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Cephalosporins  Antimicrobials refer to a group of agents that share the common aim of reducing the possibility of infection and sepsis. Antibiotics are often derived from moulds or are made synthetically and are absorbed into the body with the aim of killing bacteria (bactericidal) or pre...

Cephalosporins  Antimicrobials refer to a group of agents that share the common aim of reducing the possibility of infection and sepsis. Antibiotics are often derived from moulds or are made synthetically and are absorbed into the body with the aim of killing bacteria (bactericidal) or preventing their multiplication (bacteriostatic). Antibiotics can be given parenterally (intramuscularly, intravenously), orally, or applied topically to the skin in the form of a cream or ointment. Antiseptics on the other hand are substances that are applied to the skin but not absorbed significantly and which are able to reduce the possibility of infection. Disinfectants can destroy micro-organisms including bacteria on non- living objects such as toilets. Antifungal agents are drugs that share the common property of killing or inhibiting the growth of fungi, including yeasts. Antifungals can be given intravenously, orally or topically. Cephalosporins The cephalosporins were isolated from the fungus Cephalosporium acremonium in 1948 by Pro Tzu, Newton, and Abraham (1953). The main product being cephalosporin-C, the molecular modification of cephalosporin-C gave origin to semisynthetic substances. They are β-lactam antibiotics with same fundamental structural requirements as penicillins, the main difference between the two is that cephalosporins contain di-hydro meta thiazine ring, while penicillin contains a tetra hydro thiazole (thiazolidine) ring. The cephalosporins are much more acid stable than the corresponding penicillins and also have a mechanism of action similar to that of penicillins; they mainly inhibit the cross-linking of the peptidoglycan units in bacterial cell walls by inhibiting transpeptidase enzyme. However, they bind in the target proteins other than penicillins binding proteins. Cephalosporins Cephalosporins can be divided into three classes 1. Cephalosporin N: It has a penicillin-like structure being a derivative of 6-aminopenicillanic acid. 2. Cephalosporin P: An acidic antibiotic, which is steroidal in nature. 3. Cephalosporin-C: It is a true cephalosporin and it is a derivative of 7 amino-cephalosporanic acid. In cephalosporin N In cephalosporin C Cepahlosporin C contains a side-chain derived from D-α-aminoadipic acid, which is attached to 7-aminocephalosporanic acid A compound structurally similar to cephalosporin P is called fusidic acid Nomenclatures Cephalosporins are named in the following ways: 1. Chemical abstracts: 5-Thia-1-azobicyclo (4.2.0) octanes. Accordingly, cephalothin is 3-(Acetoxy methyl)-8-oxo-7-(2-thienyl) acetamido-5thia-1-aza-bicyclo[4.2.0]-oct-2ene-2-carboxylic acid. 2. Cepham derivatives: Cepham is the name given to the unsubstituted bicyclic lactam. Classification of cephalosporin Cephalosporins are classified on the basis of their chemical structure, clinical pharmacology, antibacterial spectrum, or penicillinase resistance. a. Orally administered: cephalexin, cephradine, and cefaclor b. Parentrally administered: cephalothin, cephapirin, cephacetrile, and cefazedone. These agents are sensitivity to β-lactamase c. Resistant to β-lactamase and parentrally administered: cefuroxime, cefamandole, cefoxitin d. Metabolically unstable: cephalothin and cephapirin First-generation cephalosporins These drugs have the highest activity against gram-positive bacteria and the lowest activity against gramnegative bacteria (Table 4.1) Second- generation cephalosporins These drugs are more active against gram-negative bacteria and less active against gram-positive bacteria than first-generation members. Third- generation cephalosporins These drugs are less active than first-generation drugs against gram- positive organisms, but have a much expanded spectrum of activity against gram-negative organisms. Fourth- generation cephalosporins Cefepime and cefpirome are new fourth- generation parenteral cephalosporins with a spectrum of activity which makes them suitable for the treatment of infections caused by a wide variety of bacteria. Fifth and Sixth Generations Ceftobiprole has been described as "fifth-generation" cephalosporin, though acceptance for this terminology is not universal. Ceftobiprole has anti- pseudomonal activity and appears to be less susceptible to development of resistance. Ceftaroline has also been described as "fifth-generation" cephalosporin, but does not have the activity against Pseudomonas aeruginosa or vancomycin-resistant enterococci that ceftobiprole has. Ceftolozane is an option for the treatment of complicated intra-abdominal infections and complicated urinary tract infections. It is combined with the β- lactamase inhibitor tazobactam, as multi-drug resistant bacterial infections will generally show resistance to all β-lactam antibiotics unless this enzyme is inhibited.  Cephalexin (Keflex, Keforal) Properties and uses: Cephalexin monohydrate is a white crystalline powder, sparingly soluble in water, and practically insoluble in alcohol. The α-amino group of cephalexin renders it acid stable. The 3-methyl group is responsible for the metabolic stability. It is particularly recommended for urinary tract infection. Dose: The oral dose for adults is 250–500 mg every 6 h, for children, the dose is 18–25mg/kg every 6 h. Assay: It is assayed by adopting liquid chromatography technique. Dosage forms: Cefalexin capsules I.P., B.P., Cefalexin oral suspension I.P., B.P., Cefalexin tablets I.P., B.P. Cefadroxil (Cefadrox, Droxyl, Codroxil) Properties and uses: Cefadroxil monohydrate is a white or almost white powder, slightly soluble in water, and sparingly soluble in ethanol. The antibacterial spectrum of action and therapeutic indications of cefadroxil are very similar to those of cephalexin and cephradine. The D-p- hydroxyphenylglycyl isomer is much more active than the L-isomer. Assay: It is assayed by adopting liquid chromatography technique. Dosage forms: Cefadroxil capsules I.P., B.P., Cefadroxil oral suspension I.P., B.P. Cefadroxil tablets I.P. Cefaclor Properties and uses: Cefaclor is a white or slightly yellow powder, slightly soluble in water, practically insoluble in methanol and methylene chloride. It has chloro group at C-3 position, and hence, stable in acid and achieves sufficient oral absorption. Used in the treatment of upper respiratory tract infections caused by Streptococcus pneumoniae and Haemophilus influenzae. Dose: The dose orally for adults is 250–500 mg every 8 h. Assay: It is assayed by adopting liquid chromatography technique. Dosage forms: Cefaclor capsules B.P., Cefaclor oral suspension B.P., Prolonged- release Cefaclor tablets B.P. Cefamandole (Mandol) Cefamandole nafate is a white powder, soluble in water, and sparingly soluble in methanol. It is the first compound of second-generation cephalosporin marketed in the United States. Cefamandole nafate is very unstable in solution and hydrolyzes rapidly to release cefamandole and formate. There is no loss of potency; however, these solutions are stored for 24 h at room temperature or up to 96 h by refrigeration. Dose: IV dose is 0.5–2 g every 4 to 6 h, also available as injection in strengh of 0.5 and 1 mg/10ml. Cefotaxime Sodium Properties and uses: Cefotaxime sodium exists as white solid and soluble in water, exhibits broad-spectrum activity against both gram- positive and gram-negative bacteria. Used in genitourinary infection and lower respiratory infection. Ceftriazone disodium Properties and uses: It exists as white crystals, soluble in water, exhibits broad- spectrum activity against both gram- positive and gram-negative bacteria. Cefpirome Properties and uses: Cefpirome is used to treat susceptible infections, including urinary and respiratory tract infections, skin infections, septicaemia, and infections in immuno-compromised patients. Dose: Intravenous dose for adults as sulphate is 1–2 g every 12 h over 3–5 min or infuse over 20–30 min. Ceftaroline fosamil Ceftaroline fosamil is a relatively recent approved cephalosporin from the fifth- generation Ceftaroline is an oxyimino compound based on the structure of cefozopran (a fourth-generation representative). Ceftaroline fosamil is a prodrug that turns in vivo into ceftaroline (the active metabolite). The name „fosamil” is given by an extra phosphono group in the chemical structure comparative to ceftaroline. The resulted compound has the advantage that it is more water-soluble. The oxime group in the C7 acyl moiety and a 1,3-thiazole ring attached to the central nucleus (C3 position) facilitate the increased activity against MRSA. The parenteral formulation (600 mg powder for concentrate for solution for infusion) contains an equivalent amount of ceftoraline fosamil acetic acid solvate monohydrate. SAR of Cephalosporins 1. 7-Acylamino substitution a. The addition of amino group and a hydrogen to α and α1 position produces basic compound, which is protonated under acidic conditions of stomach. The ammonium ion improves the stability of β-lactum of cephalosporins and make active orally. Activity against positive bacteria is increased and gram negative is decreased by acylation of amino group. b. When the new acyl groups are derived from carboxylic acids, it shows good spectrum of antibacterial action for gram-positive bacteria. c. Substitutions on the aromatic ring phenyl that increase lipophilicity provide higher gram-positive activity and generally lower gram-negative activity. SAR of Cephalosporins 1. 