned largely with their physiology photosynthetic capacities and potential therapeutic antibiotic and toxicological properties. This area of research remains significant particularly in view of the development of techniques enabling culture of micro age and bacteria on scales which had previously been seen an impracticable.
Recent studies have demonstrated that in addition to the useful products which can be obtained from marine microorganisms direct use of cell biomass has potential in applications ranging from pure research to agriculture and to wastewater treatment.
PHARMACOLOGICALLY ACTIVE COMPOUNDS FROM MARINE MICROORGANISMS
Most of the useful medical substances derived from microbial cultures have been antibiotics from three groups of microorganisms generally found in soils namely the filamentous fungi the spore forming bacilli and the actinomycetes. However the most common microorganisms in the marine environment are the Gram negative eubacteria the cyanobacteria and the myxobacteria groups not generally though to produce many medically useful substances.
The common groups of microorganisms in the ocean were investigated for the production of medically useful substances at the Roche Research Institute of Marine Pharmacology from 1976 to 1981. The significant results of these and related investigations are described in this section and the range of animals and the biological activity of compounds from them are shown in Table 2.
Marine Gram negative eubacteria and microalgae were found to produce a high frequency of potentially useful pharmacological activities but the marine cultures tested were not found to produce many significant antibiotic activities. In vitro antibiotic activity was observed but in nearly all cases no in vivo activity. A possible exception was the ability of extracts of the sponge Dysidea herbacea to cure mice of experimental local infections of Staphylococcus aureus. This antibiotic activity was only observed in samples of the sponge which contained large concentrations of a cyanobacterial symbiont. Conventional testing methods for microbial cultures would not have detected the pharmacological actives observed in this program because of the very low yields of biologically active compounds produced by marine microorganisms. The method which was successful in detecting the pharmacologically active compounds was to generate concentrated extracts from relatively large culture volumes (20 2001) for all initial bioassays. It was also found to be important to test these initial extracts using in vivo bioassays for both antibiotic and pharmacological activities.
Table 1 Distribution and Production of Antibiotics of the Major Microbial Groups
With few exceptions the investigation of marine microorganisms by Roche was not continued long enough to determine the chemical structures of the biologically active compounds. One series of compounds which was investigated was the group of new aromatic acids responsible for the bronchodilator activity of extracts of the marine bacterium. Alteromonas rubra. The proposed structures for the active compounds called rubrenoic acids A B and C are shown in Figure 1.
Their general superficial similarity to prostaglandin type structures attracted some interest and syntheses were achived by Hoffman La Roche in New Jersey.
In an assay for bronchodilator activity using the isolated intact guinea( ) pig trachea the rubrenoic acids A B and C each had a potency similar to the standard drug theophylline namely an ED50 of 1.5 7.5×10 5M. Preliminary in vivo tests of rubrenoic acids A and C were carried out using a modification fot he Konzett Rossler technique developed in the Roche Research Institute by Jamieson with disappointing results. At doses up to 20 mg kg 1i.v. there was no bronchodilator effect as evidenced by abolition of histamine induced bronchocon striction whereas at doses above 10mgkg 1 i.v blood pressure and heart rate were reduced. However the rubrenoic acids are a novel group of microbial metabolites and a full exploration of all three rubrenoic acids and selected analogues may yield compounds with a medically useful pharmacological profile.
It is important to note that the active metabolite purified from the marine bacterium Altero monas rubra was not one of the many known antibiotics produced by the soil microorganism. The probability of discovering a range of new medically useful compounds from terrestrial microorganism is comparatively low. This group of microorganisms has been studied extensively as evidenced by the large number of previously described antibiotics form them.
