Mechanism of action of Itraconazole:

• It acts like all other azoles i.e. it interferes with the cytochrome P 450-dependent enzyme, lanosterol 14 -demethylase.
• This results in the accumulation of 14-methylsterol and  depletion of ergosterol in fungal cells, which ultimately leads to disruptions in membrane structure and function.

Spectrum of action of Itraconazole:

• Itraconazole occupies a broad spectrum of action including Aspergillus species, Blastomyces dermatitidis, Candida species, C. immitis, C. neoformans, Histoplasma capsulatum, Malassezia furfur, Paracoccidioides brasiliensis, P. mameffei, Scedosporium apiospermum and S. schenckii.
• It is less active against Fusarium species.
• It is effective against many dematiaceous moulds and dermatophytes, but doesn’t act against the Zygomycetes.

Acquired Itraconazole resistance:

• Acquired resistance is rare, however, ketoconazole-resistant Candida albicans strains from patients with chronic mucocutaneous candidosis have been cross-resistant to itraconazole.
•  Also some fluconazole-resistant C. albicans strains from AIDS patients with chronic relapsing oropharyngeal candidosis show resistance to itraconazole.
• Itraconazole-resistant strains of Aspergillus fumigatus have been reported following treatment, but this is a rare condition.

Pharmacokinetics of Itraconazole:

• The gastrointestinal tract absorption of itraconazole is about 55%, but is enhanced if the drug is taken with food.
• Oral intake of a single 100-mg capsule will result peak serum concentrations of 0.1-0.2 mg/L about 2- 4h later.
• Higher concentrations are achieved, after repeated dosing, however it varies according to individuals.
• Itraconazole has high protein binding capacity, exceeding 99% in human serum.
• Itraconazole has been found to persist in the skin for 2-4 weeks.
• Even after the end of a 4 week course of treatment, it persists in toenails for upto 6 months after the end of a 3-month course of treatment.

Therapeutic use of itraconazole:

• Itraconazole is available in oral and parenteral forms as oral capsules, oral solution and intravenous infusion.
• Itraconazole has been employed to treat several superficial fungal infections, including the dermatophytoses, tinea versicolor, and mucosal and cutaneous forms of candidosis.
• Certain forms of phaeohyphomycosis, and subcutaneous infections such as chromoblastomycosis, sporotrichosis can be treated by itraconazole.
• Itraconazole is drug of choice in case of non-life-threatening forms of blastomycosis and histoplasmosis
• It is a beneficial alternative to amphotericin B for invasive aspergillosis.
• Itraconazole is the drug of choice for maintenance treatment to prevent relapse in AIDS patients with histoplasmosis.
• However, it is less effective than fluconazole as maintenance treatment in AIDS patients with cryptococcosis.
• Itraconazole oral suspension has helped to prevent aspergillosis in neutropenic patients, and has proved an effective treatment for oropharyngeal candidosis in HIV-infected individuals.
• However, the drug has not been adequately evaluated as treatment for deep- seated forms of candidosis.

Side effects of Itraconazole

• The most frequently reported side-effects are gastrointestinal in origin, such as nausea, abdominal discomfort and constipation.
• Less common side-effects include headache, dizziness, pruritus and allergic rashes. Isolated cases of Stevens- Johnson syndrome have been reported.
• The most frequent adverse events among patients treated with intravenous itraconazole have been nausea, diarrhoea and hypokalemia.
• There have been rare cases of liver failure and death among persons taking itraconazole, including some within the first week.
• The drug is best avoided in patients with liver disease and those with elevated liver function tests.
• If signs of liver disease develop during treatment, the drug should be discontinued and liver function tests performed.
• Itraconazole should not be used to treat nail infections in patients who have had heart failure, nor should it be used to treat more serious infections, unless the expected benefit exceeds the risk.
• If signs of congestive heart failure should appear during treatment, itraconazole should be discontinued.

Drug interactions of itraconazole:

• Itraconazole should not be administered together with drugs that reduce gastric acid secretion, such as antacids, H2-antagonists, omeprazole and lansoprazole.
• These drugs reduce absorption of itraconazole.
• Both itraconazole and its major metabolite, hydroxy- itraconazole, are inhibitors of the human hepatic cytochrome P-450-3A4 enzyme system.
• Associated administration of itraconazole with other drugs that are metabolized by this system can result in increased concentrations of the azole, the interacting drug, or both.
• Itraconazole prolongs the half- life of terfenadine, astemizole, midazolam, triazolam, lovastatin, simvastatin, cisapride, pimozide, quinidine and dofetilide. It should not be administered with these drugs.
• Itraconazole increases the serum concentrations of digoxin, cyclosporin and tacrolimus, and levels of these drugs should be observed if they are given together.
• Itraconazole can also increase the serum concentration of warfarin, enhancing its anti- coagulant effect.
• Careful observation of prothrombin time in patients receiving both drugs is advised.
• Co-administration of itraconazole with anti-HIV protease inhibitors, such as ritonavir, indinavir and saquinavir, can result in increased blood concentrations of both the azole and the interacting drug.
• Itraconazole can inhibit the metabolism of vincristine.
• Patients receiving both agents should be monitored for signs of toxic effects of the vinca alkaloid.
• In case of signs observed, the dose of vincristine should be adjusted, or itraconazole treatment discontinued.
• Associated administration of itraconazole with drugs that are potent inducers of the human cytochrome P-450 enzyme system results in a marked reduction in blood levels of the azole agent.
• Interacting drugs include phenytoin, phenobarbital, carbamazepine, rifampicin and rifabutin.