7-Acylamino substitution d. The phenyl ring in the side chain can be replaced with other heterocycles with improved spectrum of activity and pharmacokinetic properties; these include thiophene, tetrazole, furan, pyridine, and aminothiazoles. e. The L-isomer of an α-amino α1 -hydrogen derivative of cephalosphorins was 30–40 fold stable than D-isomer. Addition of methoxy oxime to α and α1increases the stability to nearly 100-fold. The presence of catechol grouping can also enhance activity, particularly, against Pseudomonas aeruginosa, and also retain some gram-positive activity, which is unused for a catechol cephalosporin. SAR of Cephalosporins 2. Modification in the C-3 substitution: The pharmacokinetic and pharmacodynamics depends on C-3 substituents. Modification at C-3 position has been made to reduce the degradation (lactone of des acetyl cephalosporin) of cephalosporins. a. The benzoyl ester displays improved gram-positive activity, but lowered gram- negative activity. b. Pyridine, imidaozle replaced acetoxy group by azide ion yields derivative with relatively low gram negative activity. c. Displacement with aromatic thiols of 3-acetoxy group results in an enhancement of activity against gram-negative bacteria with improved pharmacokinetic properties. d. Orally active compounds are produced by replacement of acetoxy group at C-3 position with CH3 and Cl. SAR of Cephalosporins 3. Other modifications a. Methoxy group at C-7, shows higher resistance to hydrolysis by β- lactamase. b. Oxidation of ring spectrum to sulphoxide or sulphone greatly diminishes or destroys the antibacterial activity. c. Replacement of sulphur with oxygen leads to oxacepam (latamoxet) with increased antibacterial activity, because of its enhanced acylating power. Similarly, replacement of sulphur with methylene group (loracavet) has greater chemical stability and a longer half-life. d. The carboxyl group position-4 has been converted into ester prodrugs to increase bioavailability of cephalosporins, and these can be given orally as well. e. The antibacterial activity depends on the olefinic linkage at C-3 and C-4 position and their activity is lost due to the ionization of double bond to 2nd and 3rd positions. Degradation of Cephalosporins In strong acid solutions In the presence of β-lactamase Degradation of Cephalosporins In the presence of acylase ANTI-TB ANTITB Tuberculosis is the most prevalent infectious disease worldwide and a leading killer caused by a single infectious agent, that is, Mycobacterium tuberculosis. According to World Health Organization (WHO) report, M. tuberculosis, currently infects over 2 billion people worldwide, with 30 million new cases reported every year. This intracellular infection accounts for at least 3 million deaths annually. Common infection sites of the tuberculosis are lungs (primary site), brain, bone, liver, and kidney. The main symptoms are cough, tachycardia, cyanosis, and respiratory failure. Depending upon the site of infection, the disease can be categorized as follows: ·Pulmonary tuberculosis (respiratory tract). ·Genitourinary tuberculosis (genitourinary tract). ANTITB ·Tuberculous meningitis (nervous system). ·Miliary tuberculosis (a widespread infection). Drugs used in the treatment of tuberculosis can be divided into two major categories (Fig. 4.1): 1. First-line drugs: Isoniazid, streptomycin, rifampicin, ethambutol, and pyrazinamide. 2. Second-line drugs: Ethionamide, p-amino salicylic acid, ofloxacin, ciprofloxacin, cycloserine, amikacin, kanamycin, viomycin, and capreomycin. ANTITB Ethambutol, Isonicotinic acid Hydrazid, Rifampacin, Thioguanine, Pyrazinamide, cycloserine, Ethunamide, Cytarabine, 5-Flourouracil and Dacarbazine.  Isoniazid Isoniazid is a prodrug that is activated on the surface of M. tuberculosis by katG enzyme to isonicotinic acid. Isonicotinic acid inhibits the bacterial cell wall mycolic acid, thereby making M. tuberculosis susceptible to reactive oxygen radicals. Isoniazid may be bacteriostatic or bactericidal in action, depending on the concentration of the drug attained at the site of infection and the susceptibility of the infecting organism. The drug is active against susceptible bacteria only during bacterial cell division. Pyrazinamide Metabolism: The metabolic route constitutes of hydrolysis by hepatic microsomal pyrazinamidase into pyrazinoic acid, which may be then, oxidized by xanthine oxidase to 5-hydroxy pyrazinoic acid. The later compound may appear free either in the urine or as a conjugate with glycine. Pyrazinamide is a white crystalline powder, sparingly soluble in water, slightly soluble in alcohol and in methylene chloride. It is a prodrug and is activated by M. tuberculosis amidase enzyme into pyrazine carboxylic acid, which has bactericidal activity. Pyrazinamide has recently been elevated to first-line status in the short-term treatment of tuberculosis regimens because of its tuberculocidal activity and comparatively less short-term toxicity. Pyrazinamide is maximally effective in the low pH environment that exists in macrophages (monocytes). It is used to treat tuberculosis and meningitis. The drug should be used with great caution in patients with hyperuricaemia or gout. Pyrazinamide Pyrazinamide is a white crystalline powder, sparingly soluble in water, slightly soluble in alcohol and in methylene chloride. It is a prodrug and is activated by M. tuberculosis amidase enzyme into pyrazine carboxylic acid, which has bactericidal activity. Pyrazinamide has recently been elevated to first-line status in the short-term treatment of tuberculosis regimens because of its tuberculocidal activity and comparatively less short-term toxicity. Pyrazinamide is maximally effective in the low pH environment that exists in macrophages (monocytes). It is used to treat tuberculosis and meningitis. The drug should be used with great caution in patients with hyperuricaemia or gout. Ethambutol HCl Mode of action: It is a bacteriostatic drug that inhibits the incorporation of mycolic acid into the mycobacterium cell wall. Properties and uses: Ethambutol hydrochloride is a white crystalline powder, soluble in water and in alcohol. It is not recommended for use as a single drug, but used in combinations with other antitubercular drugs in the chemotherapy of pulmonary tuberculosis. Dose: The administered dose is 15–25 mg/kg once a day; low doses for new cases, and high doses for use in patients who have had previous antitubercular therapy. Rifampicin It is an antibiotic obtained from Streptomyces mediterranei. Rifampicin inhibits DNA-dependent RNA polymerase of mycobacteria by forming a stable drug enzyme complex, leading to suppression of initiation of chain formation in RNA synthesis and acts as a bactericidal drug. The major metabolism of rifampicin and rifapentine is deacetylation, which occurs at the C-25 acetate. The resulting products, desacetyl rifampin, and desacetyl rifampentine are still active antibacterial agents. 3-Formylrifamycin has been reported as a second metabolite following both rifampicin and rifampentine administration. Rifampicin Rifampicin is a reddish-brown or brownish-red crystalline powder, slightly soluble in water, acetone, alcohol, and soluble in methanol. Rifampicin is the most active agent in clinical use for the treatment of tuberculosis. It is used only in combination with other antitubercular drugs, and it is ordinarily not recommended for the treatment of other bacterial infections when alternative antibacterial agents are available.  Rifabutin It is a semisynthetic rifamycin, structurally similar to rifampicin. It is used against M. avium, one of the most common causes of disseminated infections, with patients suffering with Human Immunodeficiency Virus (HIV). In vitro activity is attributed to rifabutin’s lipophilic nature and its ability to penetrate the cell wall of the organism more effectively than other agents. Rifabutin is a reddish-violet amorphous powder, slightly soluble in water and alcohol, soluble in methanol. It is used as an antitubercular drug. Streptomycin sulphate The enzymes responsible for inactivation are adenyltransferase, which catalyzes adenylation of the C-3 hydroxyl group in the N-methyl glucosamine moiety to give the O-3-adenylated metabolite and phosphotransferase, which phosphorylates the same C-3 hydroxyl to give O-3 phosphorylated metabolite. Streptomycin is a white hygroscopic powder, very soluble in water, and practically insoluble in ethanol. It was the first effective drug for the treatment of tuberculosis. It is most often used in combination with other drugs, such as ethambutol and isoniazid, to treat pulmonary infections in patients with organisms that are known to be resistant. There has been an increasing tendency to reserve streptomycin products for the treatment of tuberculosis. Ethionamide (Tridocin) Mode of action: The antimycobacterial action of ethionamide seems to be due to an inhibitory effect on the mycolic acid synthesis. Metabolism: Less than 1% of the drug is excreted in the free form, and remainder of the drug appear as six metabolites. Among the metabolites, ethionamide sulphoxide, 2-ethyl-isonicotinamide, and the N-methylated-6-oxo-dihydropyridines are the few. Properties and uses: Ethionamide is a yellow crystalline powder or crystals, practically insoluble in water, soluble in methanol, and sparingly soluble in alcohol. It is used as antitubercular drug.  