In the case where the symbiosis between the sponge Dysidea herbacea and cyanobacteria was found to produce an antibacterial metabolite hexabromodihydroxybiphenyl ether was found to be the active compound. This is also a novel compound not produced by any soil microorganisms studied to date. The purified compound was found to have activity against localized infections of mice caused by both Gram positive bacteria such as Staphylococcus aureus and Gram negative bacteria such as Escherichia coli at 30 to 100 .. In the same in vivo tests hexachlorophene was only one third as active against Staphylococcus aurous and inactive against Escherichia coli. No systemic antimicrobial action was observed. Two very surprising properties of this novel compound were (a) its relatively broad spectrum since activity against Gram negative bacteria is generally not observed for compounds with antimicrobial activity based on the phenyl moiety; and (b) its lack of observable toxicity when injected intravenously into mice since related compounds have been found to have toxicity attributable to the phenyl moiety. These properties suggested that the hexabromodihydroxybiphenyl ether be considered as a skin cleanser to replace hexachlorophene.
The structure of the hexabromodihydroxybiphenyl ether was elucidated by Norton and although the substance showed the promising activity profile as above Roche was reluctant to proceed with development because of the possible by production in synthesis or in use of one or other of the dioxins shown in Figure 3 and the possible relation of certain b products to the dioxin TCDD accidentally generated and released as Seveso in Italy.
The problem of by products has long been recognized in all branches of the production of biologically active substances for commercial utilization and this negative aspect of the by product compared with the interesting activities of the naturally occurring hexabromodihydro xybiphenyl ether should not be over accentuated In fact not all dioxins are toxic or teratogenic and the brominated dioxins above have not been fully screened for their biological activity.
PRODUCTS FROM THE CULTURE OF MICROALGAE IN COASTAL PONDS
Marine microbial technology is also being developed for the production of fine chemicals vitamins and animal feeds by the use of large coastal ponds to culture certain types of microalgae. Seawater is modified by evaporation and/or addition of inorganic nutrients to allow the growth of the desired species of microage in very larger quantities.
Until the technology of algal pond culture is further developed the cost of producing a tonne of algal biomass may be too high to allow production of cheap fuel or cheap food material. Initial prioirty is thus being given to higher value products. For example the green microalga Dunaliella salina produces high concentration of B carotene. Because of the market price of chemically synthesized B carotene the goal of achieveing a cheaper source by algal culture is more feasible than producing cheaper food. Algal pond culture is being successfully used in the specialized market of health foods. The cyanobacterium Spirulina is cultured in Mexico California and Israel and sold as a relatively high priced health food.
Not all species of algae can be grown in large open ponds because of the tendency for a succession of different types of algae to overgrow the species being cultivated. However certain algae can be maintained successfully in open ponds because of their ability to grown under conditions which severely inhibit the growth of most other species. The green microalga Dunaliella salina and the cyanobacterium Aphanothece halophytica are favoured by high salinity and high temperatures; the cyanobacteria Spirulina platensis and Spirulina maxima are favoured by high alkalinity. For any other alga proposed as a source of alga products it will be necessary either (a) to demonstrate conditions under which the alga is known to bloom (i.e. consistently grown to high concentrations as the dominant species) or (b) to consider the possibility of genetic transfer of the proposed product into one of the algal species known to grow well in open ponds.
Recent research on the genetics of cyanobacteria has made it more feasible to consider the genetic transfer of a proposed product into microage. As a model system for genetic studies of microage the cyanobacteria have several advantages. Mutant isolation is facilitated by the relative ease of handling prokaryotic cells. Most cyanobacteria can be grown in defined liquid media under rigorously controlled conditions with generation times of 5 20 hours depending on the strain. Unicellular and some flamentous species can be grown on agar medium as discrete colonies. Many bacteriological techniques for mutant isolation have been successfully adapted to the cyanobacteria. Phenotypic segregation in prokaryotes is rapid and does not require a sexual life cycle as in diploid organisms.
Of the known gene transfer mechanism in bacteria only transformation has been unequivocally domonstrated in cyanobacteria. Transformation is the term used to describe genetic recombination brought about by the uptake of DNA from the culture medium. Transformation with chemically extracted DNA has been reported for a number of unicellular cyanobacterial strains. Recombinant DNA techniques have revolutionized the study of genetics of allowing the isolation amplification and purfication of specific genes from any organism. The use of these techniques with the cyanobacteria has gained momentum over the last five years in both the development of plasmid vectors for cloning of genes in cyanobacteria and the cloning of cyanobacteria genes in the bacterium E. Coli. A number of shuttle vectors for cyanobacteria now exist which replicate stably in both E. Coli and the cyanobacterial host.
