PENICILLIN

Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium fungi. They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V. Penicillin antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases, such as syphilis, and infections caused by staphylococci and streptococci. Penicillins are still widely used today, though many types of bacteria are now resistant. All penicillins are B-lactam antibiotics and are used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms.

MEDICAL USES

The term "penicillin" is often used generically to refer to benzylpenicillin (penicillin G), procaine benzylpenicillin (procaine penicillin), benzathine benzylpenicillin (benzathine penicillin), and phenoxymethylpenicillin (penicillin V). Procaine penicillin and benzathine penicillin have the same antibacterial activity as benzylpenicillin but act for a longer time span. Phenoxymethylpenicillin is less active against Gram-negative bacteria than benzylpenicillin. Benzylpenicillin, procaine penicillin and benzathine penicillin are given by injection (parenterally), but phenoxymethylpenicillin is given orally.

ADVERSE EFFECTS

Common adverse drug reactions (>1% of patients) associated with use of the penicillins include diarrhoea, hypersensitivity, nausea, rash, neurotoxicity, urticaria, and superinfection (including candidiasis). Infrequent adverse effects (0.1–1% of patients) include fever, vomiting, erythema, dermatitis, angioedema, seizures (especially in epileptics), and pseudomembranous colitis.

MECHANISM OF ACTION

Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. B-Lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall; this is achieved through binding of the four-membered B-lactam ring of penicillin to the enzyme DD-transpeptidase. Consequently, DD-transpeptidase cannot catalyze formation of these cross-links, and an imbalance between cell wall production and degradation develops, causing the cell to rapidly die. More specifically, the enzymes that hydrolyze 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 continues to rise—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 the glycopeptide antibiotics vancomycin and teicoplanin, which are both much larger than the penicillins. Gram-positive bacteria are called protoplasts when they lose their cell walls. Gram-negative bacteria do not lose their cell walls completely and are called spheroplasts after treatment with penicillin. Penicillin shows a synergistic effect with aminoglycosides, 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 lowered MBC for susceptible organisms. Penicillins, like other B-lactam antibiotics, block not only the division of bacteria, including cyanobacteria, but also the division of cyanelles, the photosynthetic organelles of the glaucophytes, and the division of chloroplasts of bryophytes. In contrast, they have no effect on the plastids of the highly developed vascular plants. This supports the endosymbiotic theory of the evolution of plastid division in land plants.

STRUCTURE

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 a molecular weight of 243 g/mol, with larger penicillins having molecular weights near 450—for example, cloxacillin has a molecular weight of 436 g/mol. The key structural feature of the penicillins is the four-membered B-lactam ring; this structural moiety is essential for penicillin's antibacterial activity. The B-lactam ring is itself fused to a five-membered thiazolidine ring. The fusion of these two rings causes the B-lactam ring to be more reactive than monocyclic B-lactams because the two fused rings distort the B-lactam amide bond and therefore remove the resonance stabilisation normally found in these chemical bonds.

BIOSYNTHESIS

Overall, there are three main and important steps to the biosynthesis of penicillin G (benzylpenicillin).
The first step is the condensation of three amino acids—L-a-aminoadipic acid, L-cysteine, L-valine into a tripeptide. Before condensing into the tripeptide, the amino acid L-valine must undergo epimerization to become D-valine. The condensed tripeptide is named S-(L-a-aminoadipyl)-L-cysteine-D-valine (ACV). The condensation reaction and epimerization are both catalyzed by the enzyme S-(L-a-aminoadipyl)-L-cysteine-D-valine synthetase (ACVS), a nonribosomal peptide synthetase or NRPS.

The second step in the biosynthesis of penicillin G is the oxidative conversion of linear ACV into the bicyclic intermediate isopenicillin N by isopenicillin N synthase (IPNS), which is encoded by the gene pcbC. Isopenicillin N is a very weak intermediate, because it does not show strong antibiotic activity.
The final step is a transamidation by isopenicillin N N-acyltransferase, in which the a-aminoadipyl side-chain of isopenicillin N is removed and exchanged for a phenylacetyl side-chain. This reaction is encoded by the gene penDE, which is unique in the process of obtaining penicillins.