Para-amino-salicylic acid Aminosalicylic acid is an inhibitor of bacterial folate Route I. From: Anthranilic acid metabolism in a manner similar to the sulphonamide antibacterials. Aminosalicylic acid is Route II. From: m-Nitrophenol bacteriostatic and highly specific for M. tuberculosis. Side effects are anorexia, nausea, epigastric pain, diarrhoea, and making poor compliance.  Amikacin Amikacin is a white powder, soluble in water, practically insoluble in acetone and in ethanol. It is a semisynthetic aminoglycoside that was first prepared in Japan. It is extremely active against several mycobacterial species, and may become the drug of choice for treatment of diseases caused by nontuberculous mycobacteria. Antibacterial Sulphonamides Antibacterial Sulphonamides Para amino benzene sulphonamide (sulphanilamide). Firrst effective chemotherapeutic agents to be employed systemically for the prevention and treatment of bacterial infections in humans. Bacteriostatic antibiotics with a wide spectrum action against most gram-positive bacteria and many gram-negative organisms. Metabolic product of Prontosil, which is responsible for antibacterial activity, and this has given the initiation to develop sulphonamides as antibacterial agents. Antibacterial Sulphonamides Sulphonamides are total synthetic substances that are produced by relatively simple chemical synthesis. The advent of penicillin and, subsequently of other antibiotics has diminished the usefulness of sulphonamides. Antimicrobial compounds contain sulphonamide (SO2NH2) group. This group (SO2NH2) is also present in other compounds, such as antidiabetic agents (e.g. Tolubutamide), diuretics (e.g. chlorthiazide and its congeners, furosemide, and acetazolamide), and anticonvulsants such as sulthiame. The sulphonamides exists as white powder, mildly acidic in character, and they form water- soluble salts with bases. The pH of sodium salts with some exception, for example, sodium sulphacetamide, is very high when given intramuscular (IM), the marked alkalinity causes damage to the tissues. Antibacterial Sulphonamides Microorganisms that may be susceptible in vitro to sulphonamides include Streptococcus pyogens, Streptococcus pneumoniae, Haemophilus influenzae, H. ducreyi, Nocardia, Actinomyces, Calymmatobacterium granulomatis, and Chlamydia trachomatis. The minimal inhibitory concentration ranges from 0.1 μg/ml for C. trachomatis to 4–64 μg/ml for E. coli. Sulphonamides are selective drugs used to treat urinary tract infections, bacterial respiratory infections, and gastrointestinal (GI) infections. Antibacterial Sulphonamides Sulphonamides are structure analogues and competitive antagonists of para- amino benzoic acid (PABA). They inhibit dihydropteroate synthetase, the bacterial enzyme responsible for the incorporation of PABA into dihydropteric acid, and it is the intermediate precursor of folic acid. Synergistic effect is obtained by a combination of trimethoprim. The compound trimethoprim is a potent and selective inhibitor of microbial dihydrofolate reductase, the enzyme that reduces dihydrofolate to tetrahydrofolate. The simultaneous administration of sulphonamide and trimethoprim blocks the pathway of cell-wall synthesis sequentially. SAR of Sulphonamides The major features of SAR of sulphonamides include the following: ·Sulphanilamide skeleton is the minimum structural requirement for antibacterial activity. ·The aminoand sulphonyl-groups on the benzene ring are essential and should be in 1 and 4 position. ·The N-4 amino group could be modified to be prodrugs, which are converted to free amino function in vivo. ·Sulphur atom should be directly linked to the benzene ring. ·Replacement of benzene ring by other ring systems or the introduction of additional substituents on it decreases or abolishes its activity. SAR of Sulphonamides · Exchange of the –SO2NH group by –CONH reduces the activity. ·On N-1-substituted sulphonamides, activity varies with the nature of the substituent at the amino group. With substituents imparting electron-rich characters to SO2 group, bacteriostatic activity increases. ·Heterocyclic substituents lead to highly potent derivatives, while sulphonamides, which contain a single benzene ring at N-1 position, are considerably more toxic than heterocyclic ring analogues. ·The free aromatic amino groups should reside para to the sulphonamide group. Its replacement at ortho or meta position results in compounds devoid of antibacterial activity. ·The active form of sulphonamide is the ionized, maximum activity that is observed between the pKa values 6.6–7.4. SAR of Sulphonamides ·Substitutions in the benzene ring of sulphonamides produced inactive compounds. ·Substitution of free sulphonic acid (–SO3H) group for sulphonamido function destroys the activity, but replacement by a sulphinic acid group (–SO2H) and acetylation of N-4 position retains back the activity. · Sulphonamides bind to the basic centres of proteins. · The binding groups are alkyl, alkoxy, and halides. · The binding affects the activity of sulphonamides; protein binding appears to modulate the availability of the drug and its half-life. ·The lipid solubility influences the pharmacokinetic and antibacterial activity, and so increases the half-life and antibacterial activity in vitro. On the basis of the site of action Sulphonamides for general infection: Sulphanilamide, Sulphapyridine, Sulphadiazine, Sulphamethoxacine, Sulphamethoxazole. ·Sulphonamides for urinary tract infections: Sulphaisoxazole, Sulphathiazole. ·Sulphonamides for intestinal infections: Phthalylsulphathiazole, Succinyl sulphathiazole, Sulphasalazine. ·Sulphonamides for local infections: Sulpahacetamide, Mafenamide, Silver sulphadiazine. ·Sulphonamides for dermatitis: Dapsone, Solapsone. ·Sulphonamides in combination: Trimethoprim with Sulphamethoxazole. On the basis of the pharmacokinetic properties ·Poorly absorbed sulphonamides (locally acting sulphonamides)— Sulphasalazine, Phthalylsulphathiazole, Sulphaguanidine, Salicylazo sulphapyridine, Succinyl sulpha thiazole. ·Rapidly absorbed and rapidly excreted (systemic sulphanamides): Sulphamethoxazole, Sulphaisoxazole, Sulphadiazine, Sulphadimidine, Sulphafurazole, Sulphasomidine, Sulphamethiazole, Sulphacetamide Sulphachlorpyridazine. ·Topically used sulphonamides: Sulphacetamide, Mafenide, Sulphathiazole, Silver sulphadiazine. On the basis of the duration of action ·Extra long-acting sulphonamides (half-life greater than 50 h): Sulphasalazine, Sulphaclomide, Sulphalene. ·Long-acting sulphonamides (half-life greater than 24 h):Sulphadoxine, Sulphadimethoxine, Sulphamethoxy pyridazine, Sulphamethoxydiazine, Sulphaphenazole, Sulphamethoxine. ·Intermediate-acting sulphonamides (half-life between 10–24 h): Sulphasomizole, Sulphamethoxazole. ·Short-acting sulphonamides (half-life less than 20 h): Sulphamethiazole, sulphaisoxazole. ·Injectables (soluble sulpha drugs): Sulphafurazole, Sulphadiazine, Sulphamethoxine. On the basis of the chemical structure ·N-substituted sulphonamide:Sulphadiazine, Sulphacetamide, Sulphadimidine. ·N-4 substituted sulphonamides (prodrugs): Prontosil. ·Both N-1 and N-4 substituted sulphonamides: Succinyl sulphathiazole, Phthalylsulphathiazole. ·Miscellaneous: Mefenide sodium. I. N-1 Substituted sulphonamides a. Short-acting sulpha drugs b. Intermediate-acting sulphonamides c. Long-acting sulphonamides d. Extra long-acting sulphonamides II. N-4 substituted suphonamides III. Both N-1 and N-4 substituted suphonamides N-1 Substituted sulphonamides ii. Sulphacetamide (Albucid) Properties and uses: It exists as white crystalline powder, bitter in taste. Used in the treatment of bacterial infections of urinary tract. Assay: Dissolve the sample in water and hydrochloric acid. Titrate with sodium nitrite and determine the end point potentiometrically. Dose: Dose for eyes, as drops 10%, 15%, 20%, and 30%; in ointments 2.5% and 6% of Sulphacetamide. iii. Sulphasalazine Properties and uses: Sulphasalazine is a bright yellow or brownish- yellow fine powder, very slightly soluble in alcohol, practically insoluble in methylene chloride. It dissolves in dilute solutions of alkali hydroxides. It is used in the treatment of ulcerative colitis. Assay: Dissolve and dilute the sample in 0.1 M sodium hydroxide and add 0.1 M acetic acid and measure the absorbance at the maxima of 359 nm using ultraviolet spectrophotometer. Dosage forms: Sulphasalazine tablets B.P. iv. Sulphadiazine Properties and uses: Sulphadiazine is a white or yellowish-white or pinkish-white crystalline powder or crystals, insoluble in water, slightly soluble in acetone, very slightly soluble in alcohol, and soluble in solutions of alkali hydroxides and in dilute mineral acids. It is used in the treatment of canceroids and rheumatic fever. Assay: Dissolve the sample in water and hydrochloric acid. Titrate the mixture with sodium nitrite and determine the end point potentiometrically. Dose: Usual dose is 2–8 g per day Dosage forms: Sulphadiazine tablets I.P., Sulphadiazine injection B.P. v. Sulphadimidine Properties and uses: It exists as white crystalline powder with a bitter taste, insoluble in water, and sparingly soluble in alcohol. It is less effective in meningeal infection because of its poor penetration into the cerebrospinal fluid. Dose: Dose is 3 g initially and subsequent doses up to 6 g per day in divided doses. vi. Sulphamerazine (Solumedine) Uses: Used as an antibacterial agent. Dose: Dose is 4 g initially, and subsequent dose is 1 g every 6 h xi. Sulphamethoxazole Properties and uses: Sulphamethoxazole is a white or almost white crystalline powder, practically insoluble in water, soluble in acetone, sparingly soluble in ethanol, dissolves in dilute solutions of sodium hydroxide and in dilute acids. Used in the treatment of bacterial