These developments mean that algal pond culture methods may now be considered for a wider range of products. To exploit this possibility the genetic research on the cyanobacteria will have to be focused on the Spirulina and Alphanothece strains which grow well in open ponds. Extension of the latest genetic techniques to the eukaryotic microalgae particularly Dunaliella species is also a priority.
Few biochemical features unique to tumor cells have so far been selectively exploited for treatment. Cancer chemotherapy has primarily been directed at discovery of cytotoxic agents capable of inhibiting many aspects of mammalian cell division. This approach to development of human clinical anticancer agents has resulted in investigation of a tremendous variety of naturally occurring compounds produced by microorganisms plants and more recently by mammalian cells in culture.
Process development for production of this variety of anticancer agents may require more diversity of biotechnology than any other single field because of the many bioengineering problems associated with these potent agents. The demand for this process technology is increasing as more antitumor antibiotics are scaled up for commercial production and as new treatment approaches using biologicals such as interferons and monoclonal antibodies begin to be evaluated for human therapy.
Table I Biotechnological Problems in Production of Anticancer Agents
General toxicity to the biochemistry of cell division often limits the final concentration of drugs produced by microbial or cell culture systems to very low levels
Recovery of agents from dilute medium in the presence of large quantities of contaminating proteins and subsequent purification for use as a human pharmaceutical
Containment of potent chemical and biological activity of the agent throughout processing and purification to protect workers and the environment
Reduction in the risks associated with production and purification (>f experimental biological using large scale transformed mammalian cells in culture
Decontamination of process wastes for inactivation of biological and chemical activity
Anticancer drug process development and production processes are different from those for other antibiotics and biologicals in that they are primarily small scale. i.e. designed to produce small quantities of drugs for investigational use or clinical application for specific types of tumors. Anticancer antibiotics marketed for general medical practice are produced on a significantly reduced scale compared with antibacterial or antifungal antibiotics.
Some of this technology however does not differ significantly from that associated with the production and purification of antibacterial and antifungal drugs. Thus biotechnology associated with laboratory scale production of many types of antibiotics with antitumor activity large scale production of hormones alkylating agents semisynthetic antimetabolites and extraction of plant materials important in cancer treatment is not included in this chapter. Process examples of cytotoxic drug production by actinomycetes therapeutic enzyme production and biologicals production by mammalian cell culture are reviewed in this chapter in order to illustrate process scale up. Purification containment and decontamination technologies unique to the anticancer field.
The Drug Development Process
The drug development scheme used by the National Cancer Institute begins with a drug discovery program (prescreen and screening phase). Since ascites tumors are useful in identifying potentially active agents a prescreening step using the P388 mouse leukemia in vitro has been evaluated along with other tumor clonogenic microbial phage induction or enzyme inhibition screens In the next in vivo evaluation step. The potentially active agents are tested against a number of model murine tumors such as mouse colon breast lung B16 melanoma and L1210 leukemia and occasionally xenografts of human colon breast and lung cancers in immunodeficient nude mice . If the compounds are active in the animal tumor screens they are then formulated for intravenous or oral use tested for toxicity in large animals (dogs monkeys) and if toxicities are reasonable brought to Phase I clinical trials (toxicology and activity). This initial small scale clinical trial is designed to find the maximally tolerated human dose. Often the Phase I trials are concurrent with Phase 11 trials (potential usefulness and dosage) designed to evaluate the maximally tolerated dose of a drug against a panel of several common solid tumors. Phase III and I V trials (safety and efficacy) explore the activity of a new drug against established agents. Finally if the drug is found to be superior to other agents it is brought into general medical practice. This overall development scheme may be as long as seven to twelve years. during which a process for large scale production of the desired agent is developed. Extensive genetics selection of a cell line or strain that is an efficient producer of the drug or engineering optimization of fermentation scale up recovery and purification often cannot be justified until after the new compound s potential clinical effectiveness has been determined (Phase I and II clinical trials). In order for a compound to progress through the stages of evaluation however increasing quantities of purified material must be available. This can require several grams (for tumor panel evaluation) to kilogram quantities of clinical grade material for analogue development or Phase I and II trials The estimated development cost for an anticancer antibiotic is $7 15 million excluding unsuccessful drugs.
