In the United States, 10% of the population claimspenicillin allergies, but because the frequency of positive skin test results decreases by 10% with each year of avoidance, 90% of these patients can eventually tolerate penicillin. Additionally, those with penicillin allergies can usually toleratecephalosporins (another group of β-lactam) because theimmunoglobulin E (IgE) cross-reactivity is only 3%.[5]
Penicillin was discovered in 1928 by the Scottish physicianAlexander Fleming as a crude extract ofP. rubens.[6] Fleming's student Cecil George Paine was the first to successfully use penicillin to treat eye infection (neonatal conjunctivitis) in 1930. The purified compound (penicillin F) was isolated in 1940 by a research team led byHoward Florey andErnst Boris Chain at theUniversity of Oxford. Fleming first used the purified penicillin to treat streptococcalmeningitis in 1942.[7] The 1945Nobel Prize in Physiology or Medicine was shared by Chain, Fleming and Florey.
The term "penicillin" is defined as the natural product ofPenicillium mould with antimicrobial activity.[8] It was coined byAlexander Fleming on 7 March 1929 when he discovered the antibacterial property ofPenicillium rubens.[9] Fleming explained in his 1929 paper in theBritish Journal of Experimental Pathology that "to avoid the repetition of the rather cumbersome phrase 'Mould broth filtrate', the name 'penicillin' will be used."[10] The name thus refers to the scientific name of the mould, as described by Fleming in his Nobel lecture in 1945:
I have been frequently asked why I invented the name "Penicillin". I simply followed perfectly orthodox lines and coined a word which explained that the substance penicillin was derived from a plant of the genus Penicillium just as many years ago the word "Digitalin" was invented for a substance derived from the plantDigitalis.[11]
In modern usagepenicillin is used more broadly to refer to anyβ-lactam antimicrobial that contains athiazolidine ring fused to the β-lactam core and may or may not be a natural product.[12] Like most natural products, penicillin is present inPenicillium moulds as a mixture of active constituents (gentamicin is another example of a natural product that is an ill-defined mixture of active components).[8] The principal active components ofPenicillium are listed in the following table:[13][14]
Other minor active components ofPenicillium includepenicillin O,[20][21] penicillin U1, and penicillin U6. Other named constituents of naturalPenicillium, such as penicillin A, were subsequently found not to have antibiotic activity and are not chemically related to antibiotic penicillins.[8]
The precise constitution of the penicillin extracted depends on the species ofPenicillium mould used and on the nutrient media used to culture the mould.[8] Fleming's original strain ofPenicillium rubens produces principally penicillin F, named after Fleming. But penicillin F is unstable, difficult to isolate, and produced by the mould in small quantities.[8]
The principal commercial strain ofPenicillium chrysogenum (the Peoria strain) producespenicillin G as the principal component when corn steep liquor is used as the culture medium.[8] Whenphenoxyethanol or phenoxyacetic acid are added to the culture medium, the mould producespenicillin V as the main penicillin instead.[8]
6-Aminopenicillanic acid (6-APA) is a compound derived from penicillin G. 6-APA contains the beta-lactam core of penicillin G, but with the side chains stripped off; 6-APA is a useful precursor for manufacturing other penicillins. There are many semi-synthetic penicillins derived from 6-APA and these are in three groups: antistaphylococcal penicillins, broad-spectrum penicillins, and antipseudomonal penicillins. The semi-synthetic penicillins are all referred to as penicillins because they are all derived ultimately from penicillin G.
Penicillin units
One unit of penicillin G sodium is defined as 0.600 micrograms. Therefore, 2 million units (2 megaunits) of penicillin G is 1.2 g.[22]
One unit of penicillin V potassium is defined as 0.625 micrograms. Therefore 400,000 units of penicillin V is 250 mg.[23]
The use of units to prescribe penicillin is largely obsolete outside of the US. Since the original penicillin was an ill-defined mixture of active compounds (an amorphous yellow powder), the potency of penicillin varied from batch to batch. It was therefore impractical to prescribe 1 g of penicillin because the activity of 1 g of penicillin from one batch would be different from the activity from another batch. To address this problem, after manufacture, each batch of penicillin was standardised against a known unit of penicillin: each glass vial was then filled with the number of units required. In the 1940s, a vial of 5,000 Oxford units was standard,[24] but the depending on the batch, could contain anything from 15 mg to 20 mg of penicillin. Later, a vial of 1,000,000 international units became standard, and this could contain 2.5 g to 3 g of natural penicillin (a mixture of penicillin I, II, III, and IV and natural impurities). With the advent of pure penicillin G preparations (a white crystalline powder), there is little reason to prescribe penicillin in units, although units are still used forbenzathine benzylpenicillin in the United States.