infections. Assay: Dissolve the sample in dilute hydrochloric acid and add potassium bromide. Cool in ice and titrate against 0.1N Sodium nitrate. Determine the end point electrometrically. Dose: Orally 2 g followed by 1 g every 8 h. Dosage forms: Co-trimoxazole intravenous infusion B.P., Co-trimoxazole oral suspension B.P., Paediatric co-trimoxazole oral suspension B.P., Co-trimoxazole tablets B.P., Dispersible co-trimoxazole tablets B.P., Paediatric co-trimoxazole tablets B.P. i. Succinyl sulphathiazole Uses: Used in bacillary dysentery and cholera. Dose: Dose is 10–20 g per day in divided doses. ii. Phthalyl Sulphathiazole Properties and uses: Slightly soluble in alcohol and ether, but insoluble in water. It is used in the treatment of acute bacillary dysentery, bowel irregularities, and ulcerative colitis. Dose: Dose is 5–10 g per day in divided doses. i. Mafenide (Sulfamylon) Uses: It is used in the treatment and cure of gas gangrene. It is also effective against Clostridium welchii on topical application. Dose: Dose is 5% solution of mafenide hydrochloride or mafenide propionate for topical use. ii. Dapsone (Avcosulfon) Action and use: Dapsone is a white or slightly yellowish-white crystalline powder, very slightly soluble in water, soluble in acetone and dilute mineral acids, sparingly soluble in alcohol. Used as folic acid synthesis inhibitor in the treatment of leprosy and nocardiosis. Assay: Dissolve the sample in dilute hydrochloric acid, add potassium bromide, cool in ice, and titrate against 0.1N sodium nitrate. Determine the end point electrometrically. Dose: The dose as a leprostatic is 25 mg twice a week initially for 1 month followed by 25 mg per day each month. As suppressant for dermatitis herpetiformis the dose is 100–200 mg per day. Dosage forms: Dapsone tablets B.P. i. Trimethoprim Trimethoprim is a white or yellowish-white powder, very slightly soluble in water and slightly soluble in ethanol. It is used as dihydrofolate reductase inhibitor, effective against chloroquine and pyrimethamine resistant strains of Plasmodium falsiparum. Dissolve the sample in anhydrous acetic acid and titrate with 0.1 M perchloric acid. Determine the end-point potentiometrically. Dosage forms: Co-trimoxazole intravenous infusion B.P., Co-trimoxazole oral suspension B.P., Paediatric co-trimoxazole oral suspension B.P., Co- trimoxazole tablets B.P., Dispersible co-trimoxazole tablets B.P., Paediatric co-trimoxazole B.P., Tablets trimethoprim oral suspension B.P., Trimethoprim tablets B.P. ii. Pyrimethamine Properties and uses: Pyrimethamine is a white crystalline powder or colourless crystals, practically insoluble in water, and slightly soluble in alcohol. It is used in combination with sulphadoxine for the treatment of malaria. Assay: Dissolve the sample in anhydrous acetic acid by heating gently. Cool and titrate with 0.1 M perchloric acid. Determine the end point potentiometrically. Dosage forms: Pyrimethamine tablets I.P., pyrime thamine tablets B.P. IMMUNOSUPPRESSANT DRUGS  Immune system Is designed to protect the host from harmful foreign molecules. This system can result into serious problem. Allograft introduction can elicit a damaging immune response. Immune system include two main arms 1) Cell –mediated immunity. 2) Humoral (antibody –mediated immunity). Cell-mediated Immunity Involves ingestion& digestion of antigen by antigen-presenting cells. Activated TH cells secretes IL-2 IL-2 produced stimulates TH1 & TH2. TH1 produce TNF-β and IFN-γ which. Activate – NK cells (kill tumor & virus-infected cells). – Cytotoxic T cells (kill tumor & virus- infected cells). – Macrophages (kill bacteria). Cell-mediated Immunity Humoral Immunity B-lymphocytes bind to antigen and are induced by interleukins (IL-4 & IL-5) produced by TH2 which in turn causes B-cells proliferation & differentiation into: – memory cells – Antibody secreting plasma cells Humoral Immunity Mutual regulation of T helper lymphocytes TH1 interferon-γ: inhibits TH2 cell proliferation TH2 cells TH2 IL-10: inhibits TH1 cytokine production  Cytokines Cytokines are soluble, antigen-nonspecific signaling proteins that bind to cell surface receptors on a variety of cells. Cytokines include – Interleukins – Interferons (IFNs), – Tumor Necrosis Factors (TNFs), – Transforming Growth Factors (TGFs) – Colony-stimulating factors (CSFs).  IMMUNOSUPPRESSANT DRUGS I. inhibitors of cytokine (IL-2) production or action (Immunophilin ligands): 1) Calcineurin inhibitors Cyclosporine Tacrolimus (FK506) 2) Sirolimus (rapamycin). II. Inhibitors of cytokine gene expression – Corticosteroids III. Cytotoxic drugs Inhibitors of purine or pyrimidine synthesis (Antimetabolites): – Myclophenolate Mofetil – Leflunomide – Azathioprine – Methotrexate Alkylating agents Cyclophosphamide IV. Immunosuppressive antibodies that block T cell surface molecules – antilymphocyte globulins (ALG). – antithymocyte globulins (ATG). – Rho (D) immunoglobulin. – Muromonab-CD3 – Basiliximab – Daclizumab V. Interferon VI. Thalidomide I) Immunophilin ligands: – Inhibitors of cytokines (IL-2) production Calcineurin inhibitors Cyclosporine Tacrolimus (FK506) – Inhibitors of cytokines (IL-2) action Sirolimus (rapamycin).  CYCLOSPORINE Chemistry Cyclosporine is a fungal polypeptide composed of 11 amino acids. Mechanism of action: – Acts by blocking activation of T cells by inhibiting interleukin-2 production (IL-2). – Decreases proliferation and differentiation of T cells. – Cyclosporine binds to cyclophilin (immunophilin) intracellular protein receptors. – Cyclosporine- immunophilin complex inhibits calcineurin, a phosphatase necessary for dephosphorylation of transcription factor (NFATc) required for interleukins synthesis (IL-2). – NFATc (Nuclear Fcator of Activated Tcells). – Suppresses cell-mediated immunity. Pharmacokinetics: – Can be given orally or i.v. infusion – orally (25 or 100 mg) soft gelatin capsules, microemulsion. – Orally, it is slowly and incompletely absorbed. – Peak levels is reached after 1– 4 hours, elimination half life 24 h. – Oral absorption is delayed by fatty meal (gelatin capsule formulation) – Microemulsion ( has higher bioavailability-is not affected by food). – 50 – 60% of cyclosporine accumulates in blood (erythrocytes – lymphocytes). – metabolized by CYT-P450 system (CYP3A4). – excreted mainly through bile into feces, about 6% is excreted in urine. Therapeutic Uses: – Organ transplantation (kidney, liver, heart) either alone or with other immunosuppressive agents (Corticosteroids). – Autoimmune disorders (low dose 7.5 mg/kg/d). e.g. endogenous uveitis, rheumatoid arthritis, active Crohn’s disease, psoriasis, psoriasis, nephrotic syndrome, severe corticosteroid-dependent asthma. – Graft-versus-host disease after stem cell transplants Adverse Effects (Dose-dependent) Therapeutic monitoring is essential – Nephrotoxicity (increased by NSAIDs and aminoglycosides). – Hypertension, hyperkalemia. (K-sparing diuretics should not be used). – Liver dysfunction. – Hyperglycemia. – Viral infections (Herpes - cytomegalovirus). – Lymphoma (Predispose recipients to cancer) – Hirsutism – Neurotoxicity (tremor). – Gum hyperplasia. – Anaphylaxis after I.V. Drug Interactions Clearance of cyclosporine is enhanced by co- administration of Cyt P450 inducers (Phenobarbitone, Phenytoin & Rifampin ) rejection of transplant. Clearance of cyclosporine is decreased when it is co-administered with inhibitors erythromycin, ketoconazole, grapefruit juice cyclosporine toxicity. TACROLIMUS (FK506) a macrolide antibiotic produced by bacteria Streptomyces tsukubaensis. Chemically not related to cyclosporine both drugs have similar mechanism of action. The internal receptor for tacrolimus is immunophilin ( FK-binding protein, FK-BP). Tacrolimus-FKBP complex inhibits calcineurin. Kinetics Given orally or i.v. Oral absorption is variable and incomplete, reduced by fat. Half-life after I.V. form is 9-12 hours. Highly bound with serum proteins and concentrated in erythrocytes. metabolized by P450 in liver. Excreted mainly in bile and minimally in urine. USES as cyclosporine Organ and stem cell transplantation Prevention of rejection of liver and kidney transplants. Atopic dermatitis and psoriasis (topically). Toxic effects Nephrotoxicity (more than CsA) Neurotoxicity (more than CsA) Hyperglycemia ( require insulin). GIT disturbances Hperkalemia Hypertension Anaphylaxis NO hirsutism or gum hyperplasia Drug interactions as cyclosporine. What are the differences between CsA and TAC ? TAC is more favorable than CsA due to: TAC is 10 – 100 times more potent than CsA in inhibiting immune responses. TAC has decreased episodes of rejection. TAC is combined with lower doses of glucocorticoids. But TAC is more nephrotoxic and neurotoxic.  