Antibiotics II

Beta-Lactam Antibiotics They have β-lactam heterocyclic nucleus consists of a 4-membered cyclic ring with a N-atom. The highly-strained β-lactam nucleus is stabilized by means of the fusion of a variety of either 5-membered or 6-membered heterocyclic moieties to give rise to a wide spectrum of newer antibiotics. Beta-Lactam antibiotics inhibit the formation of the structure peptidoglycan of the bacterial cell wall. As this component is absent in mammalian cells, Beta-Lactam antibiotics have very low toxiciy towards mammailian.

Beta-Lactam antibiotics essentially comprise of : Penicillins Cephalosporins Imipenem, Nocardicin A, Monobactem , Clavulanic acid, Moxalactam, and Thienamycin

General structure of penicillin

Production of antibiotics In general, the antibiotics are produced on a large scale by three known methods, namely: (a) fermentation process. (b) semi-synthetic process : in which the products obtained by fermentation are modified by chemical addition of side chain . (c) synthetic process.

Production of Penicillin Now most antibiotics including penicillin are produced by staged fermentations in which strains of microorganisms producing high yields are grown under optimum conditions nutrient media fermentation tanks holding several thousand gallons. Penicillin is produced by the fungus Penicillium chrysogenum which requires sugars, and a source of nitrogen in the medium to grow well. Like all antibiotics, penicillin is a secondary metabolite, so is only produced in the stationary phase.

When penicillin was first made at the end of the second world war using the fungus Penicilium notatum, the process made 4 mg L-1. Today, using a different species (P. chrysogenum) and a better extraction procedures the yield is 40 mg L–1. Penicilium notatum P. chrysogenum

Penicillin production The microorganism culture is developed first in the lab by the addition of Penicillium chrysogenum spores into a liquid medium. When it has grown to the acceptable amount, it is inoculated into the fermenter , the inoculum sholud represent 5-10% of the volume of fermenter which contains the culture medium. culture medium consists of : Source of carbon nutrition: – e.g., lactose and glucose; Nitrogen sources: – e.g., corn steep liquor; Phosphate buffer: – to provide P in the medium and also to maintain the pH of the medium. The medium is constantly aerated and agitated. Typical parameters such as pH, temperature, stirrer speed and dissolved oxygen concentration, are observed

There are two important and distinct phases normally encountered in the fermentative process, namely: (a) Growth Phase of the Organism: It is also sometimes referred to as the ‘trophophase’; wherein the number of organisms per unit time increases progressively (30-40 hours) (b) Idiophase of the Organism: In the idiophase there is a substantial antibiotic production; and hence, invariably termed as the ‘antibiotic production phase’. (5-7 days)

In the ‘growth phase’, the culture becomes thick by virtue of the formation of ‘aggregates of fungal cells’ usually known as mycelium. Glucose is preferentially consumed as compared to lactose specifically in the ‘growth phase’, as it may be employed as a prime source of C directly, However , the glucose gets fully utilized, and subsequently the fungus makes use of ‘lactose’ as a source of C. Interestingly, no additional growth takes place as the lactose cannot be used as such unless and until it gets converted to glucose and galactose via hydrolysis. Hence the decreased availability of C in the medium obviously offers a ‘triggering mechanism’ in the production of penicillin.

After growth phase penicillin production phase occurs After growth phase penicillin production phase occurs. and penicillin secreted from the fungal cells in to the medium .In this phase growth is sharply reduced PH is kept constant at 6.5. Phenyl acetic acid or pehnoxyacetic acid is fed continuously as precursor. After about 7 days, growth is completed, the pH rises to 8.0 or above and penicillin production ceases. The resulting penicillin can be chemically and enzymatically modified by the introduction of various acids, amines or amides into the medium in which the mold is being developed thereby leading to the ultimate production of a spectrum of biosynthetic penicillins.

Batch-fed fermenter GKM/ANTIBIOTIC/2013 Tuesday, March 26, 2013

Separation and Isolation of Antibiotics In an ideal situation the first isolation process must be as selective and efficient as possible so as to achieve the maximum yield, besides to help in subsequent purification . However, the choice of isolation method depends on particular chemical characteristic feature of an antibiotic and also their accompanying metabolites . .

Separation and Isolation of Antibiotics The first step is the recovery process is the removal of mycelium or cells by filtration or centrifuging. Second step is to remove the antibiotic from the spent production medium by solvent extraction, adsorption or precipitation. Additional solvent extraction, distillation, sublimation, column chromatography or other methods accomplish purification.

The various factors that exert vital impact upon the qualitative and quantitative antibiotic production are : 􀁺 Sources of nutritional C and N 􀁺 Ratio of C/N in nutrients 􀁺 Mineral composition of medium 􀁺 Temperature of incubation 􀁺 Initial pH, control and management of pH during the entire course of fermentation. 􀁺 Aeration mode and rate 􀁺 Time-phase for addition of special growth and antibiotic enhancing materials.

Classification of penicillin Natural Penicillins (best streptococcal and narrow spectrum) Penicillinase-resistant Penicillins (antistaphylococcal) Aminopenicillins (improved Gram-negative: H. influenzae, Enterococcus, Shigella, Salmonella) Extended-spectrum (antipseudomonal) penicillins Beta Lactamase Combinations (expand spectrum to staph, beta-lactamase producers)

Penicillin G Potassium Preparation: It is prepared by the interaction of 6-amino-penicillanic acid and phenyl acetyl chloride in an inert organic solvent. Uses 1. It is still recommended as an important and useful drug for the treatment of many Gram-positive organisms, such as streptococci, pneumococci, gonococci, and meningococci infections. 2. It is mostly destroyed by gastric juice and is, therefore, not given by oral route, and is best administered as IM or IV injection. 3. The K-salt as such has no advantage over the corresponding Na-salt except when high doses are used in patients on sodium restriction e.g., blood-pressure patients. 4. The K-salt also avoids the incidence of hypokalemic alkalosis which occasionally takes place during prolonged treatment with high doses of penicillins. 5. The half-life ranges between 0.5 to 0.7 hour; except 2.5 to 10 hour in renal failure or after probenecid.