The "unit" of penicillin has had three previous definitions, and each definition was chosen as being roughly equivalent to the previous one.
Oxford or Florey unit (1941). This was originally defined as the minimum amount of penicillin dissolved in 50 ml of meat extract that would inhibit the growth of a standard strain ofStaphylococcus aureus (theOxford Staphylococcus). The reference standard was a large batch of impure penicillin kept inOxford.[25] The assay was later modified by Florey's group to a more reproducible "cup assay": in this assay, a penicillin solution was defined to contain one unit/ml of penicillin when 339 microlitres of the solution placed in a "cup" on a plate of solid agar produced a 24 millimetre zone of inhibition of growth of Oxford Staphylococcus.[26]: 107 [27][28]
First International Standard (1944). A single 8 gram batch of pure crystalline penicillin G sodium was stored at TheNational Institute for Medical Research inMill Hill, London (the International Standard). One penicillin unit was defined at 0.6 micrograms of the International Standard. An impure "working standard" was also defined and was available in much larger quantities distributed around the world: one unit of the working standard was 2.7 micrograms (the amount per unit was much larger because of the impurities). At the same time, the cup assay was refined, where instead of specifying a zone diameter of 24 mm, the zone size were instead plotted against a reference curve to provide a readout on potency.[28][13][29]
Second International Standard (1953). A single 30 gram batch of pure crystalline penicillin G sodium was obtained: this was also stored at Mill Hill. One penicillin unit was defined as 0.5988 micrograms of the Second International Standard.[30]
There is an older unit for penicillin V that is not equivalent to the current penicillin V unit. The reason is that the US FDA incorrectly assumed that the potency of penicillin V is the same mole-for-mole as penicillin G. In fact, penicillin V is less potent than penicillin G, and the current penicillin V unit reflects that fact.
First international unit of penicillin V (1959). One unit of penicillin V was defined as 0.590 micrograms of a reference standard held at Mill Hill in London.[31] This unit is now obsolete.
A similar standard was also established for penicillin K.[32]
Types
Penicillins consist of a distinct 4-memberedbeta-lactam ring, in addition to a thiazolide ring and an R side chain. The main distinguishing feature between variants within this family is theR substituent.
This side chain is connected to the 6-aminopenicillanic acid residue and results invariations in the antimicrobial spectrum, stability and susceptibility tobeta-lactamases of each type.
Natural penicillins
Penicillin G (benzylpenicillin) was first produced from apenicillium fungus that occurs in nature. The strain of fungus used today for the manufacture of penicillin G was created bygenetic engineering to improve the yield in the manufacturing process. None of the other natural penicillins (F, K, N, X, O, U1 or U6) are currently in clinical use.
Semi-synthetic penicillin
This sectionis missing information about why – need to take stuff from the PV article History section on how it magically makes more. Please expand the sectionby making an edit requestto include this information. Further details may exist on thetalk page.(December 2022)
Penicillin V (phenoxymethylpenicillin) is produced by adding theprecursor phenoxyacetic acid to the medium in which a genetically modified strain[dubious –discuss] of thepenicillium fungus is being cultured.
Antibiotics created from 6-APA
There are three major groups of othersemi-syntheticantibiotics related to the penicillins. They are synthesised by adding various side-chains to theprecursor6-APA, which is isolated from penicillin G. These are the antistaphylococcal antibiotics, broad-spectrum antibiotics and antipseudomonal antibiotics.
Antistaphylococcal antibiotics are so-called because they are resistant to being broken down by staphylococcalpenicillinase. They are also, therefore, referred to as being penicillinase-resistant.