Sirolimus (Rapamycin) SRL is macrolide antibiotic. It is not a calcineurin inhibitor. Sirolimus inhibits the response of T cells to IL-2 and thereby blocks activation of T- & B-cells SRL blocks the progression of activated T cells from G1 to S phase of cell cycle (Antiproliferative action). It does not block the IL-2 production but blocks T cell response to cytokines. Inhibits B cell proliferation & immunoglobulin production. It binds to FK-BP and the formed complex binds to mTOR (mammalian Target Of Rapamycin). mTOR is serine-threonine kinase essential for cell cycle progression, DNA repairs, protein translation. Pharmakinetics Given orally and topically, reduced by fat meal. Extensively bound to plasma proteins metabolized by CYP3A4 in liver. Excreted in feces. Pharmacodynamics Immunosuppressive effects Anti- proliferative action. Equipotent to CsA. USES Synergistic action with CsA Solid organ allografts alone or combined with (CSA, tacrolimus, steroids, mycophenolate). Hematopoietic stem cell transplant recipients. Topically with cyclosporine in uveoretinitis. In halting graft vascular disease. in conjunction with coronary stents to prevent restenosis in coronary arteries following balloon angioplasty. Toxic effects Hyperlipidaemia (cholesterol, triglycerides). Thrombocytopenia Leukopenia Hepatotoxicity Hypertension GIT dysfunction  Inhibitors of cytokine gene expression Corticosteroids – Prednisone – Prednisolone – Methylprednisolone – Dexamethasone They have both anti-inflammatory action and immunosuppressant effects. Mechanism of action Anti-inflammatory action – Induce lipocortin-1 synthesis, which binds to cell membranes preventing the phospholipase A2. This leads to diminished eicosanoid production and cyclooxygenase expression – Decrease production of inflammatory mediators as prostaglandins, leukotrienes, histamine, PAF, bradykinin. – inhibit gene transcription of many inflammatory genes. Immunosuppressant action – suppress the cell-mediated immunity decrease production of cytokines IL-1, IL-2, interferon, TNF & decrease T lymphocyte proliferation. – Glucocorticoids also suppress the humoral immunity by reducing both B cell clone expansion and antibody synthesis Kinetics Can be given orally, parenterally, topically and by inhalation (asthma). Dynamics 1. anti-inflammatory and immunosuppresant. 2. Suppression of response to infection 3. Metabolic effects. Indications – Solid organ allografts & haematopoietic stem cell transplantation. – Autoimmune diseases as refractory rheumatoid arthritis, systemic lupus erythematosus, asthma – Acute or chronic rejection of solid organ allografts. Adverse Effects – Adrenal suppression – Osteoporosis – Hypercholesterolemia – Hyperglycemia – Hypertension – Cataract – Infection III. Cytotoxic drugs  Antimetabolites (Inhibitors of purine or pyrimidine synthesis) – Leflunomide – Azathioprine – Myclophenolate Mofetil – Methotrexate  Alkylating agents Cyclophosphamide  AZATHIOPRINE CHEMISTRY: – Derivative of mercaptopurine. – Prodrug. – Cleaved to 6-mercaptopurine then to 6-mercaptopurine nucleotide, thio-inosine monophosphate (nucleotide analog). – Inhibits de novo synthesis of purines required for lymphocytes proliferation. – Prevents clonal expansion of both B and T lymphocytes. Pharmacokinetics – orally or intravenously. – Widely distributed but does not cross BBB. – Metabolized in the liver to thiouric acid (inactive metabolite) by xanthine oxidase. – excreted primarily in urine. Drug Interactions: – Co-administration of allopurinol with azathioprine may lead to toxicity due to inhibition of xanthine oxidase by allopurinol. USES Acute glomerulonephritis Systemic lupus erythematosus Rheumatoid arthritis Crohn’ s disease. Autoimmune hemolytic anemia. Adverse Effects Bone marrow depression: leukopenia, thrombocytopenia. Gastrointestinal toxicity. Hepatic dysfunction. Increased risk of infections.  MYCOPHENOLATE MOFETIL – Is a semisynthetic derivative of mycophenolic acid from fungus source. – Prodrug; is hydrolyzed to mycophenolic acid. Mechanism of action: – Inhibits de novo synthesis of purines. – mycophenolic acid is a potent inhibitor of inosine monophosphate dehydrogenase (IMP), crucial for purine synthesis deprivation of proliferating T and B cells of nucleic acids. Pharmacokinetics: – Given orally, i.v. or i.m. – rapidly and completely absorbed after oral administration. – It undergoes first-pass metabolism to give the active moiety, mycophenolic acid (MPA). – MPA is extensively bound to plasma protein. – metabolized in the liver by glucuronidation. – Excreted in urine as glucuronide conjugate – Dose : 2-3 g /d CLINICAL USES: In solid organ transplantation – hematopoietic stem cell transplant patients. – Combined with tacrolimus as prophylaxis to prevent graft versus host disease. In autoimmune disorders: – Rheumatoid arthritis, & dermatologic disorders. ADVERSE EFFECTS: – GIT toxicity: Nausea, Vomiting, diarrhea, abdominal pain. – Leukopenia, neutropenia. – Lymphoma Contraindicated during pregnancy  LEFLUNOMIDE  antimetabolite immunosuppressant.  Pyrimidine synthesis inhibitor  Can be given orally  A prodrug  Active metabolite undergoes enterohepatic circulation.  Has long duration of action.  Approved only for rheumatoid arthritis Adverse effects 1. Elevation of liver enzymes 2. Renal impairment 3. Teratogenicity 4. Cardiovascular effects (tachycardia). Methotrexate – a folic acid antagonist – Orally, parenterally (I.V., I.M). – Excreted in urine. – Inhibits dihydrofolate reductase required for folic acid activation (tetrahydrofolate) – Inhibition of DNA, RNA &protein synthesis – Interferes with T cell replication. – In treatment of many neoplastic disorders including acute lymphoblastic leukemia. – Autoimmune disorders as rheumatoid arthritis & psoriasis and Croh’n disease Adverse effects – Pulmonary fibrosis – Nausea-vomiting-diarrhea – Alopecia – Bone marrow depression – Teratogenicity (X) Cyclophosphamide – Alkylating agent to DNA. – Prodrug, activated into phosphamide. – Is given orally& intravenously – Destroy proliferating lymphoid cells. – Anticancer in lymphomas. – Effective in autoimmune diseases – e.g rheumatoid arthritis – Systemic lupus erythrematosus. – Autoimmune hemolytic anemia Side Effects – Alopecia – Hemorraghic cystitis. – Bone marrow suppression – GIT disorders (Nausea –vomiting-diarrhea) – Sterility (testicular atrophy & amenorrhea)  Antibodies are sometimes used as a quick and potent immunosuppressive therapy to prevent the acute rejection reactions Polyclonal antibodies Antilymphocyte globulins (ALG). Antithymocyte globulins (ATG). Monoclonal antibodies - Rho (D) immunoglobulin. – Basiliximab – Daclizumab Antibodies Preparation 1. by immunization of either horses or rabbits with human lymphoid cells producing mixtures of polyclonal antibodies directed against a number of lymphocyte antigens (variable, less specific). 2. Hybridoma technology produce antigen-specific, monoclonal antibody (homogenous, specific). produced by fusing mouse antibody-producing cells with immortal, malignant plasma cells. Hybrid cells are selected, cloned and selectivity of the clone can be determined. Recombinant DNA technology can be used to replace part of the mouse gene sequence with human genetic material (less antigenicity- longer half life). Antibodies from mouse contain Muro in their names. Humanized antibodies contain ZU (humanized) or XI (chimeric) in their names.  Antilymphocyte globulins (ALG) &Antithymocyte globulins (ATG) Polyclonal antibodies obtained from plasma or serum of horses hyper-immunized with human lymphocytes. Binds to the surface of circulating T lymphocytes, which are phagocytosed in the liver and spleen giving lymphopenia and impaired T-cell responses & cellular immunity. Kinetics Given i.m. or slowly infused intravenously. Half life extends from 3-9 days. Uses Combined with cyclosporine for bone marrow transplantation. To treat acute allograft rejection. Steroid-resistant rejection. Adverse Effects: – Antigenicity. – Anaphylactic and serum sickness reactions (Fever, Chills, Flu-like syndrome). – Leukopenia, thrombocytopenia. – Risk of viral infection. Monoclonal antibodies Muromonab-CD3 Is a murine monoclonal antibody Prepared by hybridoma technology Directed against glycoprotein CD3 antigen of human T cells. Given I.V. Metabolized and excreted in the bile. Mechanism of action The drug binds to CD3 proteins on T lymphocytes (antigen recognition site) leading to transient activation and cytokine release followed by disruption of T-lymphocyte function, decreased immune response. Block killing by cytotoxic T cells. Prednisolone, diphenhydramine are given to reduce cytokine release syndrome. Uses Used for treatment of acute renal allograft rejection & steroid-resistant acute allograft To deplete T cells from bone marrow donor prior to transplantation. Adverse effects Anaphylactic reactions. Fever CNS effects (seizures) Infection Cytokine release syndrome (Flu-like illness to shock like reaction).  Monoclonal antibodies Basiliximab and Daclizumab  Obtained by replacing murine amino acid sequences with human ones.  Basiliximab is a chimeric human-mouse IgG (25% murine, 75% human protein).  Daclizumab is a humanized IgG (90% human protein).  Have less antigenicity & longer half lives than murine antibodies Mechanism of action IL-2 receptor antagonists Are Anti-CD25 Bind to CD25 (α-subunit chain of IL-2 receptor on activated lymphocytes) Block IL-2 stimulated T cells replication & T- cell response system Basiliximab is more potent than Daclizumab.  Given I.V. Half life Basiliximab (7 days ) Daclizumab (20 days) are well tolerated - only GIT disorders USES Given with CsA and corticosteroids for Prophylaxis of acute organ rejection in renal transplantation.  INTERFERONS Families: Type I IFNs ( IFN-α, β ): induced by viral infections leukocyte produces IFN-α Fibroblasts & endothelial cells produce IFN-β Type II IFN (IFN-γ): Produced by Activated T lymphocytes. Interferon types and uses: IFN- α: Hepatitis B & C infections Treatment of cancer (malignant melanoma) IFN-β : Multiple sclerosis IFN- γ : treatment of chronic granulomatous diseases  VI. INTERFERONS Recombinant DNA cloning technology. Antiproliferative activity. Antiviral action Immunomodulatory effect. USES: – Treatment of certain infections e.g. Hepatitis C (IFN- α ). – Autoimmune diseases e.g. Rheumatoid arthritis. – Certain forms of cancer e.g. melanoma, renal cell carcinoma. – Multiple sclerosis (IFN- β): reduced rate of exacerbation. – Fever, chills, myelosuppression.  20 Diuretics #!