Itraconazole-mechanism, spectrum, pharmacokinetics, uses and side effect

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Mechanism of action of Fluconazole:

• Fluconazole works like other azoles and is a potent inhibitor of ergosterol biosynthesis through its action on the cytochrome P-450- dependent enzyme, lanosterol 14 -demethylase.
• Ergosterol the principal sterol in the membrane of susceptible fungal cells
• Depletion of ergosterol, and accumulation of methylated sterols leads to disruptions in membrane structure and function.

Spectrum of action:

• Fluconazole is most active against Candida species, Cryptococcus neoformans and C . immitis.
• It has more limited activity against Blastomyces dermatitidis, Histoplasma capsulatum and Sporothrix schenckii, and is used as a second-line agent in infections with these fungi.
• Fluconazole is active against the dermatophytes (Trichophyton, Microsporum and Epidermophyton species), but appears to be ineffective against most other moulds, including Aspergillus species and Zygomycetes.
• Even if it is active against many Candida species, C . krusei is intrinsically resistant to fluconazole.
• Isolates of Candida glabrata are also less sensitive, and as many as 10% of bloodstream isolates of this species are resistant.

Acquired Fluconazole resistance:

• The resistance is seen in Candida albicans or C. tropicalis during short-term fluconazole treatment in patients with mucosal or deep- seated forms of candidosis.
• In AIDS patients, resistant strains of C. albicans have appeared following repeated courses of low-dose fluconazole treatment for oral or oesophageal infection.
• Many ofthese fluconazole-resistant C. albicans strains are seen to be cross-resistant to other azoles.
• This is an unusual problem at present.
•  There are a few reports ofresistant strains of Cryptococcus neoformans from AIDS patients with relapsed infection following long-term maintenance treatment with fluconazole.
• Several molecular mechanisms of azole drug resistance in C. albicans have now been elucidated.
• These include overexpression of a number of efflux pump genes, including the ABC transporter genes, CDRl and CDR2, and the major facilitator gene, MDRl.
• Over- expression of these pumps leads to reduced drug accumulation in the cells of resistant strains.
• The second resistance mechanism is point mutations in the ERG11 gene that encodes the target enzyme, lanosterol 14 -demethylase.
• These mutations result in structural alteration of the enzyme and this leads to decreased binding to azole antifungals.
• The third resistance mechanism is overexpression of the ERG11 gene.
• This leads to overproduction of the target enzyme.
• In addition, evidence is accumulating that changes in other enzymes involved in ergosterol biosynthesis, such as C5(6)sterol desaturase, can also contribute to azole resistance.

Pharmacokinetics of fluconazole:

• Oral administration of fluconazole leads to rapid and almost complete absorption of the drug.
• Identical serum concentrations are achieved after oral and parenteral administration indicating that first-pass metabolism of the drug does not occur.
• Blood concentrations increase in proportion to dosage over a wide range of dose levels.
• Two hours after a single 50-mg oral dose, serum concentrations in the region of 1.0 mg/L can be anticipated, but after repeated dosing this increases to about 2.0-3.0 mg/ L.
• The absorption is not affected by administration of the drug with food.
• Oral or parenteral administration of fluconazole results in rapid and widespread distribution of the drug.
• Unlike other azole antifungals, the protein binding of fluconazole is low (about 12%)resulting in high levels of circulating unbound drug.
• In most tissues and fluids, levels of the drug usually exceed 50% ofthe simultaneous blood concentration.
• Unlike other azole antifungals, fluconazole is not extensively metabolized in man. More than 90% of a given dose is eliminated in the urine: about 80% as unchanged drug and 10% as inactive metabolites.
• The drug is cleared through glomerular filtration, but significant tubular reabsorption occurs.
• Fluconazole has a serum half-life of about 30 h (range 20-50 h), but this is prolonged in renal failure, necessitating adjustment of the dosage regimen in patients with glomerular filtration rates below 50ml/min.
•  In children the volume of distribution and clearance rates are increased, and the half-life is considerably shorter (15-25 h).
• In infants fluconazole has a prolonged half-life of 55-90 h.
• Fluconazole is removed during haemodialysis and, to a lesser extent, during peritoneal dialysis.
• A3-h haemodialysis session will decrease the blood concentration by about 50%.

Therapeutic use of Fluconzole:

• Fluconazole is available in oral and parenteral forms: as tablets, oral suspension and an intravenous infusion.
• The drug is supplied for parenteral administration at a concentration of 2.0mg/ml in 0.9% sodium chloride solution.
• Dosing recommendations are identical for all dosage forms
• Fluconazole can be used to treat mucosal and cutaneous forms of candidosis.
• It is also effective in various forms of dermatophytosis and pityriasis versicolor.
• It is an effective drug for treatment of deep forms of Candida albicans, C. tropicalis and C. parapsilosis infection in patients who are stable (individuals who do not have an unexplained fever, are improving, and are not hypotensive).
• Opinion is divided as to whether fluconazole should be used in patients with C. glabrata infection, but it should not be used in those with C. krusei infection.
• Fluconazole has proved to be an effective prophylactic treatment against candidosis in haematopoietic stem cell transplant (HSCT)recipients.
•  However, it is ineffective in aspergillosis and mucormycosis.
• Fluconazole is a useful drug in acute cryptococcal meningitis, however it should not be used as first-line treatment in persons with AIDS unless there are particular reasons for withholding amphotericin B.
• However, it is more effective and better tolerated than amphotericin B as maintenance treatment to prevent relapse of cryptococcosis in patients with AIDS.
• Fluconazole is the drug of choice for patients with coccidioidal meningitis. However, it must be continued for life to prevent relapse.