Broad-spectrum antibiotics
This group of antibiotics is called "broad-spectrum" because they are active against a wide range of Gram-negative bacteria such asEscherichia coli andSalmonella typhi, for which penicillin is not suitable. However, resistance in these organisms is now common.
There are many ampicillin precursors in existence. These are inactive compounds that are broken down in the gut to release ampicillin. None of these pro-drugs of ampicillin is in current use:
Pivampicillin (pivaloyloxymethyl ester of ampicillin)
Epicillin is an aminopenicillin that has never seen widespread clinical use.
Antipseudomonal antibiotics
The Gram-negative species,Pseudomonas aeruginosa, is naturally resistant to many antibiotic classes. There were many efforts in the 1960s and 1970s to develop antibiotics that are active againstPseudomonas species. There are two chemical classes within the group: carboxypenicillins and ureidopenicillins. All are given by injection: none can be given by mouth.
Penicillin G is destroyed by stomach acid, so it cannot be taken by mouth, but doses as high as 2.4 g can be given (much higher than penicillin V). It is given by intravenous or intramuscular injection. It can be formulated as an insoluble salt, and there are two such formulations in current use:procaine penicillin andbenzathine benzylpenicillin. When a high concentration in the blood must be maintained, penicillin G must be administered at relatively frequent intervals, because it is eliminated quite rapidly from the bloodstream by the kidney.
Penicillin V can be taken by mouth because it is relatively resistant to stomach acid. Doses higher than 500 mg are not fully effective because of poor absorption. It is used for the same bacterial infections as those of penicillin G and is the most widely used form of penicillin.[34] However, it is not used for diseases, such asendocarditis, where high blood levels of penicillin are required.
Bacterial susceptibility
Because penicillin resistance is now so common, other antibiotics are now the preferred choice for treatments. For example, penicillin used to be the first-line treatment for infections withNeisseria gonorrhoeae andNeisseria meningitidis, but it is no longer recommended for treatment of these infections. Penicillin resistance is now very common inStaphylococcus aureus, which means penicillin should not be used to treat infections caused byS. aureus infection unless the infecting strain is known to be susceptible.
Pain and inflammation at the injection site are also common forparenterally administered benzathine benzylpenicillin, benzylpenicillin, and, to a lesser extent, procaine benzylpenicillin. The condition is known aslivedoid dermatitis or Nicolau syndrome.[39][40]
Structure
Chemical structure of Penicillin G. The sulfur and nitrogen of the five-memberedthiazolidine ring are shown in yellow and blue respectively. The image shows that the thiazolidine ring and fused four-memberedβ-lactam are not in the sameplane.
The term "penam" is used to describe the common core skeleton of a member of the penicillins. This core has the molecular formula R-C9H11N2O4S, where R is the variable side chain that differentiates the penicillins from one another. The penam core has amolar mass of 243 g/mol, with larger penicillins having molar mass near 450—for example, cloxacillin has a molar mass of 436 g/mol. 6-APA (C8H12N2O3S) forms the basic structure of penicillins. It is made up of an enclosed dipeptide formed by the condensation ofL-cysteine andD-valine. This results in the formations of β-lactam and thiazolidinic rings.[41]
The key structural feature of the penicillins is the four-membered β-lactam ring; this structuralmoiety is essential for penicillin's antibacterial activity. The β-lactam ring is itself fused to a five-memberedthiazolidine ring. The fusion of these two rings causes the β-lactam ring to be more reactive than monocyclic β-lactams because the two fused rings distort the β-lactamamide bond and therefore remove theresonance stabilisation normally found in these chemical bonds.[42] An acyl side side chain attached to the β-lactam ring.[43]
A variety of β-lactam antibiotics have been produced following chemical modification from the 6-APA structure during synthesis, specifically by making chemical substitutions in the acyl side chain. For example, the first chemically altered penicillin, methicillin, had substitutions by methoxy groups at positions 2' and 6' of the 6-APA benzene ring from penicillin G.[41] This difference makes methicillin resistant to the activity ofβ-lactamase, an enzyme by which many bacteria are naturally unsusceptible to penicillins.[44]