%!# Diuretics are drugs, which increase the rate of urine flow. However, clinically useful diuretics also increase excretion of Na+ and an accompanying anion (negatively charged ion) like Cl–. Since NaCl is the major determinant of extracellular fluid volume, diuretics reduce extracellular fluid volume (decrease in oedema) by decreasing total body NaCl content. Although continued use of diuretic causes sustained net loss of Na+, the time course for this effect is limited by compensatory mechanisms including activation of the renin-angiotensin-aldosterone pathway and the sympathetic nervous system. When blood is filtered at the glomerulus, the fluid which enters the proximal tubule is really the developing urine. As the tubular fluid passes down the tubule, solutes (Na+, K+, Cl–) are removed from the fluid and returned to the blood (reabsorption). Diuretics inhibits the reabsorption of Na+ ions, thereby reduces the quantity of the water in body fluids. Triamterene Spironolactone K-Sparing Acetazolamide Thiazides mercurials mercurials Glomerulus Proximal tubule Distal tubule Osmotic diuretics Osmotic diuretics Furosemide Ethacrynic acid Loop of Henle Cortical collecting tubule Site of action of diuretics. 259 260 PRINCIPLES OF ORGANIC MEDICINAL CHEMISTRY $!# !$
% The following classes of diuretics are therapeutically used: Type Example Site of action Mechanism Carbonic anhydrase Acetazolamide Proximal tubule Inhibition of CA (CA) inhibitors Osmotic diuretics Mannitol Loop of henle ; Osmotic action Proximal tubule Loop diuretics Furosemide Loop of henle Inhibition of Na+-K+ 2Cl– symport Thiazides Hydrochlorothiazide Distal convoluted Inhibition of tubule Na+-Cl– symport Potassium sparing diuretics 1. Na+ channel Triamterene, Collecting tubule Inhibition of Na+ Inhibitors Amiloride channel 2. Aldosterone Spiranolactone Anti-diuretic hormone antagonist '!# #&
 67 #'!   Carbonic anhydrase (CA) inhibitors are derived from the sulphonamide antibacterials. Sulfonamide group (—SO2NH2) is essential for its activity. In 1937, Southworth observed that sulphanilamide not only had antibacterial activity but also produced systemic acidosis and an alkaline urine ( HCO 3− excretion). The carbonic anhydrase inhibitors must have unsubstituted sulphamoyl (—SO2NH2) group. Some potent CA inhibitors have an aromatic group (phenyl or heterocycle) attached to sulphamoyl group.       This class of diuretics inhibit carbonic anhydrase enzyme in the membrane and cytoplasm of the epithelial cells. The primary site of action is proximal tubules. These agents interfere with the reabsorption of HCO 3−. HCO 3− is reabsorbed in the proximal tubule and requires the activity of carbonic anhydrase. Intracellularly carbonic anhydrase (CA in the DIURETICS 261 diagram) converts H2O and CO2 to carbonic acid (H2CO3). H2CO3 dissociates into H+ and HCO3–. The HCO3– is transported across the basolateral membrane. H+ is secreted into the tubular lumen in exchange for Na+. The H+ combines with a filtered HCO3– (using CA) to form H2CO3, which immediately dissociates into H2O and CO2 that, is reabsorbed. Therefore, filtered bicarbonate is reabsorbed for every H+ secreted. Carbonic anhydrase inhibitors, by blocking the enzyme, prevent the reabsorption of HCO3–. Accumulation of HCO3– in the tubular lumen subsequently inhibits Na+ —H+ exchange and Na+ reabsorption. The increase in sodium concentration in the tubular fluid may be com- pensated partially by increased NaCl reabsorption in later segments of the tubule. Thus, the diuretic effect of the carbonic anhydrase inhibitors is mild. Synthesis of Acetazolamide. Acetazolamide is prepared by the following reactions : N N Rearrangement (CH CO) O NH2—NH2.H2O + NH4SCN → → 3 2 Hydrazine hydrate H2N S SH 5-Amino-2-mercapto 1,3,4-thiadiazole N N N N Cl2 NH → H → 3 SH H2O  SOCl2 HN S H3C—C—N S   C=O O  N N CH3 O  SO2NH2 H3C—C—HN S Acetazolamide Structure-Activity Relationships : 1. All inhibit carbonic anhydrase activity. 2. Importantly, they have no antibacterial activity. 3. Substitution on the sulphamoyl group gives inactive compounds. 262 PRINCIPLES OF ORGANIC MEDICINAL CHEMISTRY %  Carbonic anhydrase inhibitors are also used for non-diuretic indications, such as man- agement of glaucoma, and as adjuvants for anti- epileptic drugs. !!
% Chemistry. Osmotic diuretics are the agents that mobilise fluids by increasing the osmotic pressure in tubules. Some important osmotic diuretics are below : Osmotic diuretics Oral Drug Structure Absorption HO OH Glycerin Orally active OH H HO H Isosorbide O Orally active O H H OH OH H H OH OH OH Mannitol H H Negligible H OH OH H H H O Urea Negligible H2N NH2 DIURETICS 263       Osmotic diuretics are substances to which the tubule epithelial cell membrane has lim- ited permeability. When administered (often in a large dosage), osmotic diuretics significantly increase the osmolarity of plasma and tubular fluid. The osmotic force thus generated pre- vents water reabsorption, and also extracts water from the intracellular compartment, ex- pands extracellular fluid volume and increases renal blood flow resulting in reduced medulla tonicity. The primary sites of action for osmotic diuretics are the Loop of Henle and the proxi- mal tubule where the membrane is most permeable to water. !!