 Mode of administration:

• As absorption following oral administration is good, this is the preferred method of administration.
• If the patient cannot take the drug by mouth, the intravenous solution can be used.
• This should be infused at a maximum rate of 200 mg/h.
• Vaginal candidosis can be treated with a single 150- mg oral dose of fluconazole. Oropharyngeal candidosis should be treated with 200 mg on the first day followed by 100mg/day for 2 weeks.
• Oesophageal candidosis should be treated with 200 mg on the first day followed by 100mg/day for at least 3 weeks; treatment should be continued for at least 2 weeks following resolution of symptoms.
• The recommended dose for adult patients with cryptococcosis or deep forms of candidosis is 6mg/kg per day (400mg/day in a 70-kg patient).
• However, some clinicians have used higher dosages in life-threatening infections.
• The duration of treatment will differ from patient to patient, depending upon the nature and extent of the infection and the underlying illness.
• At least 6-8 weeks is usually required for successful treatment of cryptococcosis in human immunodeficiency virus (HIV)-negative persons.
• The recommended dose for children is 3mg/kg for oropharyngeal and oesophageal candidosis, and 6mgkg for cryptococcosis or deep forms of candidosis.
• Because of its more rapid clearance in children, fluconazole should be administered at 12-h intervals for the treatment of life-threatening infections
• Long term maintenance treatment with fluconazole to prevent relapse in AIDS patients with cryptococcosis should be administered at a dosage of 200mg/day.
• To reduce the risk of invasive candidosis in neutropenic HSCT recipients, prophylactic treatment with fluconazole should be given at a dosage of400 mg/day.
• Patients with renal impairment should be given the normal dose for the first 48h of treatment.
• Thereafter, in persons with a creatinine clearance of 21-40 ml/min, the dosage interval should be doubled to 48h or the dose halved.
• Persons with a clearance of 10-20 ml/min require a 72-h interval between doses.
• Patients receiving regular haemodialysis require the usual dose after each dialysis session.

Side effects of Fluconazole

• The commonest side-effects are gastrointestinal in origin, such as nausea and abdominal discomfort, but these seldom necessitate discontinuation of treatment in patients receiving up to 400 mg/day.
• Transient asymptomatic elevations of serum transaminase levels are quite common in AIDS patients treated with the drug, and treatment should be discontinued in patients who have test findings indicative of progressive or persistent hepatic dysfunction.
• There have been rare cases of serious hepatic reactions during fluconazole treatment, including hepatitis, cholestasis, and fulminant hepatic failure.
• Occasional fatal hepatic reactions have occurred, particularly in patients with serious underlying medical conditions such as AIDS or malignancies.
• Fatal exfoliative skin rashes (Stevens-Johnson syndrome) have been reported in patients with AIDS or cancer, but the causal relationship with fluconazole is unclear because of the concomitant administration of other drugs.
• It is advisable to discontinue fluconazole in a patient with a superficial fungal infection who develops a skin rash.
• Patients with deep-seated fungal infection who develop rashes should be monitored and the drug discontinued if the lesions progress.
• Unlike ketoconazole, fluconazole, when given in recommended doses, does not inhibit human adrenal or testicular steroid metabolism.

Fluconazole: mechanism, spectrum, resistance, pharmacokinetics, uses and side effects

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Mechanism of action of Amphotericin B:

• Amphotericin B binds to ergosterol, the principal sterol in the membrane of susceptible fungal cells, causing impairment of membrane barrier function, loss of cell constituents, metabolic disruption and cell death.
• In addition to its membrane permeabilizing effects, the drug can cause oxidative damage to fungal cells.
• Mammalian cell membranes also contain sterols, and it has been suggested that amphotericin B-induced damage to human and fungal cells shares common mechanisms.

 Spectrum of action:

• Amphotericin B has a broad spectrum of action including many Aspergillus species, Blastomyces dermatitidis, Candida species, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis and Penicillium mameffei.
• Aspergillus terreus, Fusarium species, Malassezia furfur, Scedosporium species and Trichosporon asahii are often resistant.

Acquiring Amphotericin B resistance:

• Treatment failure attributable to the development of amphotericin B resistance is rare.
• Resistant strains of Candida lusitaniae and C. tropicalis, with qualitative and quantitative alterations in membrane sterol composition, including reduced amounts of ergosterol, have been isolated during treatment.
• There are a few reports of resistant strains of Cryptococcus neoformans isolated from persons with the acquired immunodeficiency syndrome (AIDS) with relapsed infection.

  Formulation of Amphotericin B:

i. Conventional formulation of amphotericin B:

• Amphotericin B is used as micellar suspension formulation.
• Administration of the conventional formulation of the drug is associated with harmful side-effects and unpleasant reactions which often limit the amount that can be given.
• Parenteral administration of the conventional micellar suspension formulation of amphotericin B is often linked with treatment-limiting toxic effects, in particular renal impairment.

ii. Lipid based formulation of amphotericin B:

• Three lipid-based formulations of the Amphotericin B are in use;
  • liposomal amphotericin B (AmBisome) in which the drug is encapsulated in phospholipid-containing liposomes
  • Amphotericin B lipid complex (Abelcet, ABLC) in which it is complexed with phospholipids to form ribbon-like structures
  • Amphotericin B colloidal dispersion (Amphocil, Amphotec, ABCD) in which the drug is complexed with cholesterol sulphate to form small lipid discs.
• The lipid-based formulations of amphotericin B are better tolerated than conventional amphotericin B and higher doses can be given over shorter periods with fewer toxic reactions.
• These formulations are licensed for the treatment of serious fungal infections in patients who have failed to respond or have developed severe side-effects to conventional amphotericin B or in whom conventional amphotericin B is contraindicated because of renal impairment.
• In the USA, AmBisome is also licensed for the empirical treatment of presumed fungal infection in febrile neutropenic patients.