Pharmacology
Entry into bacteria
Penicillin can easily enter bacterial cells in the case ofGram-positive species. This is because Gram-positive bacteria do not have an outer cell membrane and are simply enclosed in a thickcell wall.[45] Penicillin molecules are small enough to pass through the spaces ofglycoproteins in the cell wall. For this reason Gram-positive bacteria are very susceptible to penicillin (as first evidenced by the discovery of penicillin in 1928[10][page needed][clarification needed][improper synthesis?]).[46]
Penicillin, or any other molecule, entersGram-negative bacteria in a different manner. The bacteria have thinner cell walls but the external surface is coated with an additional cell membrane, called the outer membrane. The outer membrane is a lipid layer (lipopolysaccharide chain) that blocks passage of water-soluble (hydrophilic) molecules like penicillin. It thus acts as the first line of defence against any toxic substance, which is the reason for relative resistance to antibiotics compared to Gram-positive species.[47] But penicillin can still enter Gram-negative species by diffusing through aqueous channels calledporins (outer membrane proteins), which are dispersed among the fatty molecules and can transport nutrients and antibiotics into the bacteria.[48] Porins are large enough to allow diffusion of most penicillins, but the rate of diffusion through them is determined by the specific size of the drug molecules. For instance, penicillin G is large and enters through porins slowly; while smaller ampicillin and amoxicillin diffuse much faster.[49] In contrast, large vancomycin can not pass through porins and is thus ineffective for Gram-negative bacteria.[50] The size and number of porins are different in different bacteria. As a result of the two factors—size of penicillin and porin—Gram-negative bacteria can be unsusceptible or have varying degree of susceptibility to specific penicillin.[51]
Gram-negative bacteria that attempt to grow and divide in the presence of penicillin fail to do so, and instead end up shedding their cell walls.[52]Penicillin and other β-lactam antibiotics act by inhibitingpenicillin-binding proteins, which normally catalyse cross-linking of bacterial cell walls.
Penicillin kills bacteria by inhibiting the completion of the synthesis ofpeptidoglycans, the structural component of thebacterial cell wall. It specifically inhibits the activity of enzymes that are needed for the cross-linking of peptidoglycans during the final step in cell wall biosynthesis. It does this by binding topenicillin binding proteins with the β-lactam ring, a structure found on penicillin molecules.[53][54] This causes the cell wall to weaken due to fewer cross-links and means water uncontrollably flows into the cell because it cannot maintain the correct osmotic gradient. This results in celllysis and death.
Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. During the last stages of peptidoglycan biosynthesis, uridine diphosphate-N-acetylmuramic acid pentapeptide (UDP-MurNAc) is formed in which the fourth and fifth amino acids are bothD-alanyl-D-alanine. The transfer ofD-alanine is done (catalysed) by theenzymeDD-transpeptidase (penicillin-binding proteins are such type).[49] The structural integrity of bacterial cell wall depends on thecross linking of UDP-MurNAc andN-acetyl glucosamine.[55] Penicillin and other β-lactam antibiotics act as an analogue ofD-alanine-D-alanine (the dipeptide) in UDP-MurNAc owing to conformational similarities. TheDD-transpeptidase then binds the four-membered β-lactamring of penicillin instead of UDP-MurNAc.[49] As a consequence,DD-transpeptidase is inactivated, the formation of cross-links between UDP-MurNAc andN-acetyl glucosamine is blocked so that an imbalance between cell wall production and degradation develops, causing the cell to rapidly die.[56]
The enzymes thathydrolyse the peptidoglycan cross-links continue to function, even while those that form such cross-links do not. This weakens the cell wall of the bacterium, and osmotic pressure becomes increasingly uncompensated—eventually causing cell death (cytolysis). In addition, the build-up of peptidoglycan precursors triggers the activation of bacterial cell wall hydrolases and autolysins, which further digest the cell wall's peptidoglycans. The small size of the penicillins increases their potency, by allowing them to penetrate the entire depth of the cell wall. This is in contrast to theglycopeptide antibioticsvancomycin andteicoplanin, which are both much larger than the penicillins.[57]