% ! *#
% 3 Chemistry. The diuretics that produce peak diuresis than other diuretics and act dis- tinctly on renal tubular function (at loop of Henle) are called loopdiuretics or high-ceiling diuretics. There are two major classes of loop diuretics: 1) sulfonamide derivatives such as furosemide, bumetanide and torsemide; and 2) non-sulfonamide loop diuretic such as ethacrynic acid. 264 PRINCIPLES OF ORGANIC MEDICINAL CHEMISTRY       Loop diuretics inhibit reabsorption of NaCl and KCl by inhibiting the Na+ —K+ —2Cl– symport in the luminal membrane of the thick ascending limb (TAL) of loop of Henle. As TAL is responsible for the reabsorption of 35% of filtered sodium, and loop diuretics are highly efficacious and are thus called high ceiling diuretics. The Na+ —K+ —2Cl– symport and sodium pump together generate a positive lumen potential that drives the reabsorption of Ca++ and Mg++, inhibitors of the Na+ -K+ -2Cl– symport also inhibit reabsorption of Ca++ and Mg++. Loop diuretics also have direct effects on vasculature including increase in renal blood flow, and increase in systemic venous capacitance. + + + + – Na Na Na K Cl Symporter + + Interstitial K K space inhibited by loop diuretics + + Cl Cl Tubular lumen Structure-Activity Relationships of Ethacrynic acid : 1. Designed to mimic mercurial diuretics. 2. Increased activity when a electron withdrawing group (i.e. Cl–) is placed ortho to the unstaturated ketone (active site for sulfhydryl reactivity). 3. Ortho and meta positions substituted with chlorine produce most active compound. Synthesis of ethacrynic acid. Ethacrynic acid is synthesized by the following chemi- cal reactions : DIURETICS 265 Synthesis of furosemide. Furosemide is synthesized by the following chemical reac- tions : COOH COOH Cl Cl (i) ClSO3H + → → (ii) NH3 H2NSO2 O CH2NH2 Cl Cl 2, 4-Dichloro 2, 4-Dichloro-5-sulphamoyl Furfurylamine benzoic acid benzoic acid COOH NHCH2 O H2NO2S Cl Frusemide (4-Chloro-N-furfuryl -5-sulphamoylanthranilic acid). Uses of Loop diuretics. Particularly useful in acute left ventricular failure and pul- monary oedema because of quick onset and powerful diuretic action. They may also be used to treat hypercalcaemia. 1
   Thiazides are also called benzothiadiazides. Thiazides are sulfonamide derivatives. 266 PRINCIPLES OF ORGANIC MEDICINAL CHEMISTRY Drug Structure Chlorothiazide R2 = H, R3 = H, R6 = Cl R2 = H, R3 = H, R6 = Cl Hydrochlorothaizide (Saturated between C3 and N4) R2 = H, R3 = H, R6 = CF3 Hydroflumethiazide (Saturated between C3 and N4) Bendroflumethiazide R2 = H, R3 = CH2 , R6 = CF3 (Saturated between C3 and N4) Some diuretics having similar pharmacological actions as thiazides but have the follow- ing structures (different from thiazides) : OH SO2NH2 Chlorthalidone NH Cl O H3C Indapamide Cl C—N—N   H2NO2S O H H  CH3 Cl N Metolazone N H3NO2S O H3C H  Cl N CH2CH3 Quinethazone NH H3NO2S O       Thiazides inhibit a Na+—Cl– symport in the luminal membrane of the epithelial cells in the distal convoluted tubule. Thus, thiazides inhibit NaCl reabsorption in the distal convo- luted tubule, and may have a small effect on the NaCl reabsorption in the proximal tubule. DIURETICS 267 Thiazides enhance Ca++ reabsorption in the distal convoluted tubule by inhibiting Na+ entry and thus enhancing the activity of Na+ —Ca++ exchanger in the basolateral membrane of epithelial cells. + Lumen Na + + – Na Na Na , Cl + symporter – K Cl Cl Interstitial Cl space Blocked by – thiazides Cl Structure-Activity Relationships of thiazides. These compounds are weakly acidic ; 1. H atom at N-2 is the most acidic due to the electron-withdrawing effects of the neigh- bouring sulfone group. 2. Sulfonamide group at C-7 provides an additional point of acidity in molecule but is less acidic than N-2 proton. A free sulfamoyl group at position 7 is essential for diu- retic activity. 3. These acidic protons make possible the formation of a water-soluble sodium salt that can be used for I.V. dosing. 4. An electron-withdrawing group is essential at position 6. 5. The diuretic activity is enhanced by substitution at position 3. 6. Replacement of 6-Cl by 6-CF3 does not change potency but allters duration of action. 7. Replacement of 6-Cl by electron-donating groups (e.g. CH3) reduces diuretic activity. 8. Saturation of thiadiazine ring to give 3, 4-dihydro derivative and replacement re- place or removal of sulfonamide group at position C-7 yields compounds with little or no diuretic activity. Synthesis of bendroflumethiazide : H2NO2S SO2NH2 Ammonia solution/DMF + CH2CHO  → F3C NH2 2, 4-Disulphamoyl-5- Phenylacetaldehyde trifluoromethylaniline O2 H2NO2S S NH CH2 F3C N H Bendroflumethiazide 268 PRINCIPLES OF ORGANIC MEDICINAL CHEMISTRY Synthesis of chlorothiazide. Chlorothiazide is synthesized by the following route : Cl NH2 Cl NH2 Cl NH2 HOSO2Cl NH4OH → → ∆ ClO2S SO2Cl NH2O2S SO2NH2 H  Cl N Cl N HCOOH → → NH Reduction with NH H2NO2S S LiAlH4 H2NO2S S O2 O2 Chlorthiazide Hydrochlorthiazide Uses of Thiazides. In mild cardiac failure, where the lesser diuretic effect may be more acceptable to the patient. Main use of thiazides is in antihypertensive therapy. !%*#
% 1. Na+ Channel Inhibitors. E.C. Taylor and J. Weinstock introduced aminopteridines as potassium-sparing diuretics. Ex : Triamterene and amiloride. Chemistry. Amiloride and triamterene are the only two drugs in this class. The most active and successful compound of the class proved to be triamterene. Mechanism of action. Amiloride and triamterene inhibit the sodium channel in the luminal membrane of collecting tubule and collecting duct. This sodium channel is critical for Na + entry into cells down the electrochemical gradient created by sodium pump in the basolateral membrane, which pumps Na+ into interstitium. This selective transepithelial trans- port of Na+ establishes a luminal negative transepithelial potential which in turn drives secre- tion of K+ into the tubule fluid. The luminal negative potential also facilitates H+ secretion via the proton pump in the intercalated epithelial cells in collecting tubule and collecting duct. Inhibition of the sodium channel thus not only inhibits Na+ reabsorption but also inhibits secretion of K+ and H+, resulting in conservation of K+ and H+. DIURETICS 269 + + Na , K ATPase + + + Na Na Na Na entry blocked – 60 mV + + K K by Na channel –75 mV antagonists + + + K K K channel + secretes K into the tubular lumen 28  /  4  Chemistry. Spironolactone is the only available aldosterone antagonist. A metabolite of spironolactone, canrenone, is also active and has a half-life of about 16 hours. O O CH3 CH3 H H H O S O CH3 Mechanism of action. Aldosterone, by binding to its receptor in the cytoplasm of epithelial cells in collecting tubule and duct, increases expression and function of Na+ channel and sodium pump, and thus enhances sodium reabsorption (see “Na+ channel inhibitors” above). Spironolactone competitively inhibits binding of aldosterone to its receptor and abolishes its biological effects. DIURETICS  Objectives of today’s Lecture Diuretics Definition Classification Names of members in classes Mechanism of action Major indications Major side effects and Precautions Major drug interactions MCQs related to Diuretics  Facts of Renal Physiology Kidney- – Weight- 0.5% of Body, – Receive 25% of cardiac output (50 times) Kidney functions – Balance of electrolytes, Plasma volume, Acid Base – Activation of Vitamin D – Synthesis of Erythropoietin, Urokinase – Excretion of Urea, Uric acid, Creatinine etc. Transport types – Passive Simple, channel mediated and facilitated diffusion, solvent drag – Active Primary and Secondary (Symports and Secondary Counter transport)  Facts related to Renal Physiology Pressure difference at Bowman’s Capsule- 20mm Hg Filter= Plasma-Proteins Volume of – Filter- 180 liters – Urine- 1.5 liters (1%) Kidneys – Renal Blood Flow- 1200ml/min – Renal Plasma Flow- 650 ml/min – GFR- 120 ml/min – Reabsorb – Sodium, Chloride and Bicarbonates > 99% while Potassium about 85%  Terminology Natriuresis- increased sodium excretion Kaliuresis- Increased Potassium excretion Diuretics- Drugs which cause a net loss of Na+ and water in urine. (Exception- Osmotic diuretics (Mannitol) don't cause natriuresis but produce diuresis Nephron Parts and Characters  Proximal Tubule Leaky- Freely permeable to water, solutes Active absorption of – Sodium Chloride, – Sodium Bicarbonate – Glucose – Amino Acids – Organic Solutes Followed by passive absorption of water  Loop Of Henle Descending limb- – Permeable to water Thick ascending limb – – Impermeable to water but – Permeable to sodium by Na+K+2Cl- Co transport – About 25% of filtered sodium is absorbed here Macula Densa and Juxtaglomerular Apparatus Contact between Ascending limb with afferent arterioles – by specialized columnar epithelial cells Macula Densa Macula Densa sense NaCl conc. in filtrate Give signal to J.G. Cells present in afferent arterioles J.G. Cells of afferent arterioles secrete Renin RAAS in response to low BP, or Low Na Renin- – Angiotensinogen - Angiotensin I ACE- – Angiotensin II- – Sympathetic, Aldosterone Vasoconstriction, Sodium and water retention,  Early Distal Tubule Active transport of sodium by NaCl symport Calcium excretion is regulated (Parathomone and Calcitriol, increase absorption of calcium) Collecting Tubule and Collecting Duct Aldosterone- On membrane receptor and cause sodium absorption by Na+/H+/ K+ Exchange ADH- Collecting tubular epithelium permeable to water (Water enters through aquaporin-2)  Nephron parts and their functions SEGMENT FUNCTION Glomerulus Formation of glomerular filtrate Proximal convoluted Reabsorption of 65% of filtered Na+/K+/ Ca2+, and Mg2+; 85% of NaHCO3, tubule (PCT) (activity of Carbonic an-hydrase enzyme) and nearly, 100% of glucose and amino acids. Iso-osmotic reabsorption of water., Secretion and reabsorption of organic acids and bases, including uric acid and most diuretics Thin descending limb of Passive reabsorption of water Henle’s loop Thick ascending limb of Henle’s loop (TAL) Active reabsorption of 25% of filtered Na+/K+/2Cl− ; , secondary re-absorption of Ca2+ and Mg2+ Distal convoluted tubule (DCT) Active reabsorption of 4–8% of filtered Na+ Cl− Ca2+ reabsorption ; under parathyroid hormone control Cortical collecting tubule Na+ reabsorption (2–5%) coupled to K+ and H+ secretion (under (CCT) Aldosterone) Medullary collecting duct Water reabsorption under Vasopressin control The relative magnitudes of Na+ reabsorption at sites PT - 65% Asc LH - 25% DT - 9% CD - 1%.  