Figure 1. Natural product preclinical drug development
The commercial market for all anticancer agents has grown dramatically in the last five to seven years. Many new drugs discovered in the early 1970s are now completing clinical trials and will soon enter medical practice. The current annual world market for antineoplastic agents is estimated to be in excess of $1 5 billion (U.S.) with projections to increase to $27 billion by the end of the century. The rate of growth of sales of anticancer drugs in individual countries can best be seen in Japan. In 1970 the total sales of anticancer agents was Y500 million 0.1% of total pharmaceutical sales. This figure has risen steadily Y10 billion in 1975 Y60 billion in 1977 Y107 billion in 1980 and Y122 billion in 1981. Total sales of anticancer agents in Japan now comprise greater than 3.1% of all pharmaceuticals an increase of over 30 fold in 10 years.
The total sales of anticancer agents in the U.S. in 1981 was $200 million with 30 45% of this market resulting from sales of fermentation derived drugs. The largest selling antitumor antibiotic continues to be adriamycin. This single anthracycline antibiotic has led U.S. sales of all anticancer agents for the past several years but may soon be eclipsed by synthetic platinum compounds.
Anticancer biologicals such as interferon and monoclonal antibodies for treatment of breast lung colon and prostate cancers and certain types of leukemia and lymphoma will be the fastest growing area of cancer therapy in the next five years. Monoclonal antibodies for cancer treatment and diagnosis are currently in the early stages of preclinical and pilot clinical evaluation. Many previously scarce human biologicals may become commercially available by 1990 as the result of gene cloning and microbial production processes. The current U.S. and world markets for all diagnostic uses of monoclonal antibodies are $58 million and $870 million respectively. This market is expected to expand dramatically during the next 10 years to greater than $8.4 billion per year (U.S. market). Anticancer monoclonal antibodies will share a significant percentage of this market.
Table 2 Some Media for production of L Asparaginase E coli A 1 (Met)
CONTAINMENT TECHNOLOGY FOR CYTOTOXIC AGENTS
Containment technology for large scale production and purification of microbially produced cytotoxic agents and biologicals from transformed human cells in suspension culture has evolved from the National Cancer Institute guidelines for design of biomedical research facilities for investigation of oncogenic viruses.
The basic concepts for design of an effective biological barrier to contain spills and large scale aerosols are summarized in Table3. Aerosols can be generated from any liquids involved in fermentor inoculation procedures sampling vessel overpressure situations and drug recovery operations. Accidental spills often occur due to leaks in fermentation equipment (seals head plates. quick disconnect fittings probe penetrations) during transfer of process liquids and in recovery operations such as filtration. Centrifugation and crystallization.
In the late 1940s the second wave of modern wonder drugs began. Just as the early 1940s ushered in the era of antibiotics the last part of that decade saw the demonstration of the use of corticosteroids for treatment of inflammatory diseases (i.e. rheumatoid arthritis) and injuries. The naturally occurring corticosteroids cortisone and crotisol (hydrocortisone) were used in the successful treatment of arthritis and other acute inflammatory and allergic diseases. Immediately there was a great demand for synthetic sources of these compounds. Various plant and animal steroids were examined as potential sources of this new class of medicinal compounds. For example cholesterol was examined as a possible steroid source but it was found to be very difficult to convert the saturated eight carbon side chain into the required two carbon cortical side chain. However a successful process starting with bile acids (deoxycholic acid) for the production of corticosteroids was developed in Europe. It soon became apparent that phytosterols especially diosgenin and stigmasterol offered excellent potential sources of pregenolone and progesterone respectively. Diosgenin was obtained from the root of the barbasco plant (Dioscorea villosa) which grows wild principally in Mexico and Central America while stigmasterol is abudant in soya sterols obtained from soybean seed oil. Other plant sterols such as hecogenin and solasodine from Agave and Solanum species respectively have been used to a much lesser extent as steroid starting materials.