Pharmacokinetics of Amphotericin B:

• Amphotericin B is not absorbed following mucosal or cutaneous application. Minimal absorption occurs from the gastrointestinal tract.
• Oral administration of a 3 g dose will produce serum concentrations in the region of 0.1-0.5 mg/L.

Mode and dose of administration of Amphotericin B:

• The dose and duration of topical treatment will differ from patient to patient and depend on the nature and extent of infection.
• The usual adult dose of the oral suspension for oral forms of candidosis is 1-2ml (100-200mg) at 6-h intervals.
• Asthe drug is not absorbed the success of treatment depends on maintaining an adequate concentration in the mouth for as long as possible.
• The recommended dosage of the oral suspension for infants and children is 1ml (100mg) at 6-h intervals.

Therapeutic use of Amphotericin B:

• Amphotericin B is present in various forms such as oral, topical and parenteral forms.
• Topical amphotericin B preparations can be used to treat mucosal and cutaneous forms of candidosis.
• Parenteral amphotericin B is still the drug of choice for many forms of deep fungal infection, including aspergillosis, blastomycosis, candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis and paracoccidioidomycosis.
• It is also effective in certain forms of mucormycosis, hyalohyphomycosis and phaeohyphomycosis.
• However, it is often ineffective in Scedosporium infection and trichosporonosis, as well as in aspergillosis and candidosis in immunocompromised patients.

Side effects of Amphotericin B:

i. Side effects of Conventional formulation of amphotericin B:

• The immediate side-effects of the intravenous infusion of amphotericin Binclude fever, chills and rigors.
• These unpleasant reactions differ from patient to patient, but usually begin 1-3 h after starting the infusion and last for about 1h.
• They are most common during the first week of treatment and often diminish thereafter.
• Nausea and vomiting are less frequent side-effects and, just as with fever and rigors, often diminish as treatment proceeds.
• The most serious toxic effect of amphotericin Bis renal tubular damage.
• Infants and children are less susceptible to the nephrotoxic effects of amphotericin B.
• The risk of amphotericin B-induced renal impairment can be reduced by pre- and post-infusion hydration and sodium repletion with 500 ml saline, provided the clinical status of the patient will allow sodium loading.
• Amphotericin B also causes renal wasting of potassium and magnesium due to renal tubular damage, which can reach symptomatic proportions.
• Patients treated for more than 2 weeks often develop a mild normochromic, normocytic anaemia.
• Pulmonary reactions, with acute dyspnoea, hypoxaemia and interstitial infiltrates, can occur when treatment with amphotericin B is combined with granulocyte transfusion.
• For this reason it is advisable to separate the infusion of the drug from the time of granulocyte transfusion.

ii. Side effects of lipid based formulation of Amphotericin B:

• The prevalence of immediate side-effects after administration of ABLC or ABCD is lower than reported for conventional amphotericin B.
• Fever and chills tend to occur during the first two infusions and are less frequent with subsequent infusions.
• Infusion-related adverse events are more common with ABLC and ABCD treatment than with AmBisome treatment.
• Infusion-related hypoxia has been documented in up to 25% of ABLC and ABCD recipients, but is usually reversible.
• Infusion- related side-effects can be attenuated or prevented by premedication with acetaminophen, antihistamines, corticosteroids and meperidine.
• Patients intolerant of one lipid-based formulation of amphotericin B may tolerate another well.
• Renal impairment is less common with all three lipid-based formulations of amphotericin B than with the conventional preparation.
• Other adverse events associated with lipid-based formulations of amphotericin B have included elevations in liver transaminases, alkaline phosphatase, and serum bilirubin concentrations.

Amphotericin B: mechanism, spectrum, pharmacokinetics, uses and side effects

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Structure of Penicillin:

• The basic structure of penicillin consists of a thiozolidine ring condensed with a B-lactum ring.
• Natural penicillin is 6-amino-penicillinic acid (6APA).

Fermentation biotechnology for penicillin production:

• By fermentation technology penicillin is produced from Penicillium spp. If penicillin fermentation is carried out without addition of side chain precursor, the natural penicillins are produced. But fermentation can be better controlled by adding a side chain precursor to obtain derived penicillin. The synthetic penicillins are produced by enzymatic hydrolysis of 6APA by penicillin acylase enzyme and then addition of desired side chain by chemical means,
• B-lactum thiozolidine ring of penicillin is constructed from l-cystine and l-valine. These two amino acids when combined with L-α-aminoadipic acid (α-AAA) the tripeptide is formed which undergoes two step cyclization process to give isopenicillin.