Control of Renal Function Sympathetic- Increase Na reabsorption, Renin RAAS- Renin in response to Low sodium, Low BP ADH – Water reabsorption at collecting duct Atrial Natriuretic Peptide/Factor- Released when atrial pressure is high and causes solute and water diuresis and reduces blood volume and BP. Inhibits synthesis of Renin, Aldosterone, ADH and overcomes the long term persistent effect of aldosterone (Opposite of RAAS) Prostaglandins- maintain renal circulation Breath for a minute Pharmacology copy by student  Diuretics Carbonic Anhydrase Inhibitors (Site I) – Brinzolamide, Acetazolamide, Dorzolamide Osmotic Diuretic (Site II) – Glycerine, Urea, Mannitol, Isosorbide Loop Diuretics (Site III)- TALH – Frusemide/ Furosemide, Bumetanide, Torasemide, Ethacrynic acid Thiazide Diuretics (Site IV) – Hydrochlorothiazide, Clopamide, Benzthiazide, Chlorthalidone, Metolazone, Xipamide, Indapamide Potassium Sparing Diuretics (Site V) – Aldosterone Antagonist Spironolactone, Canrenone, Eplerone – Direct Acting (Inhibition of renal epithelial Nq+ channel Triamterene, Amiloride (more potent) Carbonic An-hydrase Inhibitors Thiazide diuretics Osmotic Diuretics Potassium Sparing Diuretics Loop Diuretics (High Ceiling) Carbonic Anhydrase Inhibitors Carbonic Anhydrase Inhibitors Loop Diuretics Thiazides  Spironolactone Amiloride A - GM- Brings FruTE- Cuts MIXs with Big Hands- And Starts Taking- A - Acetazolamide Carbonic Anhydrase Inhibitors (Site I) GM- Glycerine, Mannitol Osmotic Diuretics (Site I, II and…) Brings FruTE- Bumetanide, Furosemide, Loop Diuretics (Site III) Torasemide, Ethacrynic acid Cuts MIXs with Big Hands- Clopamide, Chlorthalidone, Metolazone, Indapamide, Xipamide, Benzthiazide, Hydrochlorthiazide, Thiazide Diuretics (Site IV) And Starts Taking- Amiloride, Spironolactone, Triamterene Potassium Sparing Diuretics (Site V) Diuretic Site of Action Adverse Effects Special points Carbonic PTC Metabolic Acidosis Weak, Used in Glaucoma, Petit mal epilepsy, anhydrase (inhibition of CAE) Acute mountain sickness, to alkaline the urine inhibitors Osmotic PTC, LOH, DCT Shifting of fluid from Potent Diuretics (Osmotic retention of water, intracellular to Used in Glaucoma, Poisoning, Increased ICT, Dilates Afferent arterioles, extracellular, impending ARF Increased hydrostatic Hyponatremia, pressure in glomerulus Pulmonary edema Loop Thick Ascending Limb of Hyponatremia Most potent, Most Potent is Bumetanide, Diuretics Henle Hypomagnesaemia Effective even in low GFR, All except Ethacrynic (NaK2Cl inhibition) Hypocalcaemia acid are sulphonamide related, Weak CAI action Hyperuricemia Venodilatation, Decrease Left Ventricle Pressure, Hyperglycemia Used in Acute LVF, Pulmonary Edema, Nephrotic Hyperlipidemia syndrome, ARF, NSAIDS blunt effect, Cerebral Hyperuricemia edema, short term tt of Hypertension, to reduce Ototoxic (ECA) volume overload during transfusion, Thiazide DCT Hypokalemic Moderate, Chlorthalidone is Longest acting, Diuretics (NaCl) metabolic alkalosis Paradoxical effect in Diabetes Insipidus (Gitelman’s First line in Hypertension, Syndrome) Hypercalcemia Potassium CD HyperKalemia Weak, As supplement to other to counter the Sparing Antiandrogenic effect hypokalemia, Canrenone is active metabolite, used in Conn’s syndrome (Primary Hyperaldosteronism) cirrhotic Diuretics edema, polycystic ovary Disease States The diuretic drugs are used primarily to treat two medically important conditions, edema and hypertension. Both conditions are common, although some patients exhibit refractory disease states that require additional modification of the drug regimen to include alternative diuretics or addition of nondiuretic drugs. Diuretic drugs may be administered acutely or chronically to treat edematous states. When immediate action to reduce edema (e.g., acute pulmonary edema) is needed, intravenous administration of a loop diuretic often is the approach of choice. Thiazide or loop diuretics normally are administered orally to treat nonemergency edematous states. The magnitude of the diuretic response is directly proportional to the amount of edema fluid that is present. As the volume of edema decreases, so does the magnitude of the diuretic response with each dose. If concern exists about diuretic- induced hypokalemia developing, then a potassium supplement or potassium- sparing diuretic may be added to the drug regimen. The development of hypokalemia is particularly important for patients with congestive heart failure who also are taking cardiac glycosides, such as digitalis. Digitalis has a narrow therapeutic index, and developing hypokalemia can potentiate digitalis- induced cardiac effects with potentially fatal results. Diuretic drugs (thiazide and loop diuretics) are administered orally to help control blood pressure in the treatment of hyper tension. Diuretics often are the first drugs used to treat hyper tension, and they also may be added to other drug therapies used to control blood pressure with beneficial effects. Thiazide Diuretics: Examples: Hydrochlorothiazide, Chlorthalidone Disease States: Hypertension, Edema associated with heart failure, Nephrolithiasis (calcium-containing kidney stones), Diabetes insipidus Loop Diuretics: Examples: Furosemide, Bumetanide, Torsemide Disease States: Heart failure, Edema (including pulmonary edema), Hypertension, Chronic kidney disease, Liver cirrhosis with ascites, Acute kidney injury Potassium-Sparing Diuretics: Examples: Spironolactone, Eplerenone, Amiloride, Triamterene Disease States: Hypertension, Heart failure, Edema (especially in patients at risk of hypokalemia), Primary aldosteronism, Hyperaldosteronism, Hypokalemia Osmotic Diuretics: Examples: Mannitol, Glycerol Disease States: Intracranial hypertension, Cerebral edema, Acute kidney injury, Glaucoma (mannitol) Carbonic Anhydrase Inhibitors: Examples: Acetazolamide, Dorzolamide (ophthalmic) Disease States: Glaucoma (topical), Altitude sickness, Metabolic alkalosis, Epilepsy (rarely), Edema (rarely) Combination Diuretics: Examples: Hydrochlorothiazide/triamterene (Dyazide), Hydrochlorothiazide/amiloride (Moduretic) Disease States: Hypertension, Edema, Heart failure  MONITORING TEST Electrolyte Levels: Diuretics can affect electrolyte balance, leading to abnormalities such as hypokalemia (low potassium), hyponatremia (low sodium), hypomagnesemia (low magnesium), and hyperkalemia (high potassium) in some cases. Regular monitoring of electrolyte levels, including potassium, sodium, and magnesium, is crucial, especially during the initiation of therapy and dose adjustments. Renal Function Tests: Diuretics can impact renal function, particularly in patients with preexisting kidney disease. Monitoring tests such as serum creatinine and blood urea nitrogen (BUN) can help assess renal function and detect any deterioration, especially in high-risk patients. Fluid Status: Monitoring fluid status is essential, particularly in patients with conditions such as heart failure or edema. Assessments of body weight, fluid intake and output, and clinical signs of fluid overload or dehydration can help guide diuretic therapy and prevent complications. Blood Pressure: Diuretics are commonly used to treat hypertension, so monitoring blood pressure is essential to assess the medication's effectiveness in controlling blood pressure levels.  MONITORING TEST Blood Glucose Levels: Thiazide diuretics, in particular, can affect blood glucose levels and may exacerbate diabetes or increase the risk of developing diabetes in predisposed individuals. Monitoring blood glucose levels is important, especially in patients with diabetes or prediabetes. Lipid Profile: Some diuretics, such as thiazides, can affect lipid metabolism and may lead to alterations in lipid levels. Monitoring lipid profiles periodically can help identify any changes and guide appropriate management. Renin-Aldosterone Levels: In certain conditions such as primary aldosteronism, monitoring renin and aldosterone levels can help assess the response to diuretic therapy and guide treatment decisions. Symptom Assessment: Regular assessment of symptoms such as dyspnea, edema, fatigue, and dizziness can help evaluate the response to diuretic therapy and detect any adverse effects or worsening of the underlying condition. Monitoring tests should be tailored to the individual patient's clinical situation and risk factors, and the frequency of testing may vary depending on factors such as the type and dose of diuretic, the presence of comorbidities, and the overall treatment goals. Regular follow-up with a healthcare provider is essential to ensure appropriate monitoring and management of patients receiving diuretic therapy.