Pregneneolone from diosgenin is easily converted to progesterone (Oppenauer reaction) which has the proper 3 one 4 ene A ring configuration of corticosteriods. Unfortunately progesterone has female hormone activity influencing pregnancy rather than corticosteroid activity. Chemical introduction of a 21 hydroxyl group in to the progesterone gives deoxycorticosterone a naturally occuring corticosteroid which is medically useful but it does not have the desired activity of for example hydrocortisone. Figure 1 shows the structure of deoxycorticosterone as well as the carbon atom numbering system for steriods. What is still required to obtain coritsone or corticol (hydrocortisone) from deoxycorticosterone is a 17 hydroxyl which could be introduced chemically or microbiologically and an 11 hydroxyl or 11 keto function. This proved much more difficult to obtain. The important bioconversion of steroids really got started with microbiological methods for introducing an 11 hydroxyl group although the first steroid conversions were apparently reductions of 17 keto steroids that were reported by Mamoli. Somewhat later the 7 hydroxylation of cholesterol was reported by Kramli but these bioconversions were not commercial significance.
Figure 1. Structure of deoxycorticosterone
Thus the stage was set for the first report of the very important 11 a hydroxylation by Peterson and Murray. A patent covering a process employing Rhizopus was issued to Upjohn in that year (Murray and Peterson). Chemistry was subsequently developed for converting the 11a hydroxyl into the desired 11 keto or 11b hydroxyl group. It should be pointed out that in steroid stereochemistry the ring system is considered essentially planar and substituents on carbon atoms that project below the plane of the molecule as shown on the page are designed as a while those projected above the molecule are b and are represented by dotted and solid bonds respectively.
In the late 1950s and early 1960s steriod compounds with hormonal activity began to find wide use as oral contraceptives but production of these compounds did not involve fermentation. Two problems that plague steriod bioconversion studies should be considered firstly the steroid presents 19 to 29 carbon atoms available for attack; secondly most steroids are quite insoluble in the aqueous systems usually used for bioconversions.
Presently the first problem seems to have been adequately resolved since a wide range of reactions at various position on steroid substrates have been well documented. Generally these reaction are stereospecific and a given culture usually produce mainly a single product and one or more minor by products. Indeed the wide variety of bioconversions is detailed in several excellent books and reviews that have appeared during the last two decades. Therefore no attempt will be made to review the steroid conversions in general. It is sufficient to point out the classes of reactions that have been reported. Also total stertoid degradation which encompasses several of these types of reactions is known to occur with certain organisms.
Table 1 Common Microbiological Reactions of Steroids
The steroid bioconversions that are of practical importance will be covered in some detail; these are 11 hydroxylation (both and b positions) 16 hydroxylation and 1 dehydrogenation. The 17 and 21 hydroxylations are also of potential practical importance. The important aspects covered in this discussion should apply equally well to other steroid conversions of the same type. Should information be desired on other steroid bioconversions it would be necessary to consult one or more of the available refrence texts mentioned above to determine the organisms and conditions to be used. Other important topics such as steroid substrate availbility including for example solubility suspension derivatization solvent conversions etc. and current areas of for example solubility suspension derivatization solvent conversions etc. and current areas of greatest active reserch interest i.e. sterol degradation use of immobilized cells and enzymes and conversion at high substrate levels in organic solvents will be discussed.
There is an area that will not be covered in depth but is important and should at least be mentioned namely the matter of choosing the proper medium for a given bioconversion. Generally any of a wide variety of media will work but the products obtained may be dependent on medium composition. Therefore it is usually necessary to test several media before selecting the one to be used for a given bioconversion. However in general with steroid bioconversions a well balanced medium in terms of carbon nitrogen and coafactors will give reasonable results and fine tuning is not nearly as critical as in antibiotic fermentations.