Regulation of penicillin production:

• The amino acids lysine is synthesized from a pathway that involves L-α-AAA, so that penicillin and lysine share a common but branched biosynthetic pathway. Higher concentration of lysine causes feed back inhibition of homocitrate synthase, an enzyme involved in α-AAA synthesis. Either lysine level should keep low or α-AAA level should added during fermentation.
• Penicillin biosynthesis is affected by Po4—concentration and also shows a distinct catabolic repression by glucose. Therefore, either slowly metabolizable sugars such as lactose is used or fed continuously with glucose with small dose.

Penicillin Production process:

• Penicillin production is previously achieved by surface process ie. Solid state fermentation and surface liquid ferementation. Now a days acommercial production is carried out by fed batch process

• Inoculum (Organism): Penicillium chrysogenum (improved strain)

i. Inoculum preparation:

• For inoculum preparation, spore from heavily sporulated working stocks are suspended inwater or non-toxic wetting agensts (sodium sulfonate 1: 10000)
• Theses spore are then added to flask containing wheat bran and nutrient solution for heavy sporulation
• Incubate for 5-7 days at 24C
• Spore are then transferred to seed tank and incubated for 24-48 hours at 24C with aeration and agitation for sufficient mycelial growth
• These mycelia can be used for production fermenter

ii. Production fermentation:

• Method: fed-batch or batch
• Substrate: glucose, phenoxyacetic acid (fed component used for production of side chain), Corn steep liquor, Additional nitrogen source ie, soyameal, yeast extract, Lactic acid, inorganic ions, growth factors
• Fermenter: stirred tank or air lift tank
• pH: set at 5.5 t0 6.0 which increased upto 7-7.5 (optimum) due to liberation of NH3 gas and consumptionof lactic acid. If pH is 8 or more, CaCO3 or MgCO3 or phosphate buffer is added
• temperature: 25-27 C
• aeration: 0.5-1 vvm (initially more, latter less O2 )
• agitation: 120-150 rpm)
• time: 3-5 days
• antiform: edible oil (0.25%)

iii. Product recovery:

• harvest broth from fermenter tank by filtration (rotary vaccum filtration)
• chill to 5-10 C (because penicillin is highly reactive and destroyed by alkali and enzyme)
• acidify filtrate to pH 2.0-2.5 with H2SO4 ( to convert penicillin to its anionic form)
• extract penicillin from aqueous filtrate into butyl acetate or amyl acetate (at this very low pH as soon as possible in centrifugal counter current extractor)
• discard aqueous fraction
• allow the organic solvent to pass through charcoal to remove impurities and extract penicillin from butylacetate to 2% aqueous phosphate buffer at pH 7.5
• acidify the aq. Fraction to pH 2-2.5 with mineral acid and re-extract penicillin into fresh butylacetate ( it concentrated upto 80-100 times)
• add potassium acetate to the solvent extract in a crystallization tank to crystalize as potassium salt
• recover crystal in filter centrifuge
• sterilization
• further processing
• packaging

Application of penicillin:

• clinical uses of penicillin:
• naturally effective antibiotics against gram + bacteria
• used for treatment of bacterial endocarditis

Penicillin production commercially by fermentation biotechnology

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• Extended spectrum Beta-lactamase are enzymes belongs to molecular class ‘A’ or ‘D’ and functional group 2b e and 2d. These enzymes hydrolyses penicillin, cephalosporin (1^st, 2^nd and 3^rd generaion) including Aztreonam. However they are inactivated by clavulinic acid ad cephamycin (cephoxitine and cephotetan).

Types of ESBL

1. TEM type ESBL (Temoneira)

• This type of ESBL is commonly produced by coli and Klebsiella pneumonia, but also has been noted in Neisseria gonorrhoae and Hemophilus influenza.
• All TEM type ESBLs are derived from TEM-1 and TEM-2
• TEM-1 can hydrolyze ampicillin with greater extent but has negligible activity against extended spectrum cephalosporins. It is inhibited by clavulinic acid
• TEM-2 has same hydrolytic profile as TEM-1 but has different isoelectric point.
• There are more than 140 TEM type ESBl. However most common type of TEM ESBL are TEM-10, TEM-12 and TEM-26
• Amino acid substitution in active site of enzyme is responsible for ESBL, which results in allowing excess amount of Oxy-imino-β-lactams.

2. SHV type ESBL (sulfhydryl variable)

• SHV type ESBL resembles 80% to type TEM
• SHV type is usually produced by Klebsiella pneumonia and other enterobacteriaceae.
• SHV type are produced as a result of amino acid substitution at position 238 and 240.
• More than 80 different types of SHV have been identified. Most common SHV type are SHV-5 and SHV-12

3. CTX-M type ESBL (Cefotaximases)

• CTX-M type ESBL shows more affinity towards cefotaxims than other Oxyimino-β-lactam antibiotics.
• Most common type of CTX-M are CTX-M-2, CTX-M-3, CTX-M-14, CTX-M-15
• They are usually produced by coli and Salmonella enterica serovar Typhimurium

4. OXA type ESBl (Oxacillinases)

• OXA type ESBl are usually produced by Pseudomonas aeroginosa but have been detected in other gram negative bacteria also.
• This OXA type ESBL belongs to class ‘D’ and functional class 2d.
• It is characterized by hydrolysis rate for cloxacillin and oxacillin greater than 50% than for benzylpenicillin.
• They show reduced inhibition to clavulinic acid
• Most OXA type beta-lactamase do not hydrolyse the extended spectrum cephalosporin to significant degree and are not regarded as ESBLs. However OXA-10 hydrolyses cefotaxime, ceftriaxone and aztreonam.
• Most common type OXA type ESBL are is OXA-11, OXA-14, OXA-16, OXA-17, OXA-19, OXA-28, OXA-31, OXA-32, OXA_35, OXA-45