PRODUCTION

Penicillin is a secondary metabolite of certain species of Penicillium and is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. a-ketoglutarate AcCoA - homocitrate - L-a-aminoadipic acid - L-lysine B-lactam The by-product, l-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production. The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress, which is required for induction of penicillin production. The available carbon sources are also important: Glucose inhibits penicillin production, whereas lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled. The biotechnological method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques include error-prone PCR, DNA shuffling, ITCHY, and strand-overlap PCR. Semisynthetic penicillins are prepared starting from the penicillin nucleus 6-APA.

HISTORY

DISCOVERY

The discovery of penicillin is attributed to Scottish scientist and Nobel laureate Alexander Fleming in 1928. He showed that, if Penicillium rubens were grown in the appropriate substrate, it would exude a substance with antibiotic properties, which he dubbed penicillin. This serendipitous observation began the modern era of antibiotic discovery. The development of penicillin for use as a medicine is attributed to the Australian Nobel laureate Howard Walter Florey, together with the German Nobel laureate Ernst Chain and the English biochemist Norman Heatley. The first published reference appears in the publication of the Royal Society in 1875, by John Tyndall. Joaquim Monteiro Caminhoá, Professor of Botany and Zoology of the Faculty of Medicine of the Federal University of Rio de Janeiro in Brazil, also recognised the antibiotic activity of Penicillium and other fungi in 1877. In his book, Elements of General and Medical Botany (under a section titled "Useful fungi, harmful and curious"), he stated:

"The mould (Penicillium infestans, Penicillium glaucum, figure 1680, Ascophora and many others) is useful because it feeds on decaying organic matter and destroys putrifaction so that, as a rule, the odour of infection does not occur, or is produced in infinitely smaller amounts."

In 1895, Vincenzo Tiberio, physician of the University of Naples published a research about a mold (Penicillium) in a water well that had an antibacterial action. Ernest Duchesne documented it in an 1897 paper, which was not accepted by the Institut Pasteur because of his youth. In March 2000, doctors at the San Juan de Dios Hospital in San José, Costa Rica, published the manuscripts of the Costa Rican scientist and medical doctor Clodomiro (Clorito) Picado Twight (1887–1944). They reported Picado's observations on the inhibitory actions of fungi of the genus Penicillium between 1915 and 1927. Picado reported his discovery to the Paris Academy of Sciences, yet did not patent it, even though his investigations started years before Fleming's. Joseph Lister was experimenting with Penicillum in 1871 for his aseptic surgery. He found that it weakened the microbes, but then he dismissed the fungi. These early investigations did not lead to the use of antibiotics to treat infection because they took place in obscure circumstances, and the idea that infections were caused by transmissible agents was not widely accepted at the time. Sterilization measures had been shown to limit the outbreak and spread of disease; however, the mechanism of transmission of disease by parasites, bacteria, viruses and other agents was unknown. In the late 19th century, knowledge was increasing of the mechanisms by which living organisms become infected, how they manage infection once it has begun and, most importantly in the case of penicillin, the effect that natural and man-made agents could have on the progress of infection. Fleming recounted that the date of his discovery of penicillin was on the morning of Friday, September 28, 1928. It was a fortuitous accident: in his laboratory in the basement of St. Mary's Hospital in London (now part of Imperial College), Fleming noticed a Petri dish containing Staphylococcus plate culture he mistakenly left open, was contaminated by blue-green mould, which formed a visible growth. There was a halo of inhibited bacterial growth around the mould. Fleming concluded the mould released a substance that repressed the growth and lysing the bacteria. He grew a pure culture and discovered it was a Penicillium mould, now known to be Penicillium notatum. Charles Thom, an American specialist working at the U.S. Department of Agriculture, was the acknowledged expert, and Fleming referred the matter to him. Fleming coined the term "penicillin" to describe the filtrate of a broth culture of the Penicillium mould. Even in these early stages, penicillin was found to be most effective against Gram-positive bacteria, and ineffective against Gram-negative organisms and fungi. He expressed initial optimism that penicillin would be a useful disinfectant, being highly potent with minimal toxicity compared to antiseptics of the day, and noted its laboratory value in the isolation of Bacillus influenzae (now Haemophilus influenzae). After further experiments, Fleming was convinced penicillin could not last long enough in the human body to kill pathogenic bacteria, and stopped studying it after 1931. He restarted clinical trials in 1934, and continued to try to get someone to purify it until 1940.