Gram-positive bacteria are calledprotoplasts when they lose their cell walls.Gram-negative bacteria do not lose their cell walls completely and are calledspheroplasts after treatment with penicillin.[52]
Penicillin shows a synergistic effect withaminoglycosides, since the inhibition of peptidoglycan synthesis allows aminoglycosides to penetrate the bacterial cell wall more easily, allowing their disruption of bacterial protein synthesis within the cell. This results in a loweredMBC for susceptible organisms.[58]
Some bacteria produce enzymes that break down the β-lactam ring, calledβ-lactamases, which make the bacteria resistant to penicillin. Therefore, some penicillins are modified or given with other drugs for use against antibiotic-resistant bacteria or in immunocompromised patients. The use of clavulanic acid or tazobactam, β-lactamase inhibitors, alongside penicillin gives penicillin activity against β-lactamase-producing bacteria. β-Lactamase inhibitors irreversibly bind to β-lactamase preventing it from breaking down the beta-lactam rings on the antibiotic molecule. Alternatively, flucloxacillin is a modified penicillin that has activity against β-lactamase-producing bacteria due to an acyl side chain that protects the beta-lactam ring from β-lactamase.[38]
Pharmacokinetics
Penicillin has low protein binding in plasma. Thebioavailability of penicillin depends on the type: penicillin G has low bioavailability, below 30%, whereas penicillin V has higher bioavailability, between 60 and 70%.[60]
Penicillin has a short half-life and is excreted via the kidneys.[60] This means it must be dosed at least four times a day to maintain adequate levels of penicillin in the blood. Early manuals on the use of penicillin, therefore, recommended injections of penicillin as frequently as every three hours, and dosing penicillin has been described as being similar to trying to fill a bath with the plug out.[8] This is no longer required since much larger doses of penicillin are cheaply and easily available; however, some authorities recommend the use of continuous penicillin infusions for this reason.[61]
Resistance
When Alexander Fleming discovered the crude penicillin in 1928, one important observation he made was that many bacteria were not affected by penicillin.[10] This phenomenon was realised byErnst Chain andEdward Abraham while trying to identify the exact of penicillin. In 1940 they discovered that unsusceptible bacteria likeEscherichia coli produced specific enzymes that can break down penicillin molecules, thus making them resistant to the antibiotic. They named the enzymepenicillinase.[62] Penicillinase is now classified as member of enzymes called β-lactamases. These β-lactamases are naturally present in many other bacteria, and many bacteria produce them upon constant exposure to antibiotics. In most bacteria, resistance can be through three different mechanisms – reduced permeability in bacteria, reduced binding affinity of the penicillin-binding proteins (PBPs) or destruction of the antibiotic through the expression of β-lactamase.[63] Using any of these, bacteria commonly develop resistance to different antibiotics, a phenomenon calledmulti-drug resistance.
The actual process of resistance mechanism can be very complex. In case of reduced permeability in bacteria, the mechanisms are different between Gram-positive and Gram-negative bacteria. In Gram-positive bacteria, blockage of penicillin is due to changes in the cell wall. For example, resistance to vancomycin inS. aureus is due to additional peptidoglycan synthesis that makes the cell wall much thicker preventing effective penicillin entry.[46] Resistance in Gram-negative bacteria is due to mutational variations in the structure and number of porins.[51] In bacteria likePseudomonas aeruginosa, there is reduced number of porins; whereas in bacteria likeEnterobacter species,Escherichiacoli andKlebsiella pneumoniae, there are modified porins such as non-specific porins (such as OmpC and OmpF groups) that cannot transport penicillin.[64]