5. PER type ESBL:

• It share only about 25-27% homology with TEM and SHV type ESBL
• PER-1 can effectively hydrolyse penicillins and cephalosporins and is susceptible to clavulinic acid
• PER type ESBl is produced by Pseudomonas aeroginosa, Salmonella enterica serovar Typhimurium , Acinetobacter, Vibrio cholera, Klebsiella pneumonia, Proteus mirabilis

6. Other ESBL types are:

• VEB
• BES-1
• VIM-1
• GES
• TLA
• IBC
• SFO

ESBL (Extended Spectrum beta-Lactamases) and types

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• Beta-lactamase is an enzyme capable of hydrolyzing β-lactam antibiotics. β-lactamase degrade β-lactam antibiotics by opening the β-lactam ring. The microorganisms producing beta-lactamase enzyme become resistant β-lactam antibiotics.

Classification of β-lactamase enzyme on the basis of function:

Group 1 β-lactamase :

• These are class-C enzyme and are cephalosporinase which are not inhibited by clavulanic acid.
• They are also resistant to cefamycin

Group 2 β-lactamase:

• These includes class-A enzyme and are penicillinase and cephalosporinase.
• Inhibited by clavulanic acid.

Sub class:

i. Group 2b e (extended spectrum) β-lactamase:

• These are ESBL capable of hydrolyzing penicillin even 3^rd and 4^th generation cephalosporin but are hydrolyzed by clavulanic acid.

ii. Group 2b r β-lactamase:

• These includes inhibitor resistant β-lactamase. These may be resistant to sulbactum and clavulanic acid but are susceptible to inhibition by tazobactum.

iii. Group 2c β-lactamase:

• These includes carbenicillinase. These enzymes hydrolyses carbenicillin more than Benzylpenicillin. These are class-A enzyme. They also show activity towards Oxacillin.

iv. Group 2d (extended spectrum) β-lactamase:

• These are class-A or D enzyme and includes Cloxacillinase (oxacillinase). These enzymes hydrolyses cloxacillin more than Benzylpenicillin and also has activity against carbenicillin.

v. Group 2e β-lactamase:

• These are class-A enzyme. These includes cephalosporinase. These are inhibited by clavulanic acid.

vi. Group 2f β-lactamase:

• These are class-A enzyme. These includes carbapenimase. These are serine based enzyme which are also inhibited by clavulanic acid.

Group 3 β-lactamase:

• These includes class-B enzyme and are zinc based metalloenzyme. Eg. Carbapenimase
• These are not inhibited by clavulanic acid.
• They are chromosomal mediated enzymes

Group 4 β-lactamase:

• These includes penicillinase
• These are not inhibited by clavulanic acid.

Beta-lactamase: functional classification

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• Aminoglycosides are aminocyclitol derivative antibiotics. Spectinomycin is the pure aminocyclitol which does not contain amino sugars

 Antimicrobial activity of different aminoglycosides:

1. Neomycin and paromomycin:

• Neomycin is limited only for topical application
• Paromomycin is used in the treatment of systemic infection caused by Entamoeba histolytica and Leishmania.

2. Streptomycin:

• Streptomycin have activity against Gram negative, certain gram Positive as well as Staphylococci.
• It is used treatment of Tuberculosis

3. Kanamycin and Amikacin:

• Kanamycina and amikacin are 2^nd line drug for Mycobacterium
• Gentamycin, tobramycin, Netilmicin and Amiacin also offer activity against Pseudomonas. These antibiotics are recommended in the treatment of serious systemic infection and endocarditis caused by Streptococci along with broad spectrum β-lactam antibiotics.

Mode of action of aminoglycosides:

• Aminoglycosides block the initiation step of protein synthesis in bacteria. The antibiotics aminoglycodie binds to the P-site in 30S ribosome preventing binding of IF3
• Streptomycin distorts the shape of A-site and therefore interfere with the correct positioning of the incoming aminoacyl-tRNA and thus leads to the misreading of the polypeptide chain in bacteria.
• Streptomycin also causes dissociation of polysomes into monosomes in bacteria

Mechanism of resistance to aminoglycosides:

• Anaerobes are inherently resistant to aminoglycosides. Aminoglycosides requires an active transport system (oxygen dependent) for the uptake of drugs inside bacterial cell. This type of oxygen dependent transport system is absent in anaerobes.
• Mutation in the gene responsible for deletion or alteration of the receptor protein can also leads to development of resistance towards aminoglycosides.
• Alteration of cell surface that interfere with penetration or uptake of drug in bacteria can also leads to resistance.
• Production of enzyme such as aminoglycoside acetyltransferase, aminoglycoside phosphoryltransferase, aminoglycoside adenyltransferase etc by resistant bacteria that inactivates the aminoglycoside either by phosphorylation or adenylation or acetylation. This can also cause resistance to aminoglycosides.

Pharmacological properties of aminoglycosides:

• Aminoglycosides are not suitable for oral administration because they are very poorly absorbed grom GI tract.
• None of the aminoglycoside can cross blood brain barrier.
• These drugs are nephrotoxic and ototoxic.