MEDICAL APPLICATION

In 1930, Cecil George Paine, a pathologist at the Royal Infirmary in Sheffield, attempted to use penicillin to treat sycosis barbae, eruptions in beard follicles, but was unsuccessful, probably because the drug did not penetrate the skin deeply enough. Moving on to ophthalmia neonatorum, a gonococcal infection in infants, he achieved the first recorded cure with penicillin, on November 25, 1930. He then cured four additional patients (one adult and three infants) of eye infections, and failed to cure a fifth. In 1939, Australian scientist Howard Florey (later Baron Florey) and a team of researchers (Ernst Boris Chain, Arthur Duncan Gardner, Norman Heatley, M. Jennings, J. Orr-Ewing and G. Sanders) at the Sir William Dunn School of Pathology, University of Oxford made significant progress in showing the in vivo bactericidal action of penicillin. Their attempts to treat humans failed because of insufficient volumes of penicillin (the first patient treated was Reserve Constable Albert Alexander), but they proved it harmless and effective on mice. Some of the pioneering trials of penicillin took place at the Radcliffe Infirmary in Oxford, England. These trials continue to be cited by some sources as the first cures using penicillin, though the Paine trials took place earlier. On March 14, 1942, John Bumstead and Orvan Hess saved a dying patient's life using penicillin. Notably, survivors of the November 28, 1942 Cocoanut Grove fire in Boston, which killed 492 people, were treated with penicillin. Merck and Company rushed a 32-liter supply of the drug, in the form of culture liquid in which the Penicillium mold had been grown, from New Jersey to Boston in early December. The drug was crucial in combating staphylococcus bacteria which typically infect skin grafts. As a result of the success of penicillin in preventing infections, the US Government decided to support the production and distribution of penicillin to the armed forces.

HUMAN EXPERIMENTATION

In a 1946 to 1948 study in Guatemala, U.S. researchers used prostitutes to infect prison inmates, insane asylum patients, and Guatemalan soldiers with syphilis and other sexually transmitted diseases (STDs), to test the effectiveness of penicillin in treating such diseases. They later tried infecting people with "direct inoculations made from syphilis bacteria poured into the men's penises and on forearms and faces that were slightly abraded ... or in a few cases through spinal punctures". Approximately 1300 people were infected as part of the study (including orphaned children). The study was sponsored by the Public Health Service, the National Institutes of Health and the Pan American Health Sanitary Bureau (now the World Health Organization's Pan American Health Organization) and the Guatemalan government. The team was led by John Charles Cutler, who later participated in the Tuskegee syphilis experiments. Cutler chose to do the study in Guatemala because he would not have been permitted to do it in the United States. The Presidential Commission for the Study of Bioethical Issues determined that 83 people died; however, it was not possible to determine whether the experiments were the direct cause of death.

TOTAL SYNTHESIS

Chemist John C. Sheehan at the Massachusetts Institute of Technology (MIT) completed the first chemical synthesis of penicillin in 1957. Sheehan had started his studies into penicillin synthesis in 1948, and during these investigations developed new methods for the synthesis of peptides, as well as new protecting groups—groups that mask the reactivity of certain functional groups. Although the 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. Attaching different groups to the 6-APA 'nucleus' of penicillin allowed the creation of new forms of penicillin.

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, which offered a broader spectrum of activity than either of the original penicillins. Further development yielded B-lactamase-resistant penicillins, including flucloxacillin, dicloxacillin, and methicillin. These were significant for their activity against B-lactamase-producing bacterial species, but were ineffective against the methicillin-resistant Staphylococcus aureus (MRSA) strains that subsequently emerged. 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 B-lactam ring was such that related antibiotics, including the mecillinams, the carbapenems and, most important, the cephalosporins, still retain it at the center of their structures.

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