Resistance due to PBP alterations is highly varied. A common case is found inStreptococcus pneumoniae where there is mutation in the gene for PBP, and the mutant PBPs have decreased binding affinity for penicillins.[65] There are six mutant PBPs inS. pneumoniae, of which PBP1a, PBP2b, PBP2x and sometimes PBP2a are responsible for reduced binding affinity.[66]S. aureus can activate a hidden gene that produces a different PBP, PBD2, which has low binding affinity for penicillins.[67] There is a different strain ofS. aureus namedmethicillin-resistantS. aureus (MRSA) which is resistant not only to penicillin and other β-lactams, but also to most antibiotics. The bacterial strain developed after introduction of methicillin in 1959.[44] In MRSA, mutations in the genes (mec system) for PBP produce a variant protein called PBP2a (also termed PBP2'),[68] while making four normal PBPs. PBP2a has poor binding affinity for penicillin and also lacks glycosyltransferase activity required for complete peptidoglycan synthesis (which is carried out by the four normal PBPs).[66] InHelicobacter cinaedi, there are multiple mutations in different genes that make PBP variants.[69]
Enzymatic destruction by β-lactamases is the most important mechanism of penicillin resistance,[70] and is described as "the greatest threat to the usage [of penicillins]".[71] It was the first discovered mechanism of penicillin resistance. During the experiments when purification and biological activity tests of penicillin were performed in 1940, it was found thatE. coli was unsusceptible.[72] The reason was discovered as production of an enzyme penicillinase (hence, the first β-lactamase known) inE. coli that easily degraded penicillin.[62] There are over 2,000 types of β-lactamases each of which has unique amino acid sequence, and thus, enzymatic activity.[71] All of them are able to hydrolyse β-lactam rings but their exact target sites are different.[73] They are secreted on the bacterial surface in large quantities in Gram-positive bacteria but less so in Gram-negative species. Therefore, in a mixed bacterial infection, the Gram-positive bacteria can protect the otherwise penicillin-susceptible Gram-negative cells.[49]
There are unusual mechanisms inP. aeruginosa, in which there can be biofilm-mediated resistance and formation of multidrug-tolerantpersister cells.[74]
Starting in the late-19th century there had been reports of the antibacterial properties ofPenicillium mould, but scientists were unable to discern what process was causing the effect.[75] The Scottish physicianAlexander Fleming atSt. Mary's Hospital in London (now part ofImperial College) was the first to show thatPenicillium rubens had antibacterial properties.[76] On 3 September 1928 he observed by chance that fungal contamination of a bacterial culture (Staphylococcus aureus) appeared to kill the bacteria. He confirmed this observation with a new experiment on 28 September 1928.[77][78] He published his experiment in 1929, and called the antibacterial substance (the fungal extract) penicillin.[10]
C. J. La Touche identified the fungus asPenicillium rubrum (later reclassified byCharles Thom asP. notatum andP. chrysogenum, but later corrected asP. rubens).[79] Fleming expressed initial optimism that penicillin would be a useful antiseptic, because of its high potency and minimal toxicity in comparison to other antiseptics of the day, and noted its laboratory value in the isolation ofBacillus influenzae (now calledHaemophilus influenzae).[80][10]
Fleming did not convince anyone that his discovery was important.[80] This was largely because penicillin was so difficult to isolate that its development as a drug seemed impossible. It is speculated that had Fleming been more successful at making other scientists interested in his work, penicillin would possibly have been developed years earlier.[80]
The importance of his work has been recognised by the placement of anInternational Historic Chemical Landmark at the Alexander Fleming Laboratory Museum in London on 19 November 1999.[81]
The first successful use of pure penicillin was in 1942 when Fleming cured Harry Lambert of an infection of the nervous system (streptococcalmeningitis) which would otherwise have been fatal. By that time the Oxford team could produce only a small amount. Florey willingly gave the only available sample to Fleming. Lambert showed improvement from the very next day of the treatment, and was completely cured within a week.[89][90] Fleming published his clinical trial inThe Lancet in 1943.[7] Following the medical breakthrough, the BritishWar Cabinet set up the Penicillin Committee on 5 April 1943 that led to projects formass production.[91][92]
Mass production
A lab technician spraying penicillin mould into flasks of corn steep liquor, England, 1943A World War II-era poster from a construction site for a penicillin factory. A military grave emphasizes the site's importance, as penicillin was urgently needed in wartime.