Aminoglycosides: antimicrobial activity, mode of action, mechanism of resistance and pharmacological properties

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structure of cephalosporin

• Cephalosporin is a β-lactam antibiotic that inhibits bacterial cell wall synthesis. In 1948 Dr. Abraham first isolated cephalosporin C from a fungus Cephalosporium acremonium. Cephalosporins have broder gram –ve coverage than penicillin yet no one of the cephalosporins is active against MRSA and enterococci. Basic structure of cephalosporin is 7-aminocephalosporanic acid

Classification of Cephalosporin:

On the basis of spectrum of antimicrobial activity cephalosporin are classified into several generations.

i. First generation Cephalosporin:

• 1^st generation cephalosporin have broad spectrum of activity and low toxicity.
• 1^st generation cephalosporin are very active against gram +ve cocci such as Pneumococci, viridans streptococci, β-hemolytic streptococci and staphylococci.
• They are effective against gram –ve rods such as coli, Klebseilla pneumoniae, proteus mirabilis but have very little activity against Pseudomonas aerogenosa, Proteus vulgaris, Enterobacter, Citrobacter, Serratia and Acinetobacter.
• They are also active against anaerobes like Peptococcus and Streptococcus but ineffective against Bacteroids fragillis.
• None of the 1^st generation cephalosporin can cross brain barrier. Therefore they cannot be used for treatment of meningitis.

List of 1^st generation cephalosporin;

• Cephalexin:
• Cephradine
• Cephalothin
• Cefadroxil
• Cefuzolin: drug of choice for surgical prophylaxis
• Cephapirin

ii. Second generation Cephalosporin:

• 2^nd generation cephalosporin have extended gram-ve coverage than that of 1^st generation cephalosporin.
• They are heterogenous groups of antibiotics with different antimicrobial activity.
• They are active against Enterobacter, Proteus vulgaris, Klebsiella, H. influenzae

List of 2^nd generation cephalosporin:

• Cefamandole
• Cefonicid
• Cefmetazole
• Cefaranide
• Cefprozil
• Cefuroxime
• Cefpodoxime
• Cefoxitin
• Cefotetan
• Cefaclor
• Loracarbef

** cefaclor is used in treatment of sinusitis and otitis caused by β-lactamase producing strain of H. influenza, especially in patient allergic too Ampicillin and Amoxicillin

iii. Third generation cephalosporin:

• 3^rd generation cephalosporin have extended spectrum of activities and they have extended gram –ve coverage including Citrobacter, Enterobacter, Provedeneia, Pseudomonas aerogenosa and β-lactamase producing meningococci and H. influenzae.
• This generation cephalosporin are usually used in treatment of sepsis of unknown origin especially in immunocompromised patients in in those individuals where cephalosporin possess least toxicity.

List of 3^rd generation cephalosporin:

• Cefoperazone
• Ceftizoxime
• Ceftazidime
• Cefixime
• Cefotaxime
• Moxalactame
• Ceftriaxome

** All 3^rd generation cephalosporin except cefoperazone and cefixime can cross blood brain barrier and therefore can be used to treat meningitis.

iv. Fourth generation Cephalosporin:

• 4^th generation cephalosporin have extended gram –ve coverage and they have increased activity against Streptococci and MRSA.
• The activity of 4^th generation cephalosporin can be comparable to Ceftazime against Pseudomonas.

List of 4^th generation cephalosporin

• Cefepime
• Cefpirome

v. Fifth generation Cephalosporin:

• They are extended cephalosporin with extended spectrum of activities against MRSA

List of 5h generation cephalosporin;

• Ceftaroline
• Ceftabiprole

Mode of action of Cephalosporin:

i. Binds to penicillin Binding Protein (PBP):

• Many of the cephalosporin eg. Cephalexin, cefotaxime and ceftazidime binds to Penicillin binding protein 3 (PBP3) similar to action of penicillin and results in formation of elongated and filamentous cell. Transpeptidase is the enzyme present in bacteria that cross linked with existing peptidoglycan chain and maintain integrity of cell wall. Binding of cephalosporin on PBP3 receptor deforms cell wall and kills bacteria.

ii. Activation of autolysin:

• Cephalosporin also activates autolytic enzymes in bacteria and causes lysis of bacteria

Cephalosporin: structure, classification, clinical use and mode of action

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classification of penicillin:

• There are four clinically important class of penicillin classified into four groups on the basis of substitution of R group.

Group I: Natural penicillin

• These are active against Gram positive bacteria, very less against gram negative rods but act against gram negative cocci (gonococci and Meningococci)
• Susceptible to hydrolyse by β-lactamases.
• Examples: penicillin G( Benzyl penicillin), penicillin VK

Group II: β-lactamase resistant penicillin

• These penicillin are relatively stable to Staphylococcal β-lactamase enzyme.
• They have little activity against Gram positive bacteria
• Inactive against Gram negative bacteria
• Examples: Methicillin, Nafcillin, Oxacillin, Cloxacillin, Dicloxacillin

Group III: Aminopenicillins, Carboxypenicillins and Ureidopenicillins

• These antibiotics have higher activity against Gram Positive and gram Negative bacteria but are destroyed by β-lactamases
• Examples: Aminopenicillin (Ampicillin, Amoxicillin) Carboxypenicillins (Carcenicillin, Ticarcillin) and Ureidopenicillins (Mezlocillin, Piperacillin).
• Ampicillin and Amoxycillin are used against Enterobactericeae family but not against Klebsiella and Pseudomonas.
• Carboxypenicillins and Ureidopenicillinas are active against Piperacillin is drug of choice for Psudomonas

Group IV: Penicillin for oral administration

• These antibiotics are relatively stable to gastric acid and are suitable for oral administration.
• Examples: Penicillin V (Phenoxy methyl penicillin).