Penicillin V Biological Source: It is obtained by the addition of phenoxyacetic acid to the Penicillium chrysogenum culture Uses 1. Phenoxymethylpenicillin (Penicillin V) is ‘acid-resistant, due to the introduction of an electron-withdrawing heteroatom (i.e., O-atom of phenoxy-moiety) into the side-chain. 2. It is, therefore, suitable for oral administration. 3. It is specifically recommended for respiratory tract infections and tonsilitis.

Ampicillin and Amoxicillin Biological Source: orally active, semi-synthetic antibiotics related to penicillin with side-chain containing a basic amino moiety uses They have an antibacterial spectrum similar to that of penicillin G but are more effective against gram negative bacilli These agents are also widely used in the treatment of respiratory infections. Amoxicillin is found to be more acid stable than ampicillin and absorption is not affected appreciably by food intake Amoxicillin is employed prophylactically by dentists in high-risk patients for the prevention of bacterial endocarditis Ampicillin is the drug of choice for the gram-positive bacillus Listeria monocytogenes and susceptible enterococcal species.

Cephalosporin In 1948, the first chemical compounds of the cephalosporin group was isolated from Cephalosporium acremonium. Today, cephalosporin antibiotics are classified into three manners: spectrum of activity, resistance to β- lactamase, their activity differences against gram- positive/ negative organisms. The important difference between penicillin and cephalosporin structures can be seen in the enlargement from a five- membered to six- membered ring attached to the common β- lactam core. The backbone of the cephalosporin is cephem ,which consists of a bicycle system with a four- membered β- lactam ring and a hydrocyclothiazide ring.

Cephalosporins are more stable under acidic conditions and exhibit fewer allergic reactions; these cephalosporins have a prominent place in antibiotic therapy in modern times MOA: Similar to that of the penicillins , interfere with bacterial peptidoglycan synthesis and subsequently cell wall synthesis . cephalosporin

Cephalosporin classification First generation Cephalosporins: Cephalothin , Cephalexin Cefadroxil, Cephapirin, Cephradine, Cefazolin Second Generation Cephalosporins: Cefaclor; Cefamandole; Cefuroxime; Cefonicid Third Generation Cephalosphorins: Cefotaxime; Cefoperazone ;Ceftazidime; Ceftriaxone; Cefmonoxime, Moxalactam; Prodrugs: Cefpodoxime proxetil; Cefuroxime axetil; Cephamycins: Cephamycin C; Cefoxitin

Prodrugs A major disadvantage of the current cephalosporins is that they are not rapidly and effectively absorbed through oral route. This specific serious drawback is perhaps due to the nature of the side-chain present at C-3. An attempt has been made to design orally active prodrugs, namely: cefuroxime-axetil cefpodoxime-proxetil, which have been developed by providing an additional ester group on the C-4 carboxyl moiety. Nevertheless, these prodrugs are designed in such a manner that they are easily hydrolysed to the active agents i.e., drugs by the esterases. Cephamycins represent another group of cephalosporin antibiotics that are characterized by a 7α-methoxy function, and are usually produced by two consecutive reactions, namely: hydroxylation and methylation.

First Generation Cephalosporins Cefadroxil Biological Source It is an orally active semi-synthetic cephalosporin antibiotic obtained from the species Cephalosporium aeremonium. Uses 1. It is intermediate acting and quite effective against Staphylococcus and certain enteric Gram negative bacilli. 2. Because of its prolonged exeretion criterion, it has an added advantage of catering for more sustained serum and urine concentrations than are usually obtained with other oral cephalosporins.

Second Generation Cephalosporins Cefuroxime Several antibiotics belonging to the category of ‘second generation antibiotics’ are absorbed orally. Interestingly, cefuroxime is available in two different versions first—as its sodium salt secondly— as its prodrug cefuroxime axetil that are hydrolyzed once they are absorbed and its absorption rate increased by the intake of food in-take

Uses 1. Its activity against H. influenzae and ability to penetrate into the CSF makes it specifically useful for the treatment, control and management of meningitis caused by this organism. 2. It exhibits an excellent and super activity against all species of gonococci, hence it is recommended for the treatment of gonorrhea. 3. It may also be employed to treat lower respiratory tract infections normally caused by H. influenzae and parainfluenzae, Klebsiella species, E. coli, Strep pneumoniae and pyrogenes and Staph aureus. 4. It is also approved for use against UTIs caused by E. Coli and Klebsiella. 5. It is also recommended for use in bone infections, septicemias and surgical prophylaxis.

Third Generation Cephalosporins Cefotaxime Biological Source : It is a broad spectrum third generation cephalosporin antibiotic derived from Cephalosporium acremonium. Uses It is found to be active against a good number of Gram-negative bacilli . It is highly resistant to the β-lactamases.. It is a recognized and preferred third generation cephalosporin for Gram-negative meningitis and other serious Gram-negative bacillary infections outside the CNS. It is also recommended widely for surgical prophylaxis..