As the medical application was established, the Oxford team found that it was impossible to produce usable amounts in their laboratory.[87] Failing to persuadeHis Majesty's Government, Florey and Heatley travelled to the US in June 1941 with their mould samples in order to interest theUS federal government for large-scale production.[93][94] They approached the Northern Regional Research Laboratory (NRRL, now theNational Center for Agricultural Utilization Research) of theUS Department of Agriculture atPeoria, Illinois, where facilities for large-scale fermentations were established.[95][94] Mass culture of the mould and search for better moulds immediately followed.[93]
On 14 March 1942 the first patient was treated for streptococcal sepsis with US-made penicillin produced byMerck & Co.[96] Half of the total supply produced at the time was used on that one patient, Anne Miller.[97] By June 1942, just enough US penicillin was available to treat ten patients.[98] In July 1943 theWar Production Board drew up a plan for the mass distribution of penicillin stocks toAllied troops fighting in Europe.[99] The results of fermentation research oncorn steep liquor at the NRRL allowed the United States to produce 2.3 million doses in time for theinvasion of Normandy in the spring of 1944. After a worldwide search in 1943, a mouldycantaloupe in aPeoria, Illinois market was found to contain the best strain of mould for production using the corn steep liquor process.[100] Six times as much penicillin could be produced compared to using Fleming's mould.[94]Jasper H. Kane, a scientist atPfizer, suggested using a deep-tank fermentation method for producing large quantities of pharmaceutical-grade penicillin.[101][26]: 109 Large-scale production resulted from the development of a deep-tank fermentation plant by the chemical engineerMargaret Hutchinson Rousseau.[102] As a direct result of the war and the War Production Board, by June 1945 over 646 billion units per year were being produced.[99]
G. Raymond Rettew made a significant contribution to the American war effort by his techniques to produce commercial quantities of penicillin, wherein he combined his knowledge of mushroom spawn with the function of the Sharples Cream Separator.[103] By 1943 Rettew's lab was producing most of the world's penicillin. During theSecond World War penicillin made a major difference in the number of deaths and amputations caused by infected wounds amongst Allied forces, saving an estimated 12–15% of lives.[104] Availability was severely limited, however, by the difficulty of manufacturing large quantities of penicillin and by the rapidrenal clearance of the drug, necessitating frequent dosing. Methods for mass production of penicillin were patented byAndrew Jackson Moyer in 1945.[105][106][107] Florey had not patented penicillin, having been advised by SirHenry Dale that doing so would be unethical.[87]
Penicillin is actively excreted, and about 80% of a penicillin dose is cleared from the body within three to four hours of administration. Indeed, during the early penicillin era, the drug was so scarce and so highly valued that it became common to collect the urine from patients being treated, so that the penicillin in the urine could be isolated and reused.[108] This was not a satisfactory solution, so researchers looked for a way to slow penicillin excretion. They hoped to find a molecule that could compete with penicillin for the organic acid transporter responsible for excretion, such that the transporter would preferentially excrete the competing molecule and the penicillin would be retained. Theuricosuric agentprobenecid proved to be suitable. When probenecid and penicillin are administered together, probenecid competitively inhibits the excretion of penicillin, increasing penicillin's concentration and prolonging its activity. Eventually, the advent of mass-production techniques and semi-synthetic penicillins resolved the supply issues, so this use of probenecid declined.[108] Probenecid is still useful, however, for certain infections requiring particularly high concentrations of penicillins.[109]
After the Second World War Australia was the first country to make the drug available for civilian use. In the United States penicillin was made available to the general public on 15 March 1945.[110]
Fleming, Florey and Chain shared the 1945 Nobel Prize in Physiology or Medicine for the development of penicillin.
A technician preparing penicillin in 1943.
Penicillin was being mass-produced in 1944.
Second World War poster extolling use of penicillin.
Dorothy Hodgkin determined the chemical structure of penicillin.
The chemistJohn C. Sheehan at theMassachusetts Institute of Technology (MIT) completed the first chemicalsynthesis of penicillin in 1957.[112][113][114] Sheehan had started his studies into penicillin synthesis in 1948, and during these investigations developed new methods for the synthesis ofpeptides, as well as newprotecting groups—groups that mask the reactivity of certain functional groups.[114][115] Although the initial synthesis developed by Sheehan was not appropriate for mass production of penicillins, one of the intermediate compounds in Sheehan's synthesis was 6-aminopenicillanic acid (6-APA), the nucleus of penicillin.[112][113][114][116]
6-APA was discovered by researchers at the Beecham Research Laboratories (later theBeecham Group) in Surrey in 1957 (published in 1959).[117] Attaching different groups to the 6-APA 'nucleus' of penicillin allowed the creation of new forms of penicillins which are more versatile and better in activity.[118]
Developments from penicillin
The narrow range of treatable diseases or "spectrum of activity" of the penicillins, along with the poor activity of the orally active phenoxymethylpenicillin, led to the search for derivatives of penicillin that could treat a wider range of infections. The isolation of 6-APA, the nucleus of penicillin, allowed for the preparation of semisynthetic penicillins, with various improvements over benzylpenicillin (bioavailability, spectrum, stability, tolerance).