Mode of action of penicillin:

1. Inhibition of cell wall synthesis by blocking transpeptidation:

• Penicillin binds to penicillin binding protein (PBP) receptor on the surface of bacterial cell wall. PBP is the receptor for substrate peptidoglycan precursor in bacteria. Antibiotics penicillin acts as alternative substrate and binds to PBP receptor and then inhibits transpeptidase which results in inhibition of cell wall synthesis.

2. Activation of autolytic enzymes:

• Penicillin causes activation of autolytic enzymes of bacteria which creates lesion sin bacteria causing their death.
• Autolysins are present in bacterial cell wall which maintains appropriate shape and size of cell and also helps in cell division. The activity of autolysin is regulated by components such as cell wall and teichoic acid.
• Use of antibiotics penicillin causes destruction of cell wall and disintegration of teichoic acid as a result of which autolysin is activated and cause cell lysis.

Penicillin resistance:

Mechanism of resistance of bacteria towards penicillin is dependent on following conditions.

1. Ability of bacteria to produce β-lactamases enzyme:

• Bacteria like Staphylococcus, H. influenza, Gonococci and Gram –ve rods are resistant to penicillin because they produces β-lactamases enzyme which can hydrolyse the antibiotic penicillin.

2. Altered PBP receptor:

• Certain bacteria may be resistant to penicillin not because they produce β-lactamases but either due to altered PBP receptor or due to impermeability of their cell membrane to antibiotics.

3. Inability of penicillin to activate autolysin:

• In certain bacteria penicillin cannot activate autolysins to kill bacteria. In such case bacteria are only inactivated but not killed. Eg. Streptococcus, Listeria

4. Absence of cell wall:

• Bacteria which lacks cell wall are spontaneously resistant to penicillin. Eg. Mycoplasma

5. Mutation in gene:

• Certain bacteria such as Staphylococcus aureus are resistant to penicillin (β-lactamase resistant penicillins ie. Methicillin) known as MRSA. This resistant is due to mutation in gene which code altered penicillin binding protein (PBP).

Pharmacokinetics of penicillin:

• About 50% individuals are allergic towards penicillin. Allergy occurs in the form of type I hypersensitivity reaction.

Penicillin: classification, mode of action and penicillin resistance

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1. Penicillin G:

• Penicillin G is the drug of choice in infection caused by Pneumococci, Streptococci, Meningococci, Gonococci and Staphylococci (Non β-lactamase producing Staphylococci).
• They are effective against Spirochaetes, Bacillus anthracis and other gram positive rods, Clostridium spp and other anaerobes but inactive against Bacteroides fragillis (gram –ve rod).
• They are also active against Listeria spp and Actinomycetes.

2. Penicillin V:

• Penicillin V is used for the treatment of mild respiratory tract infection such as Pharyngitis, otitis media, sinusitis etc.
• It is oral drug.

3. Benzathine Penicillin G:

• Benzathine penicillin G is the insoluble salt of penicillin G and it is used for intramuscular injection. This is slow acting drug for prolong use.
• A single dose of 1.2 million units of benzathine penicillin G is suitable for treatment of β-hemolytic Streptococcal pharyngitis.
• 4 million units of benzathine penicillin G injected once in 1-3 weeks in an appropriate proportion measure for primary and latent Syphilis.

4. Ampicillin, Amoxycillin, Carbenicillin, Ticarcillin, azolocillin, Mezlocillin and Piperacillin:

• Ampicillin is active against coli, Salmonella, Shigella and Haemophilus influenzae but not active against indole +ve Proteus (P.vulgaris), Pseudomonas, Klebsiella and Enterobacter.
• Amphicillin and Amoxycillin are commonly used for treatment of common UTI caused by coliforms bacteria and for the treatment of mixed secondary bacterial infection of respiratory tract.
• Ampicillin was a drug of choice in meningitis caused by influenzae but now a days Ceftriazone is used because of emergence of β-lactamase producing strain of H. influenzae.
• Ampicillin and Amoxyccillin are alternative drug in typhoid fever but should not be used in non-invasive Salmonella gastroenteritis.
• Carbenicillin is active against Pseudomonas and Proteus but not against Klebsiella.
• Ticarcillin also active against Pseudomonas and proteus in lower dose than Carbenicillin.
• Azolocillin, Mezlocillin and Piperacillin have greater activities against Pseudomonas than Carbenicillin.

5. Β-lactamase resistant penicillin: Methicillin, Nafcillin, Oxacillin, Cloxacillin, Dicloxacillin

• These penicillins are used to prevent infection caused by β-lactamase producing streptococci.
• These drugs are highly protein bound therefore they consumed 1 hour before and after meals.
• Except Methicillin all β-lactamase resistant penicillin are suitable for oral administration. However methicillin is nephrotoxic and is not suitable for theurapeutic use.

6. Combination of Penicillin with β-lactamase inhibitors:

• β-lactamase inhibitors are also β-lactam group of antibiotics but with very little antimicrobial activity.
• β-lactamase inhibitors are usually used in combination with β-lactam antibiotics (penicillin) to treat infection caused by β-lactamase producing strain of bacteria.
• Commonly used β-lactamase inhibitors are; sulbactum, tazobactum and clavulanic acid.

Combination drugs commonly used are:

• Zosyn: piperacillin + tazobactum
• Unasyn: amphicillin + sulbactum
• Angmentin: amoxicillin + clavulanic acid

Clinical use of penicillin antibiotics

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