The first major development was ampicillin in 1961. It offered a broader spectrum of activity than either of the original penicillins. Further development yielded β-lactamase-resistant penicillins, including flucloxacillin, dicloxacillin and methicillin. These were significant for their activity against β-lactamase-producing bacterial species, but were ineffective against themethicillin-resistantStaphylococcus aureus (MRSA) strains that subsequently emerged.[119]
Another development of the line of true penicillins was the antipseudomonal penicillins, such as carbenicillin, ticarcillin, and piperacillin, useful for their activity against Gram-negative bacteria. However, the usefulness of the β-lactam ring was such that related antibiotics, including the mecillinams, the carbapenems, and, most importantly, the cephalosporins, still retain it at the center of their structures.[120]
Production
A 1957 fermentor (bioreactor) used to growPenicillium mould
Penicillin is produced by the fermentation of various types of sugar by the fungusPenicillium rubens.[121] The fermentation process produces penicillin as asecondary metabolite when the growth of the fungus is inhibited by stress.[121] The biosynthetic pathway outlined below experiencesfeedback inhibition involving the by-productl-lysine inhibiting the enzymehomocitrate synthase.[122]
ThePenicillium cells are grown using a technique calledfed-batch culture, in which the cells are constantly subjected to stress, which is required for induction of penicillin production. While the usage ofglucose as a carbon source represses penicillin biosynthesis enzymes,lactose does not exert any effect and alkalinepH levels override this regulation. Excessphosphate, availableoxygen, and usage ofammonium as anitrogen source repress penicillin production, whilemethionine can act as a sole nitrogen/sulfur source with stimulating effects.[123]
The biosynthetic gene cluster for penicillin was first cloned and sequenced in 1990.[124] Overall, there are three main and important steps to the biosynthesis ofpenicillin G (benzylpenicillin).
The first step is the condensation of three amino acids—L-α-aminoadipic acid,L-cysteine,L-valine into atripeptide.[125][126][127] Before condensing into the tripeptide, the amino acidL-valine must undergo epimerization to becomeD-valine.[128][129] The condensed tripeptide is named δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV). The condensation reaction and epimerisation are both catalysed by the enzyme δ-(L-α-aminoadipyl)-L-cysteine-D-valine synthetase (ACVS), anonribosomal peptide synthetase or NRPS.
The second step in the biosynthesis of penicillin G is theoxidative conversion of linear ACV into thebicyclic intermediate isopenicillin N byisopenicillin N synthase (IPNS), which is encoded by the genepcbC.[125][126] Isopenicillin N is a very weak intermediate, because it does not show strong antibiotic activity.[128]
The final step is atransamidation byisopenicillin N N-acyltransferase, in which the α-aminoadipyl side-chain of isopenicillin N is removed and exchanged for aphenylacetyl side-chain. This reaction is encoded by the genepenDE, which is unique in the process of obtaining penicillins.[125]
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Further reading
Dürckheimer W, Blumbach J, Lattrell R, Scheunemann KH (March 1, 1985). "Recent Developments in the Field of β-Lactam Antibiotics".Angewandte Chemie International Edition in English.24 (3):180–202.doi:10.1002/anie.198501801.
Greenwood, David.Antimicrobial Drugs: Chronicle of a twentieth century medical triumph (Oxford University Press, 2008)summary
Hamed RB, Gomez-Castellanos JR, Henry L, Ducho C, McDonough MA,Schofield CJ (January 2013). "The eEnzymes of β-lactam Biosynthesis".Natural Product Reports.30 (1):21–107.doi:10.1039/c2np20065a.PMID23135477.
Lax E (2004).The Mold in Dr. Florey's Coat: The Story of the Penicillin Miracle. Henry Holt and Co.ISBN978-0-8050